Drug Discovery and Development

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DRUG DISCOVERY AND DEVELOPMENT

293 2-(4-HYDROXY-3-PROP-2-ENYL-PHENYL)-4-PROP-2-ENYLPHENOL INHIBITS N-FORMYL-L-METHIONYL-L-LEUCYLL-PHENYLALANINE-INDUCED SUPEROXIDE ANON PRODUCTION AND CATHEPSIN G RELEASE BY SPECIFIC MODULATE LYN KINASE ACTIVITY IN HUMAN NEUTROPHILS Liao H., Chien C., Chen J., Lee T., Lin S., Tseng C. Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4prop-2-enyl-phenol (honokiol), a lignan extracted from the stem bark of Magnolia officinalis Rehd. et Wils (Magnoliaceae), on N-formyl-Lmethionyl-L-leucyl-L-phenylalanine (fMLP)-induced human neutrophils activation. Signaling pathways regulated by honokiol to oppose fMLP-induced respiratory burst were evaluated by Lyn kinase (a member of Src kinase family) activity in an in vitro kinase assay and by immunoblotting analysis of downstream phosphorylation targets of Lyn. Briefly, honokiol specific inhibited fMLP-induced superoxide anion production and cathespin G release in a concentration-dependent manner (IC50 = 9.80 0.21; 14.23 1.43 lM respectively). Further, honokiol specific suppressed fMLP-induced Lyn phosphorylation and Lyn kinase activity. Moreover, honokiol attenuated the signaling followed by Lyn kinase, such as Tec translocation from the cytosol to the inner leaflet of the plasma membrane, phosphorylation of AKT, P38, PLCc2, protein kinase C and membrane localization of p47phox, however, honokiol did not affect fMLP-induced Hck phosphorylation, Fgr kinase activity, and the signaling followed by Hck/Fgr kinases, such as Vav1 and extracellular signal-regulated kinase phosphorylation. Moreover, honokiol, like PP2, attenuated intracellular calcium concentration. However, honokiol neither inhibited NADPH oxidase activity nor increased cyclicAMP levels. Honokiol is not a competitive or allosteric antagonist of fMLP. Additionally, in an in vivo study, honokiol prevents fMLP-induced neutrophil infiltration in mice. Conclusion: honokiol opposes fMLP-mediated neutrophil activation by inhibiting Lyn activation and subsequent interfered with the activation of PLCc2, AKT, p38, protein kinase C, and p47phox.

294 4-HYDROXYBENZALDEHYDE ISOLATED FROM GASTRODIA ELATA, ENHANCED SLEEPING BEHAVIORS THROUGH GABAA-ERGIC SYSTEMS Choi J., Han J., Oh E., Hong J., Yun Y., Yoo H., Oh K. Gastrodia elata (GE), one of famous oriental herb medicine, has been used for the treatment of headache, blood pressure and convulsion. Recent studies have been suggested it has sedative and inhibitory effects of platelet aggregation. 4-Hydroxybenzaldehyde (4-HD) is one of the major compounds of GE, which is well known as an inhibitor of GABA-transaminase. This research was investigated to know whether 4-HD enhanced sleeping behaviors through GABAAergic systems in mice. 4-HD (50, 100, and 200 mg/kg, p.o.) inhibited locomotor activity in mice. 4-HD not only increased total sleep time, but also decreased sleep latency in pentobarbital induced sleeping mice. 4-HD showed synergistic effects with muscimol, suggesting interactions with GABAAergic drugs. 4-HD increased intracellular chloride in primary cultured hypothalamic granular cells at the dose-dependent manner. The GABAA receptors subtypes by western blot partly were over-

expressed. In conclusion, these experiments provide that 4-HD increases sleeping behaviors through GABAA receptors- chloride channel complex.

295 A COMPARATIVE STUDY FOR THE TOPICAL TREATMENT OF ATOPIC DERMATITIS WITH ALOE FEROX AND ALOE VERA IN BALB/C MICE Finberg M., Muntingh G. Background: Atopic dermatitis (AD) typically develops in patients with a history of allergic ailments, and is characterised by an itchy, inflammatory skin condition with scaling, lichenification, papules, excoriations and pruritus. In AD patients a chronic relapsing inflammatory condition is seen, associated with IgE hyper production. AD flares are largely triggered by environmental factors. There is a pressing need for new treatment regimens in AD. Historically Aloe has been used to treat skin conditions as well as a variety of other diseases. Method: The therapeutic efficacy of Aloe ferox vs. a comparator Aloe vera was investigated as a topical treatment for Dinitrofluorobenzene (DNFB)-induced atopic eczema/dermatitis syndrome (AEDS) in Balb/c mice. To explore the pathogenesis and treatment of AD, Balb/c mice were sensitized and challenged with 2,4-dinitrochlorobenzene (DNCB) for atopic dermatitis induction. Thereafter, mice were treated with either Aloe ferox or Aloe vera applied daily on the dorsal skin for 10 consecutive days. A placebo gel was used for the control mice. At the end of the treatment period serum IgE levels were measured. Results: Serum IgE levels were significantly lower in the Aloe ferox group and the Aloe vera group compared to the placebo group. Superiority of Aloe ferox vs. Aloe vera was demonstrated following application of the appropriate pair-wise t-test (P = 0.002) as well as from the fact that the upper limit of the 95% confidence interval *(0.090; 0.028) for the difference between Aloe ferox and Aloe vera was 0.05). Following the administration of dofetilide, QT and QTc intervals were significantly prolonged in both groups to a similar extent (QTc: 169.4 3.37 in WT and 167.8 3.17 ms in TG). However, the short-term variability of the QT interval, a novel ECG parameter suggested for the better estimation of repolarization temporal instability and proarrhythmic risk, was higher in KCNE1 transgenic rabbits compared to wild type at baseline (4.8 0.26 in TG vs. 2.8 0.15 ms in WT, P < 0.05). Following the administration of dofetilide, the incidence of the typically drug induced Torsades des Pointes arrhythmia was significantly higher in transgenic rabbits compared to wild type (77% in TG vs. 51.8% in WT, P < 0.05). In summary, our results strongly suggest KCNE1 transgenic rabbits have higher susceptibility to drug induced arrhythmias and may represent a useful model for testing the proarrhythmic potential of new drugs under development.

Methodology: In the acute oral toxicity trial single dose of 2000 mg/ kg was administered to five nulliparous female rats. In the sub chronic study 48 rats (24 males and 24 females) were grouped into 4 groups of 12 animals (six males, six females) and treated with P. kotschyi extract at a dose of 40, 200 and 1000 mg/kg respectively. Result: The acute toxicity study showed no signs of toxicity such as general behaviour changes and mortality. Assessment for signs of chronic toxicity indicated no abnormalities in the test groups as compared to the controls. Haematological and biochemical values in treated groups were normal in comparison with the control group. Insignificant changes in body weight, internal organ weight and general behaviour were considered to be incidental. Conclusion: Therefore, the stem bark methanol extract of P. kotschyi given orally to female and male rats did not induce acute and chronic toxicity in the rats at the doses administered.

304 ACTIVITY OF SECURINEGA VIROSA IN BEHAVIOURAL MODELS OF PARKINSON’S DISEASES

306 ACUTE AND SUB-CHRONIC TOXICITY STUDIES OF METHANOL ROOT EXTRACT OF SECURIDACA LONGEPEDUNCULATA IN MICE

Magaji M., Bello B., Hussaini S., Magaji R.

Haruna Y., Kwanashie H., Anuka J., Atawodi S., Hussaini I.

Background: Parkinson’s disease (PD) is a complex multifactorial disease and the second most common neurodegenerative disorder of the central nervous system after Alzheimer’s disease (AD).Current initial therapies are associated with loss of effectiveness over time and untoward effects. Securinega virosa is a commonly used medicinal plant in African Traditional Medicine for the management of mental illnesses. In this study the activity of the methanol root bark extract of Securinega virosa in behavioural models of Parkinson’s disease was evaluated. Methods: Methanol root bark extract of Securinega virosa (50 and 100 mg/kg, p.o) was administered 1 h prior to the administration of reserpine (1 mg/kg) and haloperidol (1 mg/kg). Cataleptic score was obtained in each mouse 1 h after haloperidol and 4 h after reserpine administration by gently placing the fore paws of each animal on a 4 cm high block at 1 h interval over a period of 4 h. The effect of the extract on motor coordination was also investigated using beam walking assay test in mice. The time to complete the task as well as the number of foot slips was determined in each mouse 1 h after administration of the extract. Results: The extract at the higher dose of 100 mg/kg significantly (P < 0.05) decreased the mean cataleptic score induced by haloperidol by a factor of about 3 at the end of 120 min. This was comparable to that of the standard agent, benzhexol (0.5 mg/kg) used. Against reserpine, the extract (100 mg/kg) significantly (P < 0.05) reduced the mean cataleptic score at the end of 180 min. Benzhexol significantly decreased the scores at both 180 and 240 min when compared with control. Conversely, the extract at all the doses tested did not significantly alter the time taken to complete the walking task as well as the number of the foot slips in the beam walking assay study. Conclusion: These findings suggest that the methanol root bark extract of Securinega virosa possess activity in behavioural model of Parkinson disease and a potential candidate for further evaluation.

Background: Securidaca longepedunculata (SL) is a popular medicinal plant believed to have over 100 medicinal uses, some for long-term management of chronic illnesses such as sleeping sickness, tuberculosis and mental diseases. Despite this widespread use, not much has been reported about its toxicity and hence this study investigated acute and sub-chronic toxicity of its methanol root extract in mice, after appropriate institutional ethical approval. Methods: Roots of SL were extracted with methanol; and LD50 was determined in acute toxicity testing. In the sub-chronic study, 96 male albino mice were divided into four groups of 24 each, with three of the groups receiving 20%, 10% and 5% of the extract’s LD50 (equivalent to 0.56, 0.28, 0.14 mg/kg respectively), while the control group received normal saline. All drugs were administered i.p. daily for 28 days. Six mice from each group were sacrificed on days 14, 28 and 56 only (as they did not survive to the proposed 90 days) and parameters to assess effects on blood, liver and kidney were determined. Results: The LD50 was found to be 2.83 mg/kg which was regarded as quite toxic. Data for day 28 showed that the extract significantly (P < 0.05) decreased RBC (9106/lL) to 6.020.19 (0.56 mg/kg) and 6.740.43 (0.28 mg/kg) when compared to the control 10.680.34. PCV (%) was similarly reduced to 41.805.84 and 42.004.72 compared to control of 48.603.93. Hb and WBC were however not affected. The extract significantly (P < 0.05) and dose-dependently increased ALT and AST while ALP was decreased. The corresponding pair values in U/L for control and highest extract dose for the three liver enzymes were 26.00/41.83, 17.83/29.50 and 59.67/43.83 respectively (SEM omitted). Total albumin (G/L) also decreased significantly (P < 0.05) and dose-dependently from 41.50 to 31.67, while urea (mM) increased from 2.23 to 4.08 and total protein (G/L) was unaffected (SEM omitted). Changes in these parameters on days 14 and 56 were similar indicating toxicity to blood, liver and kidney with the day 56 data being the most profound. Conclusion: SL may induce anaemia, and damage the liver and kidney; and hence should be used with caution for management of chronic illnesses.

305 ACUTE AND SUB CHRONIC (28-DAY) ORAL TOXICITY OF PSEUDOCEDRELA KOTSCHYI METHANOL EXTRACT IN SPRAGUE-DAWLEY RATS Abubakar K. Abstract: Pseudocedrela kotschyi has been extensively used in traditional medicine for the treatment of rheumatism, malaria, dysentery and epilepsy. Purpose: The purpose of the research is to determine the safety profile of the methanolic extract of Pseudocedrela kotschyi stem bark through acute and sub chronic toxicity in female and male rats.

307 ADVANCE OF PROGESTERONE RECEPTOR ANTAGONISTS IN CHINA Zhu Y. Progesterone (P) exerts a range of important biological effects by interacting with progesterone receptor (PR), which distributes in many crucial organs, including reproductive, bone,cardiovascular and nervous

© 2014 The Authors Basic & Clinical Pharmacology & Toxicology © 2014 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 115 (Suppl. 1), 1–374

96 system. The key role of P in the female reproductive system has led to the development of much synthetic PR ligand, including agonists and antagonists. Some of them are called selective progesterone receptor modulators (SPRM) because they demonstrate different agonistic or antagonistic activity according to context-dependent cells and tissues. The development of the SPRM has created new therapeutic method and has great potential in a number of gynecologic indications. Undoubtedly, the compelling SPRM in recent years is ulipristal acetate, which has already authorized by the European Commission and used for emergency contraception and the treatment of uterine fibroids. Ulipristal reduces excessive uterine bleeding and total fibroid volume and has equivalent efficacy to and fewer side effects than gonadotropin-releasing hormone analogues. Mifepristone is firstly developed PR antagonist in the late 70s, and also belongs to selective progesterone receptor modulators (SPRM) and has mixed agonist-antagonist properties. Mifepristone is the most widely used PR antagonists in China. As a progesterone receptor antagonist, mifepristone is used for termination of early pregnancy in combination with prostaglandins. In regardless of abortion-related image, mifepristone exhibits other advantages in therapy of a number of gynecological diseases, including uterine fibroid,endometriosis and gynecologic cancer. In recent years, the efficacy of mifepristone in the therapy of neurologic diseases, including Cushing’s disease and Alzheimer disease is being investigated. Gestrinone is another strong PR antagonist and has an anti-estrogen and subtle androgenic activity. Gestrinone is widely used in therapy of endometriosis in China since its effectiveness and low price and it also exhibits satisfied therapeutic effect on inhibiting the growth of the uterine fibroid. We found that gestrinone suppressed the level of the phosphorylated tyrosine kinase of c-Src in the guinea pig model of estrogen-induced uterine fibroid and the cultured human uterine fibroid cells. In addition, the result of a randomized double-blind trial showed that the effectiveness of 10 mg gestrinone is not significantly different from 10 mg mifepristone as an emergency contraceptive method.

308 AG4, A COMPOUND ISOLATED FROM RADIX ARDISIAE GIGANTIFOLIAE, INDUCES APOPTOSIS IN HUMAN NASOPHARYNGEAL CANCER CNE CELLS THROUGH CASPASE-DEPENDENT PATHWAY AND GENERATION OF REACTIVE OXYGEN SPECIES Guo D., Dong X., Zheng X., Mu L., Liu P. Aim: 3b-O-{a-L-pyran rhamnose-(1?3)-[b-D-xylopyraose-(1?2)]-bD-glucopyranose-(1?4)- [b-D -lucopyranose-(1?2)]-a-L-pyran arabinose}-cyclamiretin A (AG4) is a saponin component obtained from the Giantleaf Ardisia Rhizome. The aim of this study was to investigate whether exposure to AG4 induced cell apoptosis in human nasopharyngeal carcinoma cells (CNE) and the possible mechanism. Materials and methods: We exposed tumor cells to AG4 in order to investigate which cell line was the most sensitive to AG4. Cell viability was assessed by using the MTT reduction assay, effects of AG4 on apoptosis, ROS content, mitochondrial membrane potential and cellcycle were detected by flow cytometer; the GSH, SOD and MDA activities were measured using colorimetric methods. Relative expression of Bax, Bad, Bid, Bcl-2, Fas mRNA were calculated using the 2DDCt comparative method using real-time PCR studies. Results: AG4 remarkably inhibited growth of CNE cells by decreasing cell proliferation, induced apoptosis and the cell cycle was blocked in S phase by AG4. Caspase-3, caspase-8 and caspase-9 release were stimulated by AG4 in CNE, but the inhibitor of caspase (Ac-DEVDCHO) could not block the apoptosis induced by AG4. Moreover, the mitochondrial membrane potential was decreased in AG4 treated cells, and AG4-induced cell apoptosis was accompanied by rapid and lasting increasing of reactive oxygen species (ROS), which was abolished by NAC; GSH, SOD and MDA were regulated by AG4. AG4 inhibited Bcl-2 mRNA expression and stimulated Bax, Bad, Bid, Fas mRNA expression in CNE cultures, suggesting an effect at transcriptional level.

Conclusion: AG4-induced apoptosis in CNE cells via the decrease of mitochondrial membrane potentials through a ROS-dependent manner and regulation on genes relative to apoptotic, and regulation on cellcycle may also participate in the antitumor mechanism of AG4.

309 ALLOSTERIC MODULATION OF THE 5-HT3 RECEPTOR; A NOVEL STRATEGY WITH THERAPEUTIC POTENTIAL Barnes N., Newman A., Batis N., Myerson R., Palandri J., Gambassi C., Chiarenza A., Powell A., Grainger R., R. S., Grafton G. Background: The human (h) 5-HT3A receptor subunit forms a functional homomeric receptor that is a useful model to study cys-cys loop ligand-gated ion channels, including the pharmacology of allosteric modulators. To date various compounds have been identified as positive allosteric modulators of 5-HT3 receptors, including alcohols, volatile anaesthetics and indoles, although these lack potency. Methods: In the present study we have used radioligand binding assays, fluorescence indicators and patch clamp electrophysiological recording techniques to explore the allosteric modulation of human 5HT3A stably expressed in HEK293 cells with the aim to identify potent modulators. Results: 5-Chloroindole and other halogen-substituted indole derivatives (concentrations up to 100 lM) did not compete with [3H]-granisetron for the orthosteric site of the 5-HT3A receptor or alter the affinity of the competitive antagonist, ondansetron, for the 5-HT3A receptor. In contrast, 5-chloroindole and other halogen-substituted indole derivatives provoked concentration dependent increases in the affinity of agonists and partial agonists for the 5-HT3A receptor e.g. quipazine’s affinity for the 5-HT3A receptor was 19 3, 11 1 and 6 1 nM in the absence and presence of 5-chloroindole (10 and 30 lM), respectively. DDP 7330 s affinity was 2.3 0.5 and 1.6 0.05 nM in the absence and presence of 5-chloroindole (30 mM), respectively. Determination of functional responses (increase in intracellular calcium assayed by fluorescence dye on Flex Station and current recorded by whole cell patch clamp) demonstrated the ability of 5-chloroindole (10–30 lM) and other halogen-substituted indoles to potentiate responses to a range of 5-HT3 receptor agonists and partial agonists. Conclusions: In this study we have demonstrated the usefulness of 5chloroindole and other halogen-substituted indoles as a pharmacological tools to explore the allosteric modulation of the h5-HT3A receptor. Further investigations with mutant receptors to map the binding site(s) for these allosteric modulators will help rational design of potential therapeutic agents that modulate 5-HT3 receptor function.

310 ANALYSIS AND COMPARISON OF THE MOLECULAR SCAFFOLDS OF CURRENTLY APPROVED ANTIMALARIAL DRUGS AND NATURAL PLANTS PRODUCTS THAT HAVE SHOWN ANTIPLASMODIAL ACTIVITIES Egieyeh S., Syce J., Christoffels A., Malan S. Background: There is an urgent need to discover or design new class of antimalarial drugs with unique mechanism of action that will circumvent the current resistance profile of Plasmodium falciparum, the causative organism of malaria. Molecular scaffolds of compounds determine their spatial orientation within the binding pockets of drug targets. Therefore identification of unique scaffolds from compounds with antiplasmodial activities may be the starting point for a new class of antimalarial drugs. We therefore conducted scaffold diversity and uniqueness analysis of currently approved antimalarial drugs (CMD) and natural plants products that have shown antiplasmodial activities (NPP). Method: Analyses were performed on two data sets containing NPP (215 compounds) and CMD (26 compounds). Molecular Operating


97 Environment (MOE) software and Scaffold hunter software were used to generate scaffolds and visualize scaffold trees from the datasets. Scaffold from the NPP were compared with those from CMD. Scaffold diversity and the occurrence of unique scaffolds were determined using a python script developed in-house. Results: When analyzed using Level 1 scaffolds, the NPP data set showed lower ratio of scaffolds to molecules (0.33) when compared to the CMD data set (0.44). This indicate that the NPP data set contain slightly more represented scaffolds (one scaffold for every three molecule) while the CMD data set had close to one scaffold for every two molecules. The distribution of the occurrence of scaffolds from NPP in CMD showed that over 80% of scaffolds in NPP are not in the CMD data set. This indicates that there are unique scaffolds in NPP data set Conclusions: The NPP had slightly lower scaffold diversity than the CMD but is comparable to larger small molecule databases. The NPP data set of 215 active antiplasmodial compounds also contains scaffolds that are distinct from known antimalarial pharmacophores.

311 ANTAGONISTIC ACTION OF SEVERAL NEW PPADS ANALOGUES ON RESPONSES MEDIATED BY P2X AND P2Y RECEPTORS Ziganshin A., Kalinina O., Strelnik A., Yurii S. Background: Pyridoxalphosphate-6-azophenyl-20 ,40 -disulfonic acid (PPADS) is a widely used P2 receptor antagonist without selectivity to any subtypes of P2X or P2Y receptors. The aim of this study was to search for more effective and/or selective antagonist of these receptors among several analogues of PPADS. Methods: Isolated smooth muscle preparations of rat urinary bladder, vas deferens and duodenum were used for this study. Contractions of urinary bladder and vas deferens were triggered either with electrical field stimulation (EFS, 1–32 Hz) in the presence of atropine and phentolamine or with a,b-methyleneATP (1–10 mM). Relaxant responses of carbachol-precontracted duodenum were induced by EFS (2–8 Hz) or by ATP (10–100 mM). Mechanical activity of smooth muscle preparations was recorded isometrically before and after incubation of tissues with a given tested compounds at a concentration of 10 mM. Analogues of PPADS by laboratory codes of AC1-AC10 were synthesized in Kazan Federal University. As a reference antagonist we used PPADS (10 mM). Results: Initially were tested all compounds against contractile responses of rat urinary bladder and vas deferens induced by EFS. We found that all compounds have antagonism against EFS-induced contractions, but only effects of compounds AC3 and AC7 were comparable with that of PPADS. In the next series of experiments we tested AC3 and AC7 against contractions of urinary bladder and vas deferens induced by a non-selective P2X agonist, a,b-methyleneATP. We found that both compounds, similar to PPADS, antagonize contractions of these tissues elicited by a,b-methyleneATP. In the third series of experiments we tested AC3 and AC7 against relaxant responses of carbachol-precontracted longitudinal muscle of rat duodenum induced by EFS or ATP (P2Y receptor-mediated responses). We found that both AC3 and AC7 do not significantly affect relaxations of duodenum induced by EFS or ATP. We also found that at similar experiment conditions PPADS antagonizes the relaxant responses of duodenum mediated by P2Y receptors. Conclusions: Analogues of PPADS, compounds AC3 and AC7, have a similar to PPDS antagonism against P2X receptor-mediated contractions in rat urinary bladder and vas deferens, but unlike PPADS do not antagonize P2Y receptor-mediated relaxations in rat duodenum.

312 ANTI-DIABETIC EFFECT OF ANTHOCLEISTA VOGELII ETHANOLIC ROOT EXTRACT AND FRACTIONS IN WISTAR RATS Sunday R., Ilesanmi O., Obuotor E. Background: Diabetes mellitus is a common metabolic disorder that affects both the young and old in the developed and developing countries. The potential anti-diabetic effect of Anthcleista vogelii ethanolic root extract and fractions in Wistar rats due to its use in ethno-medicine was investigated in this work. Methods: Acute toxicity study of the ethanolic root extract was determined orally (p.o.). A. vogelii ethanolic root extract (100, 200 and 400 mg/kg, p.o.) and fractions (200 mg/kg, p.o.) were administered acutely to glucose loaded rats (10 g/kg, p.o.) and alloxan-induced diabetic rats (150 mg/kg, i.p.) for 14 days to five Wistar rats per group. Fasting blood glucose levels (FBGLs) of the diabetic rats were monitored at intervals of minutes and days throughout the duration of the treatment. On the 15th day of the experiment, blood samples collected by cardiac puncture was used for determination of serum cholesterol (CHOL), triglyceride (TRIG), high density lipoprotein (HDL), creatinine (CRT), alanine transaminase (ALT) and aspartate transaminase (AST) levels. Data was analyzed using one way analysis of variance and posthoc analysis using Bonferroni t-test at 95% level of significance. Results: The LD50 of A. vogelii ethanolic root extract was ≥5000 mg/ kg in rats (p.o.). The ethanolic extract at 100 and 200 mg/kg caused 12.0 % and 55.7 % significant (P < 0.05) reduction in fasting blood glucose level (FBGL) at 30 and 240 min more than that of the standard drug; glibenclamide (7.7 % and 51.3 %). The ethanolic root extract and fractions also exerted a dose dependent significant (P < 0.05) reduction in FBGL on day 3, 7, 10 and 14 more than the standard drug. The ethanolic root extract and fractions also exerted a dose dependent significant (P < 0.05) decrease in CHOL, TRIG, LDL, ALT, AST and an increase in HDL and CRT when compared to the control. Conclusion: The study concluded that the ethanolic root extract of A. vogelii is safe when administered acutely (p.o.). It has anti-diabetic, anti-lipideamic properties and has no toxic effect when administered for fourteen days in alloxan induced diabetic rats.

313 ANTI-INFLAMMATORY AND ANALGESIC ACTIVITY OF GLUCOSE ASPIRIN IN ANIMALS Badyal D., Jacob J., Bala S. Aspirin is a widely used drug with many pharmaceutical and biochemical properties. Its application in the prevention of heart disease and cancer is increasingly being realized. Aspirin is taken orally as it is not very water soluble. Some of the undesirable side effects of aspirin results from undissolved particles in the gastrointestinal mucosa causing ulcers and bleeding. We describe the synthesis of glucose conjugate of aspirin at carbon-3 of glucose, 3-O-(20 -acetoxy) benzoyl-a-D-2-glucopyranose also called glucose-aspirin (GA). Hence, this study was planned to evaluate the anti-inflammatory and analgesic activity of GA in animals. All animals were acclimatized for at least one week before the experimental session. The drugs were injected by intraperitoneal route. Three methods were used to evaluate analgesic and anti-inflammatory activity. Analgesic activity was evaluated by tail immersion and tail flick methods and anti-inflammatory activity was evaluated by carrageenan induced paw edema . Animals were divided into five groups with each group consisting of eight animals. Glucosyl acetyl salicylate was used in two dose levels. Aspirin at two dose levels and normal saline were used as controls. The human serum protease activity on the ester showed slower hydrolysis rate, 3.3 9 107 moles/L/min for GA compared to 1.57 9 10-6 moles/L/min for Aspirin. Percent inhibition of paw edema was 63 and 69 for Aspirin and GA respectively, at 100 mg/kg while at 200 mg/kg it was 69 vs. 64 for Aspirin and GA. With GA there was significant increase in tail flick latency at both dose levels, 100 and 200 mg/kg. The increase was maximum with all drugs at 60 min. For tail immersion test


98 the increase in reaction time was significantly longer with both doses of GA as compared to aspirin 100 mg/kg at 60 min. In conclusion, while the serum hydrolysis for glucose aspirin was slow, there was significant anti-inflammatory and analgesic activity of GA at the doses studied under the experimental conditions.

314 ANTI-INFLAMMATORY AND ANTIPYRETIC ACTIVITIES OF THE LEAF METHANOL EXTRACT OF RUTA GRAVEOLENS L. (RUTACEAE) IN RATS

ameliorates the articular score from day 3 to the end (P < 0.01), reduced nociception at days 2 and 10 (P < 0.05) and improved histophatological parameters (P < 0.03). M 1.5 mg/kg was similar to vehicle. In vitro, treatment was not toxic. M inhibited lymphocyte proliferation with ConA in 54.78% (P < 0.01), although it had no effect on LPS. M 1 mM decreased the number of invasive FLS in 48.6% (P = 0.006). Conclusion: M improve experimental arthritis attenuating joint damage and nociception in both models. Moreover, M decreased lymphocytes proliferation and fibroblasts invasion. Its indicate M as a potential anti-inflammatory treatment for immune-mediated arthritis.

Loonat F., Amabeoku G. The leaf methanol extract of Ruta graveolens was evaluated for antiinflammatory and antipyretic activities using the carrageenan-induced oedema and E. coli-induced pyrexia tests in rats. The leaf methanol extract of R. graveolens (50–400 mg/kg, i.p.) significantly reduced carrageenan- induced oedema over the 4 h period of testing. Combined treatment of the lowest doses of R. graveolens (25 mg/kg, i.p.) and indomethacin (2 mg/kg, i.p.) produced a significant reduction in carrageenan-induced oedema over the 4 h period of testing. R. graveolens (100–400 mg/kg, i.p.) significantly reduced E. coli-induced pyrexia over the 5 h period of testing. Given together, the lowest dose of R. graveolens (25 mg/kg, i.p.) and pentoxifylline (10 mg/kg, i.p.) produced a significant reduction in pyrexia induced by E. coli (50 lg/kg, i.m.) over the 5 h period of measurement. The LD50 value obtained for R. graveolens was >4000 mg/kg (p.o), suggesting that the plant species may be safe in or nontoxic to mice. The qualitative phytochemical analysis of the plant species revealed the presence of alkaloids, cardiac glycosides, flavonoids, saponins, tannins, and triterpene steroids. The data obtained indicate that R. graveolens has anti-inflammatory and antipyretic activities, justifying the use of the plant species by traditional medicine practitioners in the management and treatment of inflammation and fever.

315 ANTI-INFLAMMATORY AND IMMUNOMODULATORY PROPERTIES OF MONTANINE, AN ALKALOID ISOLATED FROM RHODOPHIALA BIFIDA Oliveira P., Farinon M., Spies F., Clarimundo V., Ramos G., Zuanazzi J., Xavier R. Montanine (M) is an alkaloid isolated from Rhodophiala bifida, a plant used in folk medicine and never before tested in inflammatory diseases. We evaluate the effect of M in vitro on lymphocytes and fibroblasts-like synoviocytes (FLS) and as an in vivo anti-inflammatory therapy in Antigen-induced Arthritis (AIA) and Collagen-induced Arthritis (CIA) models. Methods/Results: Male BALB/c mice had AIA induced with antigen methylated bovine serum albumin (mBSA) and divided into 4 groups (n=6): vehicle (saline) and M (0.3; 1 and 3 mg/ kg - twice a day). Treatment started one day before intraarticular (ia) injection (ij) of mBSA. Was evaluated: paw nociception in 0, 3, 5 and 24 h and leukocytes migration into the knee joints 24 h after ia ij of mBSA. Male DBA/1J mice had CIA induced and were divided into 3 groups (n=7): vehicle (saline) and M (0.5 and 1.5 mg/kg - twice a day/10 days). Mice were monitored daily for signs of arthritis and treatment started after onset of disease. Was evaluated: articular score, paw nociception and histophatology of ankle joints and liver. Lymphocyte and FLS was treated with M 0.01-100 mM for 24 h viability. M 1 mM was chosen for lymphocyte proliferation with ConA and LPS (n=7). FLS invasion in 24 h was assayed in Matrigel kit and treated M 1 mM (n=3). ANOVA/t-test. AIA: M inhibited leukocytes migration at doses 0.3; 1 and 3 mg/kg (8.041.65 9 104, 4.160.99 9 104, 4.151.46 9 104 leukocytes/cavity, respectively) compared with vehicle (43.59.73 9 104 leukocytes/cavity) (P < 0.01). Nociception was reduced compared with vehicle in all doses at 5 and 24 h (P < 0.01). CIA: M was not hepatotoxic. Compared with vehicle, M 0.5 mg/kg

316 ANTI-METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS ACTIVITY AND CHEMICAL COMPOSITIONS OF ETHER EXTRACTS FROM RHIZOMA POLYGONI CUSPIDATI Zhou H., Peng W., Liu M., Qin R. Aim of the study: This study is designed to investigate the anti-methicillin resistant staphylococcus aureus (MRSA) activity of ether extract from Rhizoma Polygoni Cuspidati and then its chemical compositions is assayed. Materials and methods: Firstly, ether extract of Rhizoma Polygoni Cuspidati (ET-RPC) was prepared. Subsequently, inhibition zone diameter (IZD) test, minimal inhibitory concentration (MIC) test and dynamic bacterial growth assay were used to evaluate the anti-MRSA activity of ET-RPC against international standard MRSA 252 strain (MRSA 252) and seven clinical MRSA strains. Furthermore, morphologic change of MRSA 252 treated with ET-RPC was observed using transmission electron microscope (TEM). Finally, the compositions of the ET-RPC were determined by LC-MS/MS in order to make sure which chemicals played the antibacterial activity. Results: Our results showed that ET-RPC could significantly inhibit bacterial growth of MRSA strains; this antibacterial activity was related to the integrality destruction of cell wall and cell membrane of MRSA. There were seven major compounds existing in ET-RPC, they were identified as polydatin, resveratrol4-O-D-(60 -galloyl)-glucopyranoside, resveratrol, torachryson-8-O- glucoside, emodin-8-O-glucoside, 6hydroxyl-emodin, and emodin using LC-ESI-MS/MS. Conclusions: ET-RPC has significantly antibacterial activity. More experiments are necessary to elucidate which chemical(s) plays this antibacterial activity and how to play the effect in the future.

317 ANTI-NEOPLASTIC ACTIVITY OF BIOTECHNOLOGICALLY PRODUCED JUSTICIDIN B IN DIFFERENT TUMOR TEST SYSTEMS Marinov M. Background: The cytotoxic lignans (e.g. etoposide, teniposide) comprise an important class of antinoplastic agents. Besides these commercialised agents a considerable research interest is focused on other plant-derived lignans as potential anticancer drugs. Among these a perspective lead compound is the arylnaphtalene agent justicidin B, originally extracted from Justicia procumbens. The presented survey is focused on a comprehensive onco-pharmacological evaluation of biotechnologically produced justicidin B, in vitro. Methods: The cytotoxic lignan justicidin B was produced by genetically transformed cultures of Linum leonii and subjected to a detailed pharmacological survey, which included evaluation of the cytotoxicity of the compound in a panel of human tumour cell lines (MTT-assay), its apoptogenic effects (flow cytometry, ‘Cell death detectionTM’ ELISA) and its effects on caspase 3, 8 and 9 activation (using Western immunoblot).


99 Results: The tested arylnaphtalene lignan exerted potent cytotoxic and proapoptotic agent against the malignant cell lines, applied at low micromolar concentrations. The induction of apoptosis is crucial for the cytotoxicity mode of action of this agent and proceeds via activation of the intrinsic mitochondrial cell-death signalling pathways. Conclusions: The potent activity at low micromolar concentration and the feasibility of biotechnological production of justicidin B implies that there is enormous scope in its further evaluation as a possible antineoplastic drug candidate.

318 ANTICONVULSANT EFFECTS OF KAURENOIC ACID ISOLATED FROM THE ROOT BARK OF ANNONA SENEGALENSIS Okoye T.C., Akah P.A. The herbal preparations of Annona senegalensis Pers. (Annonaceae) root bark are used in Nigerian ethnomedicine for the treatment of epilepsy and febrile seizures. The scientific evidence to this effect has been reported[1]. This study aims to identify and characterize the active constituent(s) responsible for the anticonvulsant effect. Bioactiveguided fractionation of the methanol-methylene chloride root bark extract (MME) of A. senegalensis using pentylenetetrazole (PTZ)induced seizures in mice, afforded a potent anticonvulsant ethyl-acetate fraction (EF). Further fractionation of the EF yielded eight sub-fractions (F1-F8) which were tested for anticonvulsant activity. The sub fraction F2 yielded white crystals that were purified to obtain A. senegalensis crystals, AS2. The AS2, which exhibited potent anticonvulsant effects, was characterized by 1D and 2D NMR spectroscopy, mass spectroscopy and X-ray crystallography. The AS2 was characterized as kaur-16-en-19-oic acid (KA), a diterpenoid phytoconstituent and exhibited the most potent anticonvulsant effect. The AS2 indicated an estimated LD50 of 3800 mg/kg. The results showed that the MME, EF and AS2 significantly (P < 0.05) and dose dependently delayed the onset of myoclonic spasms and tonic-clonic phases of seizures induced by PTZ and maximal electroshock seizure (MES). The anticonvulsant effects of the MME, EF and AS2 indicated the possible mediation of the anticonvulsant activity through central inhibitory mechanisms. Kaurenoic acid was identified as the anticonvulsant principle in the root bark extract of A. senegalensis. Reference: 1. Okoye TC and Akah PA. (2010). Anticonvulsant and sedative effects of root bark extract and fractions of Annona senegalensis. Inventi Impact: Ethnopharmacology. 1(2): 100-103. Fig. 1: Kaur-16-en-19-oic acid

3

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Voronina T., Kalinina T. Background: Derivatives pyrazolopyridine (tracozolate, cartazolate) and pyrazolo quinolone (CGS-8216, CGS-9886) are known to exert anxiolytic effects, although less pronounced than those of benzodiazepines. This study was undertaken in order to examine the antidepressant and anxiolytic effects of 4,6-dimethyl-2-(4-chlorophenyl)-2,3dihydro-1H-pyrazolo[4,3-c]pyridin-3-one (GIZH-72) (RF patent number 2394034). Methods: Behavioral helplessness (Porsolt’s test), forced swimming in the pool with wheels (by Nomura) were used for studying of antidepressant effects; Vogel conflict test, elevated plus maze were applied for detecting of anxiolytic properties; exploratory behavior and motor activity were investigated in open field test and activity meter Opto Varimex, coordination ability - in rota-rod test. Albino male rats weighing 260–280 g were used. GIZH-72 was synthesized at Zakusov State Institute of Pharmacology RAMS. Results: GIZH-72 (10 and 20 mg/kg, intraperitoneally) decreased the immobility time in Porsolt’s test and increased the number of turns in Nomura’s test (P < 0.05). In term of this activity, GIZH-72 was equal to amitriptyline (10 mg/kg, intraperitoneally) and exceeded fluoxetine (10, 20 mg/kg, intraperitoneally). Anxiolytic effect of GIZH-72 is characterized by the increase of punished water taking number in conflict test and by increase of time spent in the open arms in elevated plus maze (P < 0.05). According to anxiolytic activity, GIZH-72 was more potent than diazepam (0.5, 2 and 5 mg/kg), while fluoxetine exerted the anxiogenic effects. Anxiolytic and antidepressant effects of GIZH-72 were detected both in case of single and chronic administration (20 mg/kg, 30 days long) via perioral and intraperitoneal routes. Cessation of long-term treatment with GIZH-72 did not cause any withdrawal symptoms. GIZH-72 anxiolytic activity in conflict test was attenuated by bicuculline (1 mg/kg, s.c.). GIZH-72 (20 and 50 mg/kg) didn’t disturb the motor coordination and provoked no sedation. LD50 for GIZH-72 in rats was 825.3 mg/kg (intraperitoneally). Conclusion: Novel pyrazolopyridine derivative, GIZH-72 possesses antidepressant and anxiolytic activities, devoid of sedative or muscle relaxation effects combined with a low toxicity. GIZH-72 has certain advantages over fluoxetine, amitriptyline and diazepam.

320 ANTIDEPRESSANT EFFECT OF THE HYDROETHANOLIC LEAF EXTRACT OF ALCHORNEA CORDIFOLIA SCHUM (EUPHORBIACEAE) IN MICE: INVOLVEMENT OF THE MONOAMINERGIC SYSTEM Adeyemi O.

12 11

319 ANTIDEPRESSANT AND ANXIOLYTIC EFFECTS OF A NOVEL PYRAZOLOPYRIDINE

15

17

CH2

Background: The leaves of Alchornea cordifolia Schum (Euphorbiaceae) is used in ethnomedicine for ulcers, rheumatic pains, febrile convulsions and for enhancing physical performance. Hence, this study was carried out to investigate antidepressant property of hydroethanolic leaf extract of Alchornea cordifolia (HeAC) in forced swimming test (FST). Also, the mechanisms involved in this antidepressant action and the effects of the combination of the extract with conventional antidepressants; imipramine and fluoxetine in the FST were investigated. Results: Alchornea cordifolia (200 and 400 mg/kg, p.o.) produced dose dependent and significant (P < 0.001) antidepressant-like effect, in the FST, without accompanying changes in spontaneous locomotor activities assessed in the open-field test. The anti-immobility effect of HeAC (200 mg/kg, p.o.) in the FST was prevented by pre-treatment of mice with SCH23390 (0.05 mg/kg, s.c., a dopamine D1 receptor antagonist), sulpiride (50 mg/kg, i.p., a dopamine D2 receptor antagonist), prazosin (1 mg/kg, i.p., an a1-adrenoceptor antagonist), yohimbine (1 mg/kg, i.p., an a2-adrenoceptor antagonist), or GR127993 (5-HT1B receptor antagonist). Also, four days intraperitoneal injection of pchlorophenylalanine (PCPA, 100 mg/kg, i.p., an inhibitor of serotonin


100 synthesis) prevented the antidepressant-like effect elicited by HeAC. The combination of sub-effective doses of imipramine (5 mg/kg, p.o.) or fluoxetine (5 mg/kg, p.o.), with HeAC (25 mg/kg, p.o.) produced a synergistic antidepressant effect in the FST. However, pre-treatment with propranolol (2 mg/kg, i.p., a b2-adrenoceptor antagonist), WAY100135 (10 mg/kg, i.p., 5-HT1A receptor antagonist), metergoline (4 mg/kg, i.p., non-selective 5-HT2 receptor antagonist), ketanserin (5 mg/kg, i.p., preferential 5-HT2A receptor antagonist), or RS127445 (5 mg/kg, i.p., 5-HT2B receptor antagonist) failed to reverse the antidepressant-like effect elicited by HeAC. Conclusion: The extract from A. cordifolia produces an antidepressant-like effect in mice that is dependent on the monoaminergic system.A. cordifolia could be an alternative therapeutic tool that could help the conventional pharmacotherapy of depression.

321 ANTIDIABETIC EFFECT OF BOSWELLIA DALZILELII STEM BARK EXTRACT IN ALBINO RATS

against clinical isolates of C. albicans resistant to FCZ was further confirmed by time-growth curves and agar diffusion test. Our study firstly discovered that FRC alone or in combination with FCZ effectively inhibited the growth of C. albicans. FRC alone had efficient antifungal activities against C. albicans within a MIC80 ranging from 20 lg/ml to 40 lg/ml. FRC failed to enhance the effect of FCZ against C. albicans sensitive to FCZ, though it made FCZ resistant C. albicans more sensitive. This finding was further confirmed by the result of time-growth curves and agar diffusion test. We conclude that FRC can inhibit the growth of C. albicans only at a certain degree. FCZ and FRC combination may prove to be an effective option for overcoming the problem of C. albicans resistance to FCZ.

323 ANTIMALARIAL PROPERTIES OF SALTS AND COCRYSTALS OF AMINOQUINOLINES WITH HYDROXYAROMATIC ACIDS Holland S., Van Zyl R., Khakhlary P., Baruah J.

Idem I., Gyang S., Anyanwu A. Natural remedies and alternative medicines have enjoyed the support of the World Health Organization (WHO) in treating some ailments including diabetes mellitus (DM). According to World Health Organization projection, the prevalence of DM is likely to increase from 171 million to 366 million or more in the year 2030. There are several reports of wide range of plants and plant constituents that are active hypoglycaemic agents. Boswellia dalzielii hutch is a tree plant, 13 m high of the forest savanna, with characteristically pale papery bark, peeling and ragged. It is abundantly found in Northern Nigeria where the Hausa ethnic group refer to it as ‘hano’ (to prevent or obstruct) or ‘arrabi’. The common English name is Frankincense tree. The stem bark of the plant has been investigated for therapeutic properties including antidiarrhea, antibacterial and antifungal. The toxicological properties have also been assayed. The present study was carried out to evaluate the hypoglycaemic effect of the ethanolic extract of B. dalzielii stem bark on blood glucose concentration of normoglycaemic and alloxan-induced hyperglycaemic mice. Hypoglycaemic activity of the acute and repeated dose administration of the extract evaluated in normoglycaemic and alloxan-induced hyperglycaemic mice was compared with the activity of tolbutamide, a sulphonylurea. There was a significant reduction in the BGC of experimental animals after 1 h of acute administration of 675 mg/kg extract and 5 days of repeated dose administration of 225 mg/kg of extract. This showed reduction of 43.8% from the first hour to the second hour and a constant decrease of 86% after 8 h of treatment with extract as compared with decrease of 82% from first hour to the eighth hour in tolbutamide treated group. The results indicate that the ethanolic extract of B. dalzielii stem bark has hypoglycaemic effect hence justifying its use in ethnomedicine for the treatment of diabetes mellitus.

322 ANTIFUNGAL ACTIVITY OF ETHANOL EXTRACTS FROM FLOS ROSAE CHINENSIS Zhang L., Yan L., Lin H., Liu W., Dai B., Yan T., Jiang Y., Cao Y. The study was designed to investigate antifungal activities of the ethanol-extraction from Flos Rosae Chinensis (FRC) combined with fluconazole (FCZ) against clinical isolates of Candida albicans resistant to FCZ. FRC is included in Chinese Pharmacopoeia for the treatment of menstrual disorders. The minimum inhibitory concentration (MIC) was determined using checkerboard microdilution assay. MIC80 was determined as the lowest concentration of the drugs (alone or in combination) that inhibited fungal growth by 80%, compared with that of drug-free wells. The synergistic effect of FRC and FCZ combination

Background: Malaria remains a scourge on the health status of many continents on the global map, with the disease taking the life of an African child every minute. The increase in the development of resistance by Plasmodium falciparum to antimalarial treatment necessitates the search for new alternatives. In order to multiply in host cells, malaria parasites require iron for DNA synthesis and iron/copper for metabolically important cellular enzymes. 8-Hydroxyquinolines form stable complexes with transition metals and are proposed to be ideal chelators to investigate the role metals play in malaria. Methods: The antimalarial activity of a series of salts or cocrystals of 5-aminoquinoline and 8-aminoquinoline with different hydroxyaromatic acids were evaluated against a chloroquine-resistant P. falciparum (FCR-3) strain using the tritiated hypoxanthine incorporation assay. The effect of the compounds on the host red blood cells membrane integrity was determined. To elucidate a possible mechanism of action, the free radical scavenging DPPH● and b-haematin inhibition assays were performed. The concentration required to inhibit 50% of the effect (IC50) was determined from a log sigmoid dose response curve. The Mann–Whitney statistical test was performed with a P value ΣFIC < 1.25) or antagonist (ΣFIC ≥ 1.25) interaction]. Results: All the antifungal agents, except itraconazole and fluconazole, inhibited beta-haematin formation (IC50 value range: 130.53– 240.07 mM), but were approximately four fold less active than chloroquine. Clotrimazole was the most potent azole to inhibit both haemozoin formation (IC50:130.537.26 mM) and in vitro parasite growth (IC50:0.290.07 mM); which was approximately three fold less active than chloroquine. In combination, synergistic interactions were observed between mefloquine and econazole (ΣFIC: 0.730.02), miconazole and quinine (ΣFIC: 0.670.14) and clotrimazole and quinine (ΣFIC: 0.740.22). However, there was a slight antagonist interaction between clotrimazole and chloroquine (ΣFIC: 1.280.12), which was also observed when evaluated on the in vitro parasite (ΣFIC: 1.290.46). Conclusions: These results indicate that the simple structure of clotrimazole facilitated a more potent inhibition of beta-haematin formation compared to the larger azole antifungal structures and this is a possible mechanism of action for this azole. The favourable synergistic interactions suggest that clotrimazole combined with quinine has potential for further investigations.

342 CNS DEPRESSANT ACTIVITY OF ACUTE LEAD ADMINISTRATION IN MICE Magaji R., Magaji M., Yusha’u Y., Faruk F., Adam M., Muhammad F. Background: Lead (Pb) is a widespread neurotoxic agent found in many environments with a high potential danger to human health due to its multifaceted action with a broad range of physiological and biochemical dysfunctions The objective of this study is to evaluate the general CNS depression in mice. Methods: Lead acetate was administered at the doses of 120, 60 and 30 mg/kg to three groups of five mice each respectively while the control group was administered distilled water. The Depressant activity of the lead acetate was evaluated using Ketamine-induced sleep, Diazepam-induced Sleep and Hole-Board Tests. Results: There was a statistical significant increase (P > 0.05) in the onset and duration of sleep in all the groups treated with lead acetate when compared to control in the ketamine-induced sleep test; with no subsequent statistical difference (P < 0.05) between the lead treatedgroups and the control in the onset sleep but a significant increase (P > 0.05) in the duration of sleep in the group treated with 120 mg/ kg lead acetate as compared to the control in the diazepam-induced sleep test. The hole-board test yielded a significant increase (P > 0.05) in the number of head dips only in the 30 mg/kg lead-treated group (P < 0.05) when compared to the control in the hole board test. Conclusion: It is concluded that lead acetate has depressant activity in the range of the lead acetate doses administered most especially at the highest dose of 120 mg/mg. This well set the path in exploring the ameliorative effect of some medicinal plants in our environment on lead acetate induced neurobehavioral changes.

343 COMPARATIVE ANTIDIARRHOEAL ACTIVITY OF FRUIT EXTRACT OF PHOENIX DACTYLIFERA L. (DATE PALM) AND LOPERAMIDE HYDROCHLORIDE ON CASTOR OILINDUCED DIARRHOEA IN WISTAR RATS Agbon A.N., Kwanashie H.O., Hamman W.O. Background: Phoenix dactylifera L. (date palm) fruit has been claimed to have medicinal properties and used in folk medicine for


107 relief of gastrointestinal complaints and diarrhoea. This study assessed and compared the antidiarrhoeal activity of prophylactic and therapeutic administrations of aqueous fruit extract of Phoenix dactylifera L. (AFEP) to loperamide, an established antidiarrhoeal drug, using castor oil-induced diarrhoea model in Wistar rats. Methods: Forty Wistar rats (150–170 g) of either sex were categorized for prophylactic and therapeutic treatments; twenty rats in each category were divided into four groups (A–D) of five rats each. Group A served as control and received distilled water (0.5 ml), groups B and C were administered AFEP (1000 mg/kg and 2000 mg/kg, respectively), while group D was administered loperamide hydrochloride (5 mg/kg). Diarrhoea was induced by administration of castor oil (1 ml); 1 h after extract / loperamide treatment in the prophylactic category, but 30 min before, in the therapeutic category. All administrations were by oral route. The rats were observed for 4 h post-diarrhoea induction for frequencies of characteristic diarrhoeal droppings and the percentage inhibitions were calculated. Data were analysed using one way ANOVA with LSD post hoc test for significance. Results: Results obtained revealed that, AFEP (1000 and 2000 mg/ kg) and loperamide (5 mg/kg) elicited, statistically equivalent significant (P < 0.001) decreases in severity of diarrhoea with therapeutic treatment. However, with prophylactic treatment, loperamide (5 mg/ kg) elicited statistically higher significant decrease (P < 0.001) in the severity of diarrhoea when compared with that produced by AFEP (P < 0.05). Conclusion: AFEP and loperamide hydrochloride possess antidiarrhoeal activity in prophylactic and therapeutic treatments, with equivalent efficacy in therapeutic treatment. Therefore, AFEP is potentially of therapeutic value as an antidiarrhoeal agent.

344 COMPARATIVE EFFECTS OF GINGER (ZINGIBER OFFICINALE) AND GARLIC (ALLIUM SATIVUM) ON ALBINO RATS Nneli R., Nneli R. Background: It has been shown in a previous study that ginger contains gingerol, which increased the motility of the gastrointestinal tract, and has analgesic, sedative, antipyretic and antibacterial properties in laboratory animals (Chem and Hasnah, 2002). Garlic has been long employed for culinary and medicinal purposes (Ch and Mustafa 2001; Aieksunes and Mustafa, 2008; Desar and Borek, 1990). This study compared the analgesic effects of ginger and garlic on albino rats using standard experimental models: hot plate latency assay and formalin-induced paw - licking test. Methods: Aqueous extracts of the oven-dried garlic (200 mg/kg) and ginger (200 mg/kg) were given orally to both eighteen male and female albino rats (100–150 g) which were randomly selected and divided into three groups, namely control (saline- 200 mg/kg), garlic and ginger respectively. The animals were allowed free access to water ad libitum. The garlic and ginger groups were given the extracts orally prior to the administration of 0.03 ml of formalin into the right leg and pain sensitivity was observed for 5, 15, and 30 min intervals. In the hot plate assay, it was maintained at a constant temperature of 56 2°C by cyclotherm inserted into the length. The reaction time was measured and recorded before ginger and garlic extracts were given orally. The percentage increase was recorded. Results: In the formalin test, there was delayed response by the ginger and garlic fed groups when compared with the control animals which responded to pain immediately at the various time intervals. In the hot plate assay, the control animals licked their paws immediately when they were placed on the plate and jumped off while the ginger and garlic groups responded more slowly (P < 0.05). However, the effects in both ginger and garlic groups were similar in both models except in the 5 min phase where the analgesic effect of ginger was prolonged more than that of garlic (P < 0.05).

Conclusion: Ginger and garlic extracts delayed the response of rats to pain sensitivity. Ginger extract could provide a better analgesic effect than garlic especially in acute states.

345 COMPARISON OF 5-METHYLTETRAHYDROFOLIC ACID WITH FOLIC ACID FOR RAPID FOLATE REPLETION TO REDUCE THE RISK FOR NEURAL TUBE AND OTHER BIRTH DEFECTS Bailey S., Ayling J. Background: Women of child bearing age are recommended to consume folic acid supplements for at least one if not two months prior to attempting pregnancy. This minimizes the risk for incomplete neural tube closure (e.g., spina bifida) which begins about 21 days postconception. However, many women are either unaware of or do not comply with this advice. We have recently reported that 5‑methyltetrahydrofolic acid (5-MTHF) can rapidly replete body stores and elevate plasma folate concentrations in folate deficient women within about 2 days. Since there are no examples of incipient human embryonic dysraphism earlier than Carnegie stage 11, this approach can reduce the risk for folate dependent birth defects if administered soon after conception. We have now examined whether folic acid has the same capacity for rapid repletion. Methods: Women ages 19–45 years having no intention to become pregnant and screened for total serum folate 1 mM) whereas mexiletine was approximately 2-fold less potent in blocking the three states on Nav 1.6 subtype cells than Nav 1,2 subtype cells. Thus HFI-1 is clearly a Nav 1.2 selective blocker compared to mexiletine. As HFI-1 is highly lipophilic compared to mexiletine (Log D 4.0 vs. 0.4), we have synthesised analogues of HFI-1 in order to reduce lipohilicity as well as optimise other parameters that facilitate entry into brain (ionisation, polar surface area, H-bond acceptor/donor). These analogues vary in their selectivity and potency for blocking Nav 1.2 and 1.6 subtypes. It will be interesting to now compare their actions in vivo in rat models of neuropathic pain and epilepsy.

356 DEVELOPMENT OF PHARMACOLOGICAL TOOLS FOR THE STUDY OF ORPHAN GPCR GPR22 Hanson J., Gilissen J., Geubelle P., Dupuis N., Derj A., Soni A., Bernard P. Background: G Protein-Coupled Receptors (GPCR) represent the largest family of membrane receptors. They are currently targeted by 30% of marketed drugs. Nevertheless, around 100 GPCRs remain orphan and have never been investigated for a potential therapeutic use. The elusive understanding of their physiology is the consequence

of a lack of selective small molecule pharmacological tools. Such paucity of investigative tools precludes further validation of orphans as drug target. In this study, we identified synthetic agonists for GPR22, a Class A orphan GPCR that have a protective effect on the heart and constitutes an attractive potential drug target. Methodology: Many GPCR ligands are now described as being able to discriminate between signal pathways by displaying functional selectivity. Therefore, there is also a clear interest for screening with more specific assays directed toward clearly defined signaling pathways rather than unbiased assays. Accordingly, we have determined which type of G protein GPR22 was coupled to using a luciferase based pharmacological assay (GloSensorTM cAMP Assay, Promega corp). In order to identify ligands, we chose to screen with our assay a SOSA (Selective optimization of side activities) library comprising 1280 active compounds. Results: We have generated stable cell lines expressing Flag-tagged GPR22 together with the Glo sensor plasmid (HEK.GPR22.pGlo). We showed on this cell line that the receptor is correctly expressed at the cell membrane. We demonstrate in a ligand-independent fashion (based on constitutive activity) that GPR22 is coupled to Gi. We subsequently screened the Sigma Lopac library (1280 cpds) at 100 lM and identified 65 “hits” modulating receptor activity by more than 20% (selection criterion) while being inactive on WT cells. These compounds were cherry picked and underwent a secondary screening in triplicate. Compound C8 (64 4%), C9 (297%) and E9 (504%) confirmed an agonist activity on GPR22. Conclusion: We determined that GPR22 is expressed at the cell membrane and functionally coupled to Gi. After screening of a SOSA library and performing secondary screening, we identified 3 putative agonists with specific GPR22 activity. These compounds will serve as template to further design and develop small molecules pharmacological tools and investigate the functions of GPR22.

357 DIETARY PEEL FROM FRUCTUS SCHISANDRA SUPPLEMENTATION REDUCES SERUM/HEPATIC LIPID AND HEPATIC GLUCOSE LEVELS IN MICE FED A NORMAL OR HIGH CHOLESTEROL/BILE SALT DIET Pan S., Sun N., Zhang Y., Wang X., Zhu P., Yu Z., Zhou S., Ko K. Background: Recently, it has been found that Fructus Schisandra Chinensis (FSC) and its related compounds have a profound impact on lipid metabolism process. FSC can be divided into two parts, i.e., seeds and peels (seed outsider). The current study aimed to examine the effect of aqueous extracts of FSC peel (AqFSC-P) on serum/hepatic lipid and glucose levels in mice fed with a normal diet (ND) or a high cholesterol/bile salt diet (HCBD). Methods: The AqFSC-P used in the present study was fractionated into supernatant (SAqFSC-P) and precipitate (PAqFSC-P) separated by centrifugation. Male ICR mice were fed with ND or HCBD, without or with supplementation of 1%, 3%, or 9% (based on crude herbal material, w/w) SAqFSC-P or PAqFSC-P for 10 days. On the D 11 serum and hepatic triglyceride (TG), total cholesterol (TC) and glucose, as well as serum high-density lipoprotein (HDL), low-density lipoprotein (LDL) and alanine aminotransferase (ALT) activity were determined. Results: Supplementation with SAqFSC-P or PAqFSC-P significantly reduced serum and hepatic TG levels (approximately 40%) in NDand/or HCBD-fed mice. The supplementation with SAqFSC-P or AqFSC-P reduced hepatic TC level (by 2746%) in HCBD-fed mice. Supplementation with SAqFSC-P or AqFSC-P markedly lowered hepatic glucose levels (by 1330%) in ND- and HCBD-fed mice. SAqFSC-P decreased serum ALT activity by 24%, but PAqFSC-P increased hepatic protein contents (12%) in ND-fed mice. Bicylol, as a positive control, reduced ALT activity by 29 and 42% in ND- and HCBD-fed mice, respectively. In addition, mice supplemented with AqFSC-P or bicylol showed a smaller body weight gain (17–24%) and adipose tissue mass (up to 19%) as compared to the respective un-supplemented ND- or HCBD-fed mice.


111 Conclusion: FSC-P may be used for the treatment and prevention of lipid and glucose disorders in humans.

358 DIFFERENTIAL EFFECT OF RESVERATROL ON THE VASCULAR TONE OF RENAL ARTERY OF NORMAL AND DIABETIC RATS Gojkovic-Bukarica L., Markovic-Lipkovski J., Cirovic S., Novakovic R., Scepanovic R., Kanjuh V., Heinle H. Background: A polyphenol present in red wine, resveratrol is thought to be responsible for cardiovascular benefits associated with moderate wine consumption. The mechanisms by which resveratrol causes vasodilatation are not well defined. Therefore, the aim of this study was to investigate the mechanisms of resveratrol-induced vasorelaxation of rat renal artery (RA) with endothelium in normal and diabetic rats. Material and methods: Diabetes in male Wistar rats was induced by alloxan. Rings of RA were mounted in an organ bath for recording isometric tension. The experiments followed a multiple curve design. Expression of different voltage-sensitive potassium (Kv1.1, 1.2, 2.1 and 4.2) channels in a vascular wall of RA was evaluated by immunohistochemistry. Results: Interestingly, resveratrol relaxed RA of diabetic rats more potently than RA of normal rats (EC50 were 4 and 12 lM, respectively). L-NAME partly antagonized the relaxation of RA of normal animals only. A nonselective blocker of voltage-gated K+ (Kv) channels, 4-aminopyridine (4-AP) partly inhibited the relaxation of RA of normal rats. In contrast, only margatoxin, a selective antagonist of Kv1.x channels, completely antagonized the relaxation of RA of diabetic rats but not of normal rats. The vascular wall of RA of diabetic and non diabetic rats showed positivity with the following applied antibodies: Kv1.2 and Kv1.3 channels. The Kv2.1 channels were not expressed. The Kv4.2. channels are present only in a RA of normal rats. Conclusions: In conclusion, we have shown that resveratrol induces endothelium-dependent relaxation of RA of normal rats, and that 4AP-sensitive K+ channels are involved in this relaxation (probably Kv4.2). In diabetic rats, resveratrol induced potent NO-independent relaxation, probably by activation of smooth muscle Kv1.x channels.

359 DIFFERENTIAL INVOLVEMENT OF b1 REGULATORY DOMAIN AMINO ACID RESIDUES IN THE ACTIVATION OF SOLUBLE GUANYLYL CYCLASE (SGC) BY NITRIC OXIDE AND SGC ACTIVATORS Zhou Z., Marazioti A., Giannis A., Spyroulias G., Papapetropoulos A. Background: Soluble guanylate cyclase (sGC) is a heterodimeric a/b hemoprotein that regulates signalling pathways in the cardiovascular and nervous systems in response to nitric oxide (NO). The aim of the present work was to compare the contribution of specific amino acid residues within the regulatory domain of the b1 subunit in sGC activation by NO and two chemically distinct heme-independent activators, namely HMR-1766 (ataciguat) and BAY 58-2667 (cinaciguat). Methods: Using a computational approach we identified residues in the N-terminus of b1 that might influence the response to HMR-1766 and BAY 58-2667. They were then mutated and their function analyzed in transient transfection experiments in COS-7 cells. sGC expression was determined by western blotting and cGMP accumulation was measured by enzyme immunoassay. Results: Although exposure of COS-7 over-expressing sGC to HMR1766 led to higher cGMP levels, BAY 58-2667 was a much more potent sGC activator than HMR-1766. Mutating His to Phe at b1 105 abolished sGC responsiveness to the NO donor sodium nitroprusside (SNP), due to heme loss. In contrast, BAY 58-2667 and HMR1766 were capable of activating heme-free H105F sGC. Simultaneous

mutation of Y135 and R139 to Ala lead to an enzyme that was unresponsive to both NO and sGC activators. D45E and P74H mutants exhibited a marked reduction in their ability to synthesize cGMP in response to both SNP and HMR-1766, while responses of Y83G to SNP and HMR-1766 were more moderately affected. The responsiveness of L142G to SNP was reduced, while the effect of HMR-1766 remained unaffected. The R116A mutant exhibited unaltered cGMPsynthesizing ability to HMR-116, a mildly reduced response towards SNP, but increased cGMP generation after BAY 58-2667 exposure. P118A exhibited reduced responses to SNP and HMR-116, but unaltered behaviour towards BAY 58-2267. Exposure of wt enzyme to the heme oxidant ODQ reduced sGC levels; this effect was totally reversed by BAY 58-2667, but only partially by HMR-1766, suggesting that the two sGC activators have partially overlapping, yet distinct binding to the heme cavity. Conclusions: We have identified key residues that are important for the differential responsiveness of sGC to NO/heme-independent sGC activators and NO-releasing drugs.

360 DISCOREA DUMETORUM FEED: POTENTIAL FOR INDUCTION OF ANTIOXIDANT ENZYMES AND MODULATION OF LIPID PROFILE IN STZ-INDUCED DIABETES Ogbunugafor H., Ezekwesili C., Okafor C., Igbokwe G., Ajuzieogu C., Ezeonwumelu I. Method: Dioscorea dumetorum Pax (Dioscoreaceae) commonly called bitter yam is under-exploited for its food value although it has been reported as the most nutritious among the dioscorea species. This plant is neglected, even though its bioactive constituents are reported to possess hypoglycemic effect. Diabetes is known to create oxidative stress in vivo thus; the antioxidant property of this tuber on diabetic rats after thermal treatment was investigated. Method: Twenty adult Swiss albino rats in four groups of five rats each were used for the study. Diabetes was induced in rats intraperitoneally with streptozotocin at 50 mg kg-1 body weight. Group 1 animals were fed cooked D. dumetorum feed (15 g/day) for 10 days before induction of diabetes, thereafter feeding with D. dumetorum feed (15 g/day) continued for another 10 days. Group 2 (positive control) rats were fed normal rat pellet for 10 days, diabetes was induced, then glibenclamide (5 mg/kg BW) was administered and feeding on normal rat pellet continued. Group 3 (untreated) was fed normal rat pellet before and after diabetic induction while Group 4 served as nondiabetic control rats. The effect of the feed on superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), lipid peroxidation and lipid profile in the animals were evaluated. Results: Results showed that enzyme activities (IU/L) of SOD (2.580), CAT (1.451) and GPx (7.110) were higher (P < 0.05) in the D. dumetorum-fed rats compared to the glibenclamide treated, untreated and non-diabetic rats. In glibenclamide administered rats, SOD activity (0.824) was higher (P < 0.05) than non-diabetic (0.412) but CAT activity (0.808) was lower (P < 0.05) while GPx activity was unchanged (P > 0.05). There was no significant (P > 0.05) effect on lipid peroxidation and lipid profile amongst the groups, however, in HDL values; there was a non-significant increase (from 21.444 to 35.500 mg/dl) in the tuber-fed rats. Conclusion: The present study revealed that D. dumetorum exhibited anti-oxidative activity whose mode of action suggests induction of antioxidant enzymes. The ability of the tuber to ameliorate oxidative stress that accompanies type 2 diabetes is noteworthy.


112 362 DISTINCT MECHANISMS; INCLUDING HEME BIOMINERALIZATION INHIBITION, ACCOUNT FOR ANTIPLASMODIAL AND ANTIMICROBIAL EFFECTS OF PROTEA MADIENSIS LEAF EXTRACTS Ezenyi I., Aboh M. Recently, new strains of bacteria, fungi and parasites have emerged across the globe. This negatively impacts health, general well-being and seriously hinders economic development. Besides exploring small chemical molecules, continuous research on natural products must still be pursued for the discovery of new leads in the development of antiinfectives as they are widely available and are chemically diverse. The plant, Protea madiensis Oliv. (Proteaceae) is traditionally used to treat symptoms of malaria and other infectious diseases in West and central Africa. This study was aimed at investigating the ability of P. madiensis leaf extracts to suppress early malaria infection and microbial growth. P. madiensis methanol leaf extract was prepared by maceration in 80% methanol while successive extracts were prepared by consecutive extraction with n-hexane, dichloromethane, methanol and water. The crude extract was applied to cytotoxicity, antimicrobial, in vivo lethality and suppressive antimalarial tests whereas successive extracts were tested for antimicrobial and malaria suppressive activity in mice. Microorganisms used were Escherichia coli, Salmonella typhi, Proteus mirabilis, Staphylococcus aureus, Bacillus subtilis and Candida albicans. THP-1 cells were used for cytotoxicity testing while chloroquine sensitive Plasmodium berghei was used for the 4-day antimalarial test in mice. Results obtained showed the methanol extract was non-cytotoxic at100 lg/ml and non-lethal in mice at doses up to 5 g/kg. At a dose of 400 mg/kg, it significantly P < 0.05 suppressed parasitaemia by 82.43% and this effect was improved only in the aqueous successive extract 98.65%, P < 0.001, which was comparable with the effect of chloroquine. The aqueous successive extract also blocked heme conversion to malaria pigment in vitro by 51.51–65.4% in preliminary tests. In contrast, antimicrobial activity of the crude was enhanced only in the successive methanol extract, which did not suppress parasitaemia in mice. Antimicrobial effects of the methanol extract was evident by MIC values that ranged from 0.125 - 0.5 mg/ml while MICs of the successive methanol extract were less than or equal to 0.125 mg/ml, comparable with chloramphenicol. Hence, P. madiensis methanol extract possesses antiplasmodial and antimicrobial effects brought about by different chemical components acting distinctively, and can be studied as a source of new anti-infectives.

363 DISTINCT SIGNALLING PROFILES PRODUCED BY SERELAXIN IN HUMAN VASCULAR CELLS Summers R., Sarwar M., Samuel C., Bathgate R. Background: A recent phase III clinical trial (RELAX-AHF) demonstrated that infusion of serelaxin, the recombinant form of the hormone H2 relaxin, over 48 h improved short- and long-term clinical outcomes in patients with acute heart failure. However, the precise mechanism(s) associated with its cardioprotective actions are poorly understood. This study examined the effects of serelaxin administration in primary human vascular cells that may endogenously express the serelaxin receptor, RXFP1. Methods: qPCR was used to measure RXFP1 mRNA and125I relaxin to determine cell surface expression of RXFP1 protein. Alphascreen assays were used to measure cAMP, cGMP and Surefire to measure ERK1/2 phosphorylation. Results: qPCR revealed expression of RXFP1 mRNA in all cell types studied but radioligand binding studies showed cell surface RXFP1 expression in human umbilical vein endothelial (HUVECs) and smooth muscle cells (HUVSMCs), human umbilical artery smooth muscle cells (HUASMCs) and human cardiac fibroblasts (HCFs), but not human umbilical artery endothelial cells (HUAECs). In venous cells (HUVECs, HUVSMCs), serelaxin increased cAMP and cGMP accumula-

tion and ERK1/2 phosphorylation (pERK1/2) and the concentrationresponse curves (CRCs) were bell-shaped. Similar shaped CRCs for cGMP and pERK1/2 were also seen in HCFs, whereas in HUASMCs, serelaxin increased cAMP, cGMP and pERK1/2 with conventional sigmoidal CRCs. Almost all serelaxin responses involved inhibitory G proteins (Gai) and PI3 kinase (PI3K). The bell-shaped concentrationresponse curves observed for cAMP and cGMP accumulation in HUVSMC were altered by Gai/o inhibition and lipid raft disruption, but not Gas or bc inhibition. The same treatments did not qualitatively alter signaling in HUASMC. Conclusions: We conclude that human vascular cells respond to serelaxin with a variety of patterns of RXFP1 signaling that differ between cells isolated from arteries and veins. Within the group of cells examined in this study, bell-shaped CRCs that are a hallmark of serelaxin signaling experimentally and clinically were only observed in venous cells and fibroblasts. The concentration-response relationships are clearly influenced by Gai/o located within membrane lipid rafts and are cell-background dependent. Our studies identify several human cell systems that may be useful in determining the mechanisms underlying the bell-shaped concentration-response relationships to serelaxin observed clinically.

364 DL0410, A NOVEL ANTI-ALZHEIMER’S DISEASE CANDIDATE, PROTECTS NEURON AGAINST Ab-INDUCED CELL DAMAGE VIA a7NACHR MEDICATED INTRACELLULAR AKT/JNK SIGNALING PATHWAY Liu A., Zhou D., Yang R., Liu R., Du G. Since the etiology of Alzheimer’s disease (AD) is very complex and multiple pathogenetic factors are involved, more and more attentions paid to multipotent drugs that could inhibit the multiple targets implicated in AD. DL0410 was a novel synthetic dual cholinesterase inhibitor. In the present study, Ab1-42 induced amnesia mice model and cooper induced APPsw overexpression cell model were used to investigate the pharmacodynamics effects of DL0410 and explore its neuroprotective mechanism. It was found that DL0410 exerted a comparable effect to donepezil and rivastigmine on cholinesterase inhibition. In the Morris Water Maze test and step-through positive avoidance test, DL0410 obviously improved memory deficits in Ab1-42 induced amnesia mice. MTS assay showed that DL0410 significantly reduced APPsw-SY5Y cell apoptosis. The mechanistic study showed that DL0410 inhibited Ab overproduction both in vitro and in vivo, reversed the loss of mitochondria membrane potential, unregulated neurotrophic CREB/ BDNF signaling pathway. The anti-apoptosis effect of DL0410 was suppressed by selective a7nAChR antagonist methyllycaconitine, indicating that a7nAChR-medicated intracellular AKT/JNK signaling pathway may play a key role in anti-AD effects of DL0410. These results demonstrated that DL0410 could be considered as a candidate drug for the prevention and therapy of AD.

365 EFFECT AND MECHANISM OF POLYPEPTIDES OF CARAPAX TRIONYCIS ON HEPATIC FIBROSIS INDUCED BY CARBON TETRACHLORIDE IN RATS Ye X., Kong F., Li J., Li S., Liu Y. Background: Carapax trionycis is a traditional Chinese medicine, which is often used in the treatment of liver diseases. Carapax trionycis contains chemical ingredients such as amino acids, peptides, proteins, minerals, etc. Previous research results showed that the polypeptides of carapax trionycis of molecular weights less than 6000 Daltons (PL) can inhibit the proliferation of hepatic stellate cells. The purpose of this study was to investigate the effect of PL on liver fibrosis in rats induced by carbon tetrachloride (CCl4) and to explore its possible mechanism.


113 Methods: In this study, rats were divided into eight groups, including normal controls, model, colchicin as the positive control, PL (6, 12 g/ kg, equivalent to the amount of medicinal herbs), polypeptides of carapax trionycis of molecular weights greater than 6000 Daltons (PG, 12, 24 g/kg), aqueous extract of carapax trionycis (AE, 12 g/kg). Hepatic fibrosis was induced in male Sprague-Dawley (SD) rats by subcutaneous injection of CCl4 with twice a week for 12 weeks, and rats were administered with drugs or normal saline by intragastric administration, simultaneously. Antifibrotic effects of polypeptides of carapax trionycis on hepatic fibrosis in rats were estimated by assessing serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN), precollagen (PCIII), collegen type IV(IV-C), hepatic levels of superoxide dismutase (SOD), malondialdehyde (MDA), hydroxyproline (HYP), Tissue inhibitor of matrix metalloproteinases-1 (TIMP1), transforming growth factor-beta 1 (TGF-b1) and histological study by HE staining, TEM. Results: PL treatment significantly decreased serum enzymatic activities of ALT, AST (P < 0.01), and level of HA, LN, PCIII, IV-C (P < 0.01), as well as liver HYP. Moreover, histological results indicated that PL alleviated liver damage and reduced the formation of fibrous septa. PG only decreased serum enzymatic activities of ALT, AST (P < 0.01) and liver HYP (P < 0.01) in the low-dose group. Research on mechanism showed that PL could markedly reduce liver malondialdehyde (MDA) concentration, increase activities of liver superoxide dismutase (SOD), and inhibit the expression of TGF-b1, TIMP1. Conclusion: This study indicate that PL is effective and inhibits CCl4-induced hepatic fibrosis. Its action may be ascribed to its antioxidant activity and inhibiting expression of TGF-b1, TIMP1.

The main objective is to study the role of galantamine hydrobromide (Reminylâ; GAL) on the viability and invasion of fibroblast-like synoviocyte (FLS). Methods: Mice DBA/1J fibroblast-like synoviocyte (FLS) were isolated from the tarsus of the hind paws of collagen-induced arthritis. FLS (2x104 / 96-wells) viability in 24 h treated with GAL (concentration from 0.625 to 320 mM). The invasion of FLS was assayed in a transwell system using Matrigel-coated inserts and treated with GAL. Differences between experimental groups were compared by T test. Results: GAL concentrations used were not toxic on FLS, maintaining cellular viability. The dose of 320 lM was defined for other experiments because this was the highest dose with lower cell mortality (P < 0.05). Treatment of highly invasive DBA/1J FLS with GAL significantly decreased the number of cells invading Matrigel over 24 h period in 26%. Conclusion: GAL was able to decrease synovial fibroblast invasion. These findings suggest that GAL could be acting in the cholinergic anti-inflammatory pathway in the FLS and may have potential for secondary usage as new strategy for the treatment of RA.

368 EFFECT OF PHELA, A TRADITIONAL MEDICINE ON P-GLYCOPROTEIN TRANSPORTER IN THE GASTROINTESTINAL TRACT OF A RAT MODEL Binyane M., Walubo A., Lekhooa M., du Plessis J., Matsabisa M.

366 EFFECT OF DNJ ON INSULIN RESISTANCE IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELL Guoping P., Qingpu L. DNJ (1-Deoxynojirimycin) is a natural compound from Mulberry Leaf. DNJ has been reported to lower plasma lipids and glucose and to increase glucose homeostasis in type 2 diabetic animals. Insulin resistance is an important feature of type 2 diabetes. However, it is unclear whether DNJ can improve insulin resistance. We conducted the present study to determine the effect of DNJ on insulin resistance. HUVEC (Human Umbilical Vein Endothelial Cells) were divided into control group, insulin group and DNJ groups. The DNJ groups were incubated with 0.1, 0.01, 0.001 lM. The expression of proteins regulating glucose and fatty acids metabolism were detected by Western blot. DNJ upregulated expression of phosphorylated AKT in DNJ groups. DNJ can improve insulin sensitivity in HUVEC, and DNJ plays its anti-insulin resistance role, at least in part, through the PI3K-AKT pathway.

367 EFFECT OF GALANTAMINE HYDROBROMIDE (REMINYLâ) ON THE VIABILITY AND INVASION OF MICE FIBROBLAST-LIKE SYNOVIOCYTE Spies F., Oliveira P., Farinon M., Blum G., Clarimundo V., Xavier R. Background: Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease of unknown etiology that is characterized by inflammation of the synovium leading to destruction of cartilage and bone. Recently was described an involvement of the alpha7 subunit of nicotinic acetylcholine receptors (nAChR) in modulating the inflammatory response in RA. Specific alpha7nAChR agonists, or perhaps neurostimulation of the cholinergic antiinflammatory pathway, may be candidates for development as a novel therapeutic approach for the treatment of RA. Galantamine is a competitive acetylcholinesterase inhibitor and a nicotinic allosteric modulator, commercially available to treat Alzheimer’s disease.

Background: Phela is a traditional medicine made of a mixture of four African traditional medicinal plants and is under development as an immune booster. P-glycoprotein is the efflux transporter that limits absorption of drugs across the gastro-intestinal tract (GIT). The aim of the study was to investigate the effect of Phela on the intestinal p-glycoprotein in a rat model, using paclitaxel (PTX) as the selective marker for p-glycoprotein. Methods: Male Sprague-Dawley rats (120) were divided into three groups (n=40). Each group was administered orally once-off with either, PTX only 10 mg/kg (control), PTX 10 mg/kg and cyclosporin A (CyA; positive control), and PTX 10 mg/kg and Phela 15.4 mg/kg (test group). Thereafter five animals were sacrificed and blood was withdrawn at 30 min, 1, 2, 4, 6, 8, 10, and 12 h of treatment. PTX plasma concentrations were measured with a validated high performance liquid chromatography (HPLC-UV) method. Results: Phela (test group) and CyA (positive control) significantly increased the area under the plasma concentration-time curve (AUC) with P = 0.0088 and P = 0.0003 respectively. PTX AUC for the control was meanSD, 234.02 53.53 mg.h/L, vs. Phela 698.74 159.83 mg.h/L and 2976.18 409.56 mg.h /L for CyA. Whereas the PTX peak concentrations in the control and test group were similar (4.63 0.12 lg/ml), that of the positive control was increased (6.23 0.31 lg/ml; P = 0.0011). Therefore, co-administration of Phela and PTX in rats led to increased AUC of PTX indicating p-glycoprotein inhibition leading to reduced p-glycoprotein mediated efflux of PTX in the lumen. Thus dose of drugs that are transported by p-glycoprotein should be adjusted when co-treated with Phela. Conclusion: Phela moderately inhibited p-glycoprotein transporter in the GIT of the rat model, thereby indicating potential interactions with drug-substrates of p-glycoprotein.


114 369 EFFECT OF PITUITARY ADENYLATE-CYCLASE ACTIVATING POLYPEPTIDE AND ITS ANALOGUES ON THE SPECIFIC PAC1 AND VPAC1/VPAC2 RECEPTORS ON THE CELL BODIES OF PRIMARY SENSORY NEURONS AND TRANSFECTED CELL LINES Szoke E., Saghy E., Payrits M., Reglodi D., Toth G., Couvineau A., Helyes Z. Background: Pituitary adenylate-cyclase activating polypeptide (PACAP) acts on G protein-coupled receptors: the specific PAC1 and VPAC1/VPAC2. PACAP6-38 was descibed as a potent PAC1/VPAC2 antagonist in several models. Maxadilan is a selective PAC1 agonist, while its fragment, MAXA65, is a specific antagonist. Ala11,22,28VIP is a selective VPAC1 agonist, while BAY 55-9837 is a selective VPAC2 agonist. We described previously that both PACAP1-38 and PACAP638 are able to decrease the electrical-field stimulation-induced release of the sensory neuropeptide CGRP from sensory nerve endings of the isolated rat trachea. PACAP6-38 did not behave as an antagonist. We aimed to analyse the actions of peptide fragments on sensory neural and cell line responses in vitro. Methods: Ratiometric technique of [Ca2+]i measurement with the fluorescent indicator fura-2-AM, on primary cultures of trigeminal ganglia neurons and PAC1, VPAC1 and VPAC2 receptor-expressing cell lines were performed. Measurement of CGRP release by RIA on primary cultures of trigeminal neurons was performed. Results: Results on neurons: Slowly increasing [Ca2+]i was detected both after PACAP1-38 and PACAP6-38 administration. The PAC1 receptor agonist maxadilan, the PAC1 receptor antagonist MAXA65 and the VPAC2 receptor agonist BAY 55-9837 caused similar response. In contrast, the VPAC1 receptor agonist Ala11,22,28VIP had no significant effect on [Ca2+]i. TRG cells were investigated in Ca-free ECS, similar results were detected as above. Agonists and antagonists induced CGRP release from sensory neurones. Results on cell lines: Our data show that PACAP1-38 and PACAP127 increased [Ca2+]i on PAC1, VPAC1 and VPAC2 receptor-expressing cell lines. PACAP6-38 had no similar effect on these cell lines. Maxadilan and MAXA65 increased [Ca2+]i on PAC1 receptor expressing cell lines. The selective VPAC1 agonist Ala11,22,28VIP and the selective VPAC2 receptor agonist BAY55-9837 activated the VPAC1 and the VPAC2 receptor-expressing cell line, respectively. Conclusion: We are able to test the PACAP receptor-selective agonists and antagonists by Ca-imaging. Some antagonist of PACAP receptors act as agonists on the sensory neurons. Presently unknown receptors or splice variants linked to distinct signal transduction pathways might explain these differences. The VPAC1 receptor does not play a role in these processes.

370 EFFECT OF SALVIANOLIC ACID A ON DIABETIC PERIPHERAL NEUROPATHY IN TYPE 2 DIABETIC RATS Zhang L., Yu X., Yang X., Huang Z., Du G. Background: In this study, we investigated the effects of Salvianolic acid A (SalA) on diabetic peripheral neuropathy in type 2 diabetic rats. Methods: High fat/high sucrose diet combined with small dose of STZ (30 mg/kg) intraperitoneal injection was used to establish type 2 diabetic model. Animal ethologies, electrophysiology in peripheral nerve, hemodynamics in periphery microcirculation, ultramicrostructure of sciatic nerve observation and biochemical indicators in blood were used to evaluate the effect of SalA on diabetic peripheral neuropathy. Results: Eight-weeks treatment of SalA could increase the motor nerve conduction velocity, and ameliorate the deterioration of sciatic nerve ultramicrostructure. Down-regulation of MDA, AGEs, TCHO, TG levels and the decreasing tendency of FRU level in blood were observed, up-regulation of nNOS, BDNF, GDNF and decrease of AGEs expressions in sciatic nerve were also shown.

Conclusions: SalA can improve peripheral nerve function in type 2 diabetic rats by decreasing oxidative stress damage, improving vascular function in periphery microcirculation, and promoting the expression of neurotrophic factor.

371 EFFECT OF TENUIFOLISIDE A ISOLATED FROM POLYGALA TENUIFOLIA ON THE BDNF/TRKB-DEPENDENT ERK AND PI3K PATHWAYS IN C6 GLIOMA CELLS Liu P., Dong X., Yu B., Hu Y., Mu L. Tenuifoliside A (TFSA) is a bioactive oligosaccharide ester component of Polygala tenuifolia Wild, a traditional Chinese medicine which was used to manage mental disorders effectively. The neuroprotective and anti-apoptotic effects of TFSA have been demonstrated in our previous studies. The present work was designed to study the molecular mechanism of TFSA on promoting the viability of rat glioma cells C6. We exposed C6 cells to TFSA (or combined with ERK, PI3K and TrkB inhibitors) to examine the effects of TFSA on the cell viability and the expression and phosphorylation of key proteins in the ERK and PI3K signaling pathway. TFSA increased levels of phospho-ERK and phospho-Akt, enhanced release of brain-derived neurotrophic factor (BDNF), which were blocked by ERK and PI3K inhibitors respectively (U0126 and LY294002). Moreover, the TFSA caused the enhanced phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 site, the effect was revoked by U0126, LY294002 and K252a. Furthermore, when C6 cells were pretreated with K252a, a TrkB antagonist, known to significantly inhibit the activity of BDNF, blocked the levels of phospho-ERK, phospho-Akt and phosphor-CREB. Taking these results together, we suggested the neuroprotection of TFSA might be mediated through BDNF/TrkBERK/PI3K-CREB signaling pathway in C6 glioma cells.

372 EFFECT OF THE FRACTIONS OF TAMARINDUS INDICA L. (CAESALPINIACEAE) ON EXPERIMENTALLY INDUCED HYPERGLYCAEMIC WISTAR RATS Yerima M., Anuka J., Salawu O., Abdu-Aguye I., Tanko Y. World Health Organization defines diabetes mellitus as “a metabolic disorder of multiple etiologies, characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. LD50 and phytochemical screening were conducted using Lorke’s method 1983 and Trease and Evans 1989 respectively. The stem-bark extract of Tamarindus indica L. was investigated for its hypoglycaemic action on experimentally induced hyperglycaemic Wistar rats using a single dose of alloxan (150 mg/kg IP) and fructose (10% w/v ad libitum for 20 days). The oral LD50 of the extract was found to be 3800 mg/kg. The fractions of the extract lowered the elevated blood glucose significantly with the 1000 mg/kg dose at the 8th, 16th, and 24th hours. The 500 mg/kg dose also lowered the glucose level throughout the study but only significantly at the 1st, 16th, and 24th hours. The ethyl acetate fraction also lowered the elevated blood glucose with all the doses used. The 250 mg/kg dose did not show significant decrease in the blood sugar concentration. The fractions of the stem-bark extract of T. indica L. significantly lowered elevated blood glucose concentration in alloxan and fructose-induced hyperglycaemic Wistar rats.


115 373 EFFECT OF THE LONG-TERM TREATMENT WITH ETHANOL ON SPERM QUANTITY AND QUALITY OF ADULT WISTAR RATS AND THEIR DESCENDANTS Jurkiewicz N., Fabrıcio V., Bomfim G., Silva Junior E., Nichi M., Losano J., Dalmazzo A., Jurkiewicz A., Barnabe V. Background: Long term ethanol consumption is able to affect the male reproductive system in humans or rodents. The main effects related to the abusive consumption of ethanol include testicular atrophy, gynecomasty, libido decrease, fertility impairment and ejaculatory disorders. Therefore, this study was carried out in order to further evaluate the effects of long-term exposure of ethanol in rats on quantity and quality of sperm. Methods: Male (60–70 days old/ 200–250 g) Wistar rats were treated with ethanol for 8 weeks by oral gavage with the following dosage: 1st week 1.0 g/kg/day; 2nd week 1.5 g/kg/day; 3rd week 2.5 g/kg/ day; and 4th–8th week 4.0 g/kg/day. After the 8th week, the animals were euthanatized by decapitation and the sperm collected from cauda epididymis or the treated rats were mated and their descendants (60– 70 days old) were treated with ethanol as mentioned above. The control groups were treated with drug-free vehicle. Then, the sperm collected was subjected to concentration analysis, morphology evaluation, acrosome and membrane integrity, mitochondrial activity and assessment of the susceptibility to oxidative stress. Results: The long-term treatment with ethanol decreased the sperm concentration in cauda epididymis by about 50%. In addition, the number of abnormalities was increased by about 40%. The abnormality most found was curled tail and bent tail. In addition, the sperm from treated animals showed an increase in the number of spermatozoa with low mitochondrial activity. The acrosome integrity, membrane integrity or susceptibility to oxidative stress did not show any changes. On the other hand, the descendants from rats long-term exposed with ethanol did not present any alteration in the sperm quantity and quality. Conclusion: The long-term treatment with ethanol decreases the quantity and quality of the sperm from rat cauda epididymis. Moreover, the ethanol-treated descendants from rats exposed long-term with this drug showed a tolerance in the ethanol effects on sperm parameters.

374 EFFECT OF VARIOUS PARTS OF COCCINIA INDICA AS ANTIBACTERIAL, HEPATOPROTECTIVE & ANTIDIABETIC AGENTS Kaur N., Sharma S. Coccinia indica (Family: Cucurbitaceae) is sweet, acrid, cooling, and astringent to the bowels, cures kapha and pitta. It is cultivated in Africa and Asia, and is particularly used as a vegetable in India. The aim of the present research was focused on investigating the antibacterial, hepatoprotective and antidiabetic activity of various parts of Coccinia indica. The aqueous and organic solvent (Petroleum ether, chloroform and ethanol) extracts from the leaves of Coccinia indica were tested against Enterobacter aerogenes, Pseudomonas aeruginosa, Staphylococcus epidermidis, Bacillus subtilis and Salmonella typhimurium by agar well diffusion method and broth dilution method. Ethanolic and aqueous extracts were found to have a more potent inhibitory effect comparing with the other extracts. A diethyl ether extract of the leaves was studied for hepatoprotective activity against carbon tetrachloride induced liver toxicity in rats. The results shown hepatoprotective activity of Coccinia indica leave extract at dose 400 mg/kg body weight was comparable with standard treatment 125 mg/kg body weight of silymarin, a known hepatoprotective drug. Chronic administration of fruit extracts (200 mg/kg) for 14 days showed a significant decrease in the blood glucose level in the 7th and 14th days of the diabetes induction, showing antidiabetic effect of the concern fruit. Thus, The present study favours the effect of Coccinia indica as antibacterial, hepatoprotective and antidiabetic agent.

375 EFFECT OF ZINGIBER OFFICINALE ON BLOOD COAGULATION PROFILE IN THE RAT Yeoh P., Eng Hong N., Pichika M., Ramachandran V. Background: Young ginger, Zingiber officinale var Officinalis (GM), locally known as Halia Muda has been extensively studied for its medicinal properties due to its major active essential oil, gingerol. Some studies showed ginger to have anti-emetic, anti-ulcer and antiinflammatory activities. Ginger is also believed to have anticoagulant action. In the present study, we investigated the involvement of GM on the blood coagulation profile in the rat. Methods: Male Sprague Dawley rats were used for 3 studies: pilot, ginger and warfarin interaction studies. At all times they were given rat chow and water ad lib. The rats were divided into groups (n = 6) and fed (via oral tube) daily with the stated drug for 7 days: groups: 1. control (2% methylcellulose), 2. GM (100, 200 or 400 mg/kg/ml) or 3. warfarin (0.1 mg/kg/ml) or 4. warfarin (0.1 mg/kg/ml) with GM (200 mg/kg/ml). On day 0 and day 8 blood samples were obtained through retro-orbital route. In addition, rats in groups 1 and 2 were retained for another 7 days without treatment and blood samples were drawn on day 16. All blood samples were tested for coagulation profile (activated partial thromboplastin time [APTT], prothrombin time [PT], and thrombin time [TT]). The data between groups were compared using student t test. Results: GM significantly increased mean clotting time of PT and TT on a dose-dependent basis, but only an increase in APTT occurred with 100 mg/kg dose in the rats, indicating GM to inhibit the extrinsic coagulation pathway rather than the intrinsic coagulation pathway. At 200 mg/kg, GM did not increase the warfarin anticoagulant action significantly. Conclusion: GM at the doses studied significantly increase blood coagulation profile in the rats. GM did not change the anticoagulant response to warfarin at the dose studied, suggesting that consumption of ginger in a patient on warfarin might not necessary enhance its anticoagulant action. 376 EFFECTS OF BCRP DRUG EFFLUX TRANSPORTERS ON a1A-ADRENOCEPTOR PHARMACOLOGY IN ISOLATED PREPARATIONS OF MOUSE AND RAT PROSTATE GLAND Ventura S., McGuiness L., Eise N., White C. The a1L-adrenoceptor functional phenotype, as seen in urinary tract tissues, is characterized by a low affinity for the a1-adrenoceptor antagonist prazosin. Prostatic smooth muscle cells have a significant population of a1A-adrenoceptors located intracellularly. In addition, prazosin is able to permeate cell membranes and is a known substrate for the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). We hypothesize that an intracellular population of a1A-adrenoceptors mediates contraction in prostate smooth muscle cells. Furthermore, these cells are able to actively efflux intracellular prazosin thus lowering the intracellular concentration of prazosin and decreasing the concentration available for binding to intracellular a1A-adrenoceptors. This may lead to the manifestation of the a1L-adrenoceptor functional phenotype. We tested this hypothesis using isolated preparations of mouse and rat prostate. Schild analysis of noradrenaline concentration-response curves in the absence and presence of prazosin (1–300 nM) yielded affinity values of 8.80 in rats (n=6) and 8.68 in mice (n=3). Addition of the BCRP inhibitor, Ko-143 (1 lM) increased the pA2 value in rat prostate to 9.83 (n=6) while genetic deletion of the BCRP transporter in mouse prostate increased the pKB for prazosin to 8.92 (n=5). Pharmacological blockade of P-gp or extraneuronal uptake had no effect in rat prostate (n=6). These results confirm that mouse and rat prostate glands yield affinities to prazosin and tamsulosin which are consistent with the a1L-adrenoceptor functional phenotype. Moreover, blockade or deletion of the BCRP transporter, but not P-gp or extraneuronal uptake transporters, increases the affinity of prazosin towards that seen at a1A-adrenoceptors.


116 377 EFFECTS OF CINNABAR-CONTAINING MEDICINE AND MERCURY COMPOUNDS ON MOUSE LIVER AND KIDNEY PATHOLOGY AND MT EXPRESSION

in ameliorating STZ- and alloxan-induced diabetes, and metabolic disorders in animals; (ii) DNLA could decrease the FBG level in diabetic db/db mice.

Shi J., Yang H., Liu X., Xu Y., Wu Q., Liu J. Objective: To examine the expression of metallothionein and pathology in mouse liver and kidney following administration of cinnabarcontaining traditional medicine and mercury compounds. Methods: Mice were gavaged with cinnabar-containing Zhu-Sha-AnShen Wan (ZSASW, 1.8 g/kg), cinnabar (0.2 g/kg), high-dose cinnabar (2 g/kg), HgS (0.2 g/kg), HgCl2 (0.032 g/kg), MeHg (0.026 g/kg) or saline daily for 30 days. Liver and kidneys were collected and processed for pathological examination and the expression of metallothionein was determined by immunohistochemistry (IHC) and western blot (WB). Results: HgCl2 and MeHg administration produced significant pathological lesions in liver and kidney along with increases in tissue metallothionein expression. In the HgCl2 and MeHg groups, inflammatory cell infiltration, cell degeneration and cell death were evident. The most severe damage occured in the MeHg group, with necrosis in tubule cells and hepatocytes. These changes were mild in cinnabar, HgS and cinnabar-containing ZSASW groups, but were evident in 10fold higher dose cinnabar group. Conclusions: Cinnabar, HgS and ZSASW at clinical doses are safe, but a 10-fold higher dose of cinnabar produces liver and kidney damage, and more damage is seen with HgCl2 and MeHg, along with increased metallothionein expression. Metallothionein protein expression is a sensitive biomarker in mouse liver and kidneys after administration of mercury compounds.

378 EFFECTS OF DENDROBIUM NOBILE LINDL ALKALOIDS ON EXPRESSION OF SIRT1 AND SIRT1-REGULATED METABOLIC GENES IN MICE Xu Y., Jingshan S. Background: Dendrobium nobile Lindl alkloids (DNLA) are shown to have glucose-lowering and anti-hyperlipidemia effects in streptozotocin (STZ)- and alloxan-induced diabetic rats, rats fed with high-fat diets, and in mice challenged with adrenaline. The present studies further examined the effects of DNLA on Sirt 1, the key regulator of hepatic glucose and lipid metabolism gene, and its regulated genes in mouse livers. This was followed by investigation of the hypoglycaemic effects of DNLA on db/db mice. Methods: (i) Male Kunming mice were randomly divided into three groups and given DNLA at doses of 0, 120 and 240 mg/kg, PO for 8 days. The expression of genes for glucose and lipid metabolism was determined by real-time RT-PCR; (ii) Male C57BL/KsJ mice were assigned to the normal control group, and male C57BL/KsJ-db/db mice were randomly divided into five groups based on fasting blood glucose (FBG): the model group, the rosiglitazone (positive control) group, and DNLA treatment groups (20, 40 and 80 mg/kg). All mice were dosed twice daily by gavage for 4 months and FBG concentration was monitored once a week. Results: (i) DNLA activated the Sirt1-PGC1a signaling pathways and increased mRNA levels of Glut2 and FoxO1 in mouse livers. DNLA also regulated the expression of fatty acid b-oxidation genes Acox1 and Cpt1a. Moreover, the lipolysis gene Pnpla2 was increased, while the lipid synthesis regulator Srebp1 (sterol regulatory element-binding protein-1) was decreased. Interestingly, DNLA increased mRNAs of the anti-oxidative gene metallionein1 and the Nrf2-target gene Nqo1 in mouse livers. (ii) The blood glucose in the model group was significantly increased compared with the normal control group. Both DNLA and rosiglitazone significantly decreased the blood glucose compared with the model group. Conclusions: (i) DNLA increases Sirt1 and Sirt1-regulated glucose and lipid metabolism gene expression, and enhances hepatic metallothionein-1 and Nqo1 in mouse livers, which could play integrated roles

379 EFFECTS OF FENOFIBRATE CO-ADMINISTRATED WITH FRUCTUS SCHISANDRAE PEEL EXTRACT ON LIPID AND GLUCOSE METABOLISM IN NORMAL AND HIGH CHOLESTEROL/BILE SALT DIET-FED MICE, WITH ATTENTION TO HEPATOTOXICITY Pan S., Zhu P., Wang X., Zhang Y., Sun N., Yu Z., Zhou S., Ko K. Background: Currently, combined therapy using herbal and chemical drugs has become a feasible therapeutic intervention against some diseases. Researches have shown that Fructus Schisandrae (FS) has a significant impact on fat metabolism. The present study investigated the effects of combination treatment with aqueous extract of FS peel (AqFS-P)/bicylol and fenofibrate (FF) on lipids/glucose in mice fed with normal diet (ND) or a high cholesterol/bile salt (0.5/0.15%, w/w) diet (HCBD). Methods: FS peels were separated from seeds and AqFS-P was prepared. Male ICR mice were fed either ND or HFD containing 0.1% fenofibrate (FF) without or with AqFS-P at 1, 3 and 9% (based on crude herbal material, w/w) or bicylol at 0.025% for 10 days. Serum and hepatic triglyceride (TG), total cholesterol (TC), glucose (GLU), serum alanine aminotransferase (ALT) activity, and fecal TC were measured. Results: Supplementation with FF significantly reduced serum and hepatic TG, TC and GLU levels (approximately 79%) in mice fed with ND and/or HFD. SAqFS-P treatment increased the FF-induced reduction of hepatic, but not serum, TC and TG contents (up to 67 and 26%, respectively) in ND-fed mice, but not in HCBD-fed mice. However, the hepatic GLU-lowering effect of FF supplementation was enhanced by 20 and 16% in both normal and hypercholesterolemic mice, respectively. FF treatment enhanced the excretion of fecal TC by 10 and by 42% in mice on normal and hypercholesterolemic diets respectively. Fecal TC contents were increased by 12% in the combination therapy with FF and SAqFS-P in ND-fed mice, when compared with the FF supplementation alone. Serum ALT activity was elevated by 43% in HCBD-fed mice. FF caused a significant increase in ALT activity by 199 and 120% in normal and hypercholesterolemic mice, respectively. Although co-administration with SAqFS-P and FF elevated serum ALT activity by 98% in HCBD-fed mice, the difference was not statistically significant. Bicylol markedly attenuated the ALT activity by 54% in mice fed with ND supplemented with FF. Conclusions: SAqFS-P treatment enhanced the hepatic lipids/GLUlowering effect of FF supplementation in mice fed with ND and/or HCBD. The authors declare that they have no competing interests.

380 EFFECTS OF FRUCTUS SCHISANDRAE EXTRACT SUPPLEMENT ON SERUM/HEPATIC LIPIDS AND GLUCOSE IN NORMAL DIET- AND HYPERCHOLESTEROLEMIC DIET-FED MICE Pan S., Wang X., Zhang Y., Zhou S., Sun N., Zhu P., Yu Z., Ko K. Background: According to the traditional Chinese medicine theory, Fructus Schisandrae (FS, WuWeiZi in Chinese) contains all five tastes: sweet (fruit skin), sour (fleshy tissue), bitter and pungent (seed core), and salty (all parts). The current study investigated the effects of ethanolic extract of peel and seed of FS or their combination (EtFS-P, EtFS-S, EtFS-P/S, respectively) on lipids and glucose (GLU) in normal and hypercholesterolemic mice. Methods: Male ICR mice were fed with either normal diet (ND) or HCBD containing 0.3, 1, 3 and 9% EtFS (based on crude herbal material, w/w) or 0.1% fenofibrate (FF, w/w) for 10 days. On day 11


117 serum and hepatic Triglyceride (TG), total cholesterol (TC), glucose (GLU), and serum high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were determined. Results: Feeding mice with HCBD for 10 days significantly increased serum TC/LDL and hepatic TC/TG levels by 35–294%, compared with ND-fed mice. Supplementation with EtFSs (EtFS-S, EtFS-P, and EtFSS/P) significantly reduced hepatic TG and TC levels by 18–47% in both ND- and HCBD-fed mice. EtFS-S, EtFS-P and EtFS-S/P reduced serum TG levels (approximately 22–29%), whereas EtFS-P and EtFSS/P elevated serum TC (up to 26 and 44%, respectively) in HCBD-fed mice. EtFS-P and EtFS-S/P markedly elevated serum LDL levels in normal mice. Supplement with EtFS-P and EtFS-S/P, but not EtFS-S, increased serum HDL and LDL levels (up to 17 and 73%, respectively) in normal or hypercholesterolemic mice. Treatment with EtFSS, EtFS-P and EtFS-S/P decreased hepatic GLU levels (by 9–44%) in both ND- and HCBD-fed mice. Daily intake of EtFS-S decreased serum GLU levels in ND-fed mice (up to 18%). However, feeding mice with EtFS-S/P increased serum GLU levels by 10 and 17% in ND- and HCBD-fed mice, respectively. Fenofibrate treatment reduced serum/hepatic TC, TG, and GLU levels by 15–113% in both ND- and HCBD-fed mice. Conclusions: Supplementation with ethanolic extracts of FS peel and seed regulated lipid and glucose metabolism in normal and hypercholesterolemic mice. The authors declare that they have no competing interests.

381 EFFECTS OF MELATONIN ON LIPOPOLYSACCHARIDEINDUCED DISSEMINATED INTRAVASCULAR COAGULATION IN RATS Wu C., Chen S., Chou H., Liaw M., Shih C., Lue W., Chen R. Background: Disseminated intravascular coagulation (DIC) is a common complication of sepsis and leads to multiple organ failure (MOF) and death. Lipopolysacchride (LPS) induces the expression of tissue factor on monocytes and endothelium, leading to coagulation activation and microvascular thrombosis. Oxidative stress and cytokines play a major role in coagulopathy of sepsis. Melatonin is an anti-oxidant and anti-inflammatory agent in addition to its effect on circadian rhythm. Therefore, this study tried to evaluate the effects of melatonin on LPS-induced DIC rats. Methods: Wistar rats were divided into six groups: (i) Sham; (ii) Sham + EtOH (1% EtOH, at 40 min and 3 h after saline); (3) Sham + Melatonin (3 mg/kg in 1% EtOH, at 40 min and 3 h after saline); (4) LPS (10 mg/kg, i.v. infusion), (5) LPS + EtOH (1% EtOH, at 40 min and 3 h after LPS) and LPS + Melatonin (3 mg/kg in 1% EtOH, at 40 min and 3 h after LPS). The changes of hemodynamics, blood glucose, ISTH scoring parameters (i.e. platelet, fibrinogen, prothrombin time (PT) and D-dimer), cytokines, thrombin-antithrombin complex, NO, lactate dehydrogenase (LDH), hepatic function (ALT), renal function (BUN and creatinine), tissue factor pathway inhibitor and survival rate were monitored over 6 h. Aorta, lung, liver and kidney were harvested at 6 h after LPS to perform the expression of inducible NO synthase and plasminogen activator inhibitor 1 (PAI-1) and histological studies. Results: LPS caused hyper-coagulation, inhibition of fibrinolysis system and hypotension. However, these effects did not match the criteria of the ISTH for overt-DIC. Melatonin or its vehicle (1 % EtOH) attenuated the level of TNF-a and IL-6, decreased the level of LDH and ALT, improved prolongation of PT and hypotension, and increased survival rate in LPS-rats. In addition, melatonin further attenuated kidney damage, liver coagulopathy, and maintained the level of fibrinogen. Conclusions: We suggest that melatonin and EtOH seem to have beneficial effects on coagulopathy and MOF in LPS-rats. In addition, melatonin maintained the activity of the fibrinolysis system, prevented fibrin deposition and improved MOF.

382 EFFECTS OF TRICHILIA MONADELPHA BARK CREAMS ON EXCISION WOUNDS IN WISTAR RATS Ayande P., Woode E. Introduction: Wounds are epithelial defects, resulting from mechanical and thermal damage otherwise, are traceable to the presence of an underlying pathophysiological condition. Wound disorders present serious clinical problems and are likely to increase since they are associated with diseases such as diabetes, hypertension, and obesity. In much the same way, malnutrition can profoundly influence wound healing at multiple points in the phases of wound repair. Method: The excision wound model was adopted to assess molecular, cellular, and tissue movements in Wistar rats. It represents an animal model that provides access to investigate complex tissue movements associated with repair. Accordingly, repair of the injured skin required coordinated cellular movements to restore epidermal, dermal, and subcutaneous tissue as expected of the aqueous Trichilia bark creams. Results: The effect of aqueous extract cream on excised wounds showed appreciable rate of healing in the 3% and 10% mg Trichilia treated groups at P < 0.01 and P < 0.001 respectfully. But not in the 30% dose group which had not completely healed by day 13 of treatment, P > 0.05. In the ethanolic extract cream however, rate of healing was dose dependent and comparable to the standard treatment. Most wounds had completely healed by day 13, evidenced by epithelialisation scar with P > 0.05, P < 0.001 and P < 0.01 for the 3%, 10% and 30% dressings respectfully. The petroleum ether extract cream showed a relatively poor rate of healing, with sepsis as evidence of chronic wounds at P > 0.05, for the 10% and 30% dressings. The ethyl acetate extract cream was an extremely poor wound formulation, characteristic of ineffective treatment, as evidenced by increased mortality peaking to 100% in the 30% Trichilia treated group. Conclusion: The petroleum ether and ethyl acetate extracts did not proof to be effective creams for excision wound treatment. But the ethanolic extract cream showed consistent dose dependent effect on the healing of excision wounds, while the aqueous extract cream was found to be effective at minimal doses.

383 ETHNOPHARMACOLOGICAL SURVEY OF HERBAL TREATMENT OF MALARIA IN LAGOS STATE, NIGERIA Oreagba I., Ishola I., Oshikoya K., Adirije C. Background: Drug-resistant P. falciparum has been on the increase at a faster rate than new antimalarial drugs are developed. This suggests a need to search for new plants for the development of new drugs that will be useful for prevention and treatment of malaria. In view of this, an ethnobotanical survey was conducted to investigate the pattern of treatment practices and medicinal plants used for malaria therapy in Lagos State, Nigeria. Methods: Focus group discussions were initially held with traditional herbal medicine practitioners (THMPs), herb retailers (HRs), elderly people, child caregivers and the general population to identify who amongst them have ever used herbs to treat malaria fever. Participants were recruited from various local government areas in Lagos. Five hundred and fifty eligible participants were interviewed with a semistructured questionnaire purposefully designed to capture information on the type, composition, method of preparation, dosage, and mode of administration of herbal preparations used as local antimalarial therapies. Results: Herb sellers (36.4%), THMPs (27.3%), children caregivers (27.3%), undergraduate students with knowledge of herb use (5.5%), and elderly people with knowledge of herb use (3.6%) were the participants in this study. Forty one plant species belonging to 27 families were identified to be employed locally for antimalarial herbal recipes. Of these Enantia chlorantha (31.5%), Carica papaya (27.5%), Azadirachta indica (25.5%), Cymbopogon citrates (23.3%), Morinda lucida (22.7%), Mangifera indica (21.1%), and Alstonia boonei (20.5%)


118 were the most frequently used plants. The stem barks, roots, leaves or whole plants were the plant parts frequently used. These were used either alone or in combination with other plant parts. Different plant species were also used in combinations. Conclusion: There are indigenous plants with potential antimalarial properties identified in this survey. The plants could serve as good sources of new antimalarial drug development

384 ETHYLACETATE EXTRACT OF LONCHOCARPUS CYANESCENS LEAF INHIBITS DIABETES RELATED ENZYMES AND SUPPRESSES POSTPRANDIAL HYPERGLYCEMIA IN RATS Kazeem M., Ashafa A. Inhibition of some carbohydrate metabolizing enzymes such as pancreatic a-amylase and intestinal a-glucosidase is an important strategy in the management of hyperglycemia associated with Diabetes mellitus. Some studies had shown that the leaves of Lonchocarpus cyanescens possess hypoglycemic activity but the mechanism(s) by which it exerts this effect is not known. Therefore, this study is aimed at evaluating the in vitro inhibitory effects of different extracts of L. cyanescens leaf on the activities of diabetes-related enzymes (a-amylase and a-glucosidase) and the effect of its ethylacetate extract on postprandial hyperglycemia in sucrose-loaded rats. Acetone, ethylacetate and hexane extracts of L. cyanescens leaves were subjected to standard enzyme inhibition assay followed by determination of modes of inhibition of the enzymes using the most potent extract. The effect of oral administration of ethylacetate extract of L. cyanescens leaf in wistar rats was also assessed by determining blood glucose levels of the animals within 2 h of loading with 3 g/kg sucrose solution. Results of this study showed that ethylacetate extract was the most potent inhibitor of a-amylase (IC50: 7.37 mg/mL) and a-glucosidase (IC50: 0.25 mg/mL). Lineweaver-Burke plot of the enzyme kinetics showed that the mode of inhibition of a-amylase by the ethylacetate extract of this plant is competitive while it inhibited a-glucosidase non-competitively. Administration of ethylacetate extract of L. cyanescens leaf to sucrose-loaded Wistar rats significantly reduced (P < 0.05) the blood glucose level of the rats within 2 h compared to the control animals. This study suggests that the previously reported hypoglycemic effect of L. cyanescens leaf could be attributed to the inhibition of diabetes related enzymes (a-amylase and a-glucosidase). It can also be concluded that the potential of this plant may be due to the presence of phytochemicals such as alkaloids, flavonoids and terpenoids.

385 EUTERPE OLERACEA MART. (ACAI) EXTRACT PREVENTS OXIDATIVE STRESS, ENDOTHELIAL DYSFUNCTION AND VASCULAR AND RENAL CHANGES IN 2 KIDNEY, 1 CLIP HYPERTENSION Da Costa C., de Bem G.F., Barcellos Santos I., Braz de Oliveira P., dos Reis Marins de Carvalho L., Augusto Vieira de Souza M., da Cunha P., Soares de Moura R., de Castro Resende A. Studies have shown that Euterpe oleracea Mart. (acßai) is rich in polyphenols and consumption of polyphenol-rich food is associated with protection against cardiovascular risk. This study examined the effect of acßaı stone extract (ASE) on the endothelial dysfunction, oxidative stress, vascular and renal structural changes associated with 2 kidney, 1 clip (2K,1C) hypertension. The experiments were approved by the Ethics Committee of Animal Experiments of the UERJ (protocol: CEA/024/2010). Young male Wistar rats used to obtain 2K1C and control rats (2K) received daily treatment with vehicle or ASE (200 mg/kg/day) for 40 days. Systolic blood pressure (SBP) was measured by plethismography. The vasodilator effect of acetylcholine (ACh) was studied in perfused mesenteric arterial bed (MAB) pre-con-

tracted with norepinephrine (30 mM). Antioxidant enzymes, oxidative damage and nitrite levels were evaluated in mesenteric arteries and kidney by spectrophotometry. The expressions of eNOS, pro (NOX-4) and antioxidant enzymes and metalloproteinase 2 (MMP-2) were assessed by western blot and the activity of MMP-2 was analyzed by zymography in mesenteric arteries. The treatment with ASE prevented 2K,1C hypertension and the reduction of acetylcholine-induced vasodilation. The reduced antioxidant activity in samples of mesenteric arteries and kidney of 2K,1C rats were recovered by ASE. Expression of Superoxide dismutase-1, 2, and eNOS and production of NO were decreased in 2K,1C rats and recovered by treatment with ASE. The increased expression of MMP-2 and NOX-4 and MMP-2 activity in 2K,1C rats were reduced by ASE. 2K,1C rats showed an increase in medial thickness of mesenteric artery and aorta, which was prevented by ASE. The renal morphological changes in 2K,1C rats were prevented by ASE. Therefore, treatment with ASE prevents the development of hypertension, improves endothelial dysfunction and prevents the renal and vascular changes in 2K,1C rats. The reduction in antioxidant activity and increased lipid peroxidation and carbonil protein suggest the involvement of a mechanism of impaired antioxidant defense and increased oxidative damage in 2K,1C rats, which was reversed by ASE. The NO synthesis, the antioxidant action and the inhibitory effect on MMP-2 levels induced by ASE may contribute to the beneficial effects of the extract.

386 EVALUATION OF ANTI INFLAMMATORY AND ANTINOCICEPTIVE ACTIVITY OF VANILLIN IN RATS Bains R., Kaur N. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the primary chemical components extracted from the seedpods of Vanilla planifolia, a monocotyledonous orchid and widely used in foods, cosmetics, beverages and drugs.Vanillin is known to have antimutagenic, antiinvasive and metastatic suppression potential. Antinociceptive property in acetic acid, antioxidant and hepato-protective properties in carbon tetrachloride-treated rats have also been demonstrated. The objective of this study is to evaluate the anti inflammatory and antinociceptive activity of vanillin. Rats were assigned to groups of six animals each. Carrageenan induced paw edema was used to evaluate pre and post antinociceptive activity and Tail flick method was used in the evaluation of anti nociceptive activity. The result of the anti-inflammatory activity demonstrated that vanillin in higher doses was effective in the early phase of inflammation which has been reported due to the release histamine and serotonin primarily. Ethanolic extract in dose of 50 mg/ kg, orally showed antinociceptive activity which was comparable to pethidine but in higher doses of 200 and 300 mg/kg, the effect was more pronounced than the standard drug. The results obtained strengthen the claim of vanillin as anti-inflammatory and antinociceptive agent.

387 EVALUATION OF ANTIASTHMATIC ACTIVITY OF AQUEOUS AND METHANOLIC ROOT EXTRACT OF CALOTROPIS PROCERA (ASCLEPIADACEAE) ON EXPERIMENTAL GUINEA PIGS AND WISTAR RATS Aliyu I., Zezi A., Magaji M., Abdu-aguye I. Background: The decoction of the leaves, stems, latex, fruits and roots of Calotropis procera is reported to be used traditionally for many ailments including bronchial asthma. Methods: The present study deals with the effect of methanolic and aqueous extracts of roots Calotropis procera by using various in vivo animal models and in vitro studies. Animal models used include egg albumin induced passive paw anaphylaxis in rats and carageenaninduced leucocytosis in rats. Using an in vitro model, spasmolytic


119 activity on isolated guinea pig ileum (10 mg/ml extract) was used. Effects of aqueous and methanolic extracts were compared. Results: Both aqueous and methanolic Calotropis procera extract (10 mg/ml) significantly (P < 0.001) inhibited, concentration dependently histamine induced contractions of the isolated guinea pig ileum. Significant (P < 0.001, P < 0.005, and P < 0.01) and dose dependent decrease in total leucocyte count comparable to that of chlorpheniramine (CPM) (P < 0.01) at dose 200 mg/kg aqueous extract, 100 mg/ kg aqueous extract, and 100 mg/kg methanolic extract were obtained in carrageenan-induced leucocytosis model respectively. In passive paw anaphylaxis, all treatment groups (250 mg/kg and 350 mg/kg for both aqueous and methanolic extracts, 0.27 mg/kg dexamethasone) showed a significant (P < 0.05) and dose dependent inhibition of anaphylactic inflammation compared to the control group after 5 h of drug treatment (i.e. 4 h after albumin administration). Conclusion: These results provide experimental evidence suggesting that root extracts of both aqueous and methanolic C. procera possess anti-asthmatic activities in both in vitro and in vivo animal models.

388 EVALUATION OF ANTICONVULSANT ACTIVITY OF METHANOL STEM BARK EXTRACT OF BOSWELLIA DALZIEL II - HUTCH BURSERACEAE IN MICE AND CHICKS Balarabe Nazifi A., Danjuma Mohammed N., Ya’u J. Background: Boswellia dalzielii - Hutch (family Burseraceae) is used traditionally in Northern Nigeria as tranquillizer, anticonvulsant and in the treatment of mental illness, fever and rheumatism. The aim of this work therefore was to screen for the anticonvulsant effect of Boswellia dalzielii as to elucidate scientific basis for its use. Method: Phytochemical screening and LD50 described by Lorke was investigated. Elemental content of the extract was determined using atomic absorption spectrophotometer. The extract was evaluated for anticonvulsant activity using maximal electroshock test, pentylenetetrazole, strychnine, picrotoxin and 4-aminopyridine-induced seizures at doses of 20, 40 and 80 mg/kg. The results were analyzed for statistical significance using one-way ANOVA followed by Dunnett’s test. A P < 0.05 was considered significant. Results: In the maximal electroshock test, the extract at 20 mg/kg provided 40% protection and significantly (P < 0.05) increased the mean time to onset of seizure (7.031.0) compared to control (4.630.28). The extract at 40 mg/kg provided 50% protection against picrotoxin induced convulsion. It also produced a dose dependent increase in latency to convulsions with significant difference (P < 0.05) at 40 mg/ kg (19.004.00) and P < 0.01 at 80 mg/kg (20.501.59) compared to control (11.500.56). The extract produced a dose dependent increase in the mean time to onset of pentylenetetrazole-induced seizures with significant difference (P < 0.05) at 80 mg/kg (10.752.01) compared to control (5.000.73). In the strychnine-induced seizure, 33.33% of mice were protected at 40 mg/kg. It also significantly (P < 0.05) increased the latency of convulsion at the dose of 20 mg/kg (13.002.02) and 80 mg/kg (10.60.75) compared to control (6.330.49). The extract however had no effect on 4-aminopyridine-induced seizures: Thin layer chromatography analysis on the extract revealed the presence of sugars, tannins, flavonoids, cardiac glycosides and saponins, while alkaloids were absent. The intraperitoneal LD50 of the methanol extract in mice was 282.2 mg/kg. The elemental analysis revealed the presence of calcium 32.505 ppm, magnesium 5.761 ppm, iron 0.591 ppm, zinc 0.382 ppm, nickel 0.158 ppm, manganese 0.051 ppm, copper 0.102 ppm and cadmium 0.005 ppm. Conclusion: The methanol extract of Boswellia dalzielii has anticonvulsant activity and may be exerting its effect by affecting the GABAergic and glycinergic pathways. The activity may also be due to the appreciable calcium content of the plant.

389 EVALUATION OF ANTIDEPRESSANT-LIKE ACTIVITY OF CURCUMIN BY USING FORCED SWIMMING TEST COMPARED WITH IMIPRAMINE Ataee R., Ataie A. Various antidepressants, ranging from monoamine oxidase inhibitors to recently developed dual reuptake inhibitors, are prescribed for alleviating the symptoms of depression. Despite the availability of these blockbuster molecules, approximately 30% of depressed patients do not respond to the existing drug therapies and the remaining 70% fails to achieve complete remission. Moreover, antidepressants are associated with a plethora of side effects and drug-drug/drug-food interactions. In this context, novel approaches are being tried to find more efficacious and safer drugs for the treatment of major depression. Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. There is not enough information available about the antidepressant activity of curcumin. In the present study, we analyzed the effects of curcumin on depressive-like behaviors in mice, using the forced swimming test. Our results showed that curcumin at the doses of 1.25, 2.5, 5 and 10 mg/kg reduced, in a dose dependent manner, the duration of immobility in the forced swimming test, resulting in a 7.01%, 44.76%, 51.97%, 75.64% immobility reduction compared with the negative control group, respectively. Also, it was 68.98% for imipramine at 10 mg/kg. Considering that clinical antidepressant effects often appear after chronic treatment, the long term effects of curcumin should be evaluated and further studies should focus on the receptors and signal transduction to elucidate the detailed mechanisms of the antidepressant effect of curcumin.

390 EVALUATION OF ANTIHYPERLIPIDEMIC &CARDIOPROTECTIVE EFFECT OF METHANOLIC EXTRACT OF LAGENARIA SICERARIA Khare A., Khadke V. Hyperlipidemia is a highly predictive risk factor for atherosclerosis, coronary artery disease, and cerebral vascular diseases. Condiments, medicinal plants, fruits used in day-to-day preparation of food in Indian kitchens have been identified as hypolipidaemic in Ayurveda. Lagenaria siceraria commonly known as Bottle gourd, which is official in Ayurvedic Pharmacopoeia of India, and having composition of variety of essential phytoconstituents, so that the fruits are traditionally used for their cardioprotective, cardiotonic, general tonic, diuretic, aphrodisiac, antidote to certain poisons and scorpion strings, alternative purgative, and cooling effects. In the present study, extending previous work done, methanolic extract of L. siceraria was obtained by using Column Chromatography. Thirty-six healthy rabbits were used in the study in which 24 were drug induced hyperlipidemic rats using TritonX-100 and 6 served as controls.Blood samples were collected every 15 days for a period of 6 weeks to observe complete Lipid profile and C-reactive protein.The study exhibited that elevated levels of blood cholesterol, triglycerides, LDL, were significantly reduced and decreased HDL was significantly increased by administration of methanolic extract. C- reactive proteins were also seen significantly reduced in the test group than in the group controls. In the present study direct relation was observed in reduction of levels of triglycerides, LDL, Blood cholesterol, C-reactive proteins owing to the study formulation given. Thus the overall effects can be considered as a good marker of the the antihyperlipidemic effects and reduction in associated morbidity with use of Lagenaria siceraria. Clinical trial in humans is advocated to validate the effect.


120 391 EVIDENCE FROM SYNTHETIC AGONISTS OF THE CXCR4 CHEMOKINE RECEPTOR ALTERS THE DOGMA OF CHEMOTAXIS ACTIVATION Escher E., Lefrancßois M., Mona C., Marsault E., Lavigne P., Leduc R., Heveker N. The recent discovery of synthetic agonists of the CXCR4 allowed for the first time to identify potent biased ligands on this receptor (ACS Med Chem Lett. 2011;2(8):597-602). CXCL12, the endogenous agonist ligand of CXCR4, was reputed to induce chemotaxis through CXCR4 by Gi and beta-arrestin recruitment. Synthetic full agonists with low nM affinity were produced through grafting the CXCL12 pharmacophore, its N-terminal peptide portion, onto T140, the cyclopeptide antagonist of CXCR4 (see figure below). These agonists displayed chemotactic behaviour like CXCL12, both in vitro and in vivo. Subsequent signalling pathway analysis showed that these agonists couple, like CXCL12, CXCR4 to Gi with nanomolar affinities. These synthetic agonists did, however, not induce beta-arrestin coupling and consequently, significantly less CXCR4 internalization than the one observed upon CXCL12 stimulation. The availability of biased agonists for these chemokine receptors will allow dissection of the hitherto not well-understood functional interactions of CXCR4. These agonists are also of interest as tools for structural biology investigations to provide molecular details on the mechanism of CXCR4 activation, which remains unknown despite the CXCR4 crystal structure. Finally, agonistic CXCR4 ligands possess several attractive features as alternatives for CXCR4 targeted drug therapy where antagonists are actually the only option; therefore synthetic transitions to partial and fully peptidomimetic agonistic moieties are under way. Figure: Typical synthetic CXCR4 agonist with 4 nM affinity and full agonist properties on chemotaxis and Gi activation

Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Arg-Cys-Lys-OH Lys-Pro-BPA-Ser-Leu-Ser-Tyr-Arg-Ser

392 EVIDENCE THAT a1B-ADRENOCEPTORS MEDIATE CONTRACTIONS OF MOUSE SPLEEN Docherty J., Daly C. In rat spleen, adrenergic contractions are reported to involve alpha1and alpha2-adrenoceptors (for references, see Docherty, 2010), and it has been suggested that the alpha1-adrenoceptor present is an alpha1Badrenoceptor. We wished to confirm this identification in rat spleen, but in the absence of a truly selective alpha1B-adrenoceptor antagonist, it is difficult to positively identify the alpha1-adrenoceptors present pharmacologically. However, this problem can be studied by gene knock-out technology in the mouse. Hence, we have investigated isometric contractions to noradrenaline both in rat bisected and in mouse whole spleen in the presence of the noradrenaline transporter blocker cocaine (3 lM). Animals were killed by overdose of CO2 and cervical dislocation. In rat spleen, the alpha1-adrenoceptor antagonist prazosin (10-8M) or the alpha2-adrenoceptor antagonist yohimbine (10-6M) alone produced only small shifts in noradrenaline potency, but the combination produced a large shift in noradrenaline potency. In mouse spleen, noradrenaline was more potent than in rat spleen, but we have found similarly that both prazosin (10-8M) and yohimbine (10-6M) inhibit components of the contraction to noradrenaline and that the combination produced a large shift in noradrenaline potency. However, in mice lacking alpha1A and alpha1D-adrenoceptors (alpha1A/D-KO mice), the effects of prazosin and yohimbine were similar to their effects in WT mice. In particular, prazosin shifted the noradrenaline concentration-response curve (CRC) in a similar way in WT and alpha1A/D-KO mice, both in terms of degree of shift in potency and

in change of shape of CRC. In spleen from both WT and KO mice, prazosin shifted the contractile response to higher concentrations of noradrenaline more markedly than the response to low concentrations, with similar pKB values of 9.30 and 9.34, in WT and KO, respectively, respectively. Hence, in alpha1A/D-KO mice, in which the only alpha1-adrenoceptor present is the alpha1B-adrenoceptor, prazosin still antagonised contractions to noradrenaline. It is concluded that both alpha2- and alpha1-adrenoceptors mediate contractions of mouse spleen and that the major, if not exclusive, alpha1-adrenoceptor involved is the alpha1B-adrenoceptor. Docherty, JR (2010). Cell Mol Life Sci 67, 405-417.

393 EXPLORING BIOACTIVE PEPTIDES AS PHARMACOLOGICAL TOOLS FOR OXYTOCIN AND VASOPRESSIN LIGAND DESIGN Gruber C., Koehbach J., Freissmuth M. The diversity in nature has long been and still is one of the biggest resources of pharmaceutical lead compounds and many natural products often exhibit biological activity against unrelated biological targets, thus providing us with starting points for pharmacological analysis. Natural peptides of great number and diversity occur in all organisms from plants to microbes to man. Examples for such rich and yet largely untapped libraries of bioactive compounds are animal venom peptides, invertebrate peptide hormones or plant defense peptides. Our goals are to discover and characterize novel bioactive peptides, screen their pharmacological activity, determine their structureactivity relationship and synthesize optimized peptide compounds to study ligand-receptor signaling.[1] We have used a genome-mining approach or mass spectrometry to determine the occurrence and molecular structure of naturally-occurring oxytocin-like peptides and we have investigated their pharmacological profile on human oxytocin and vasopressin receptors.[2] For example, cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Their unique structural topology, high stability and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. The cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a-receptors, members of the G protein-coupled receptor (GPCR) family, as molecular targets for this cyclotide. Furthermore we show that cyclotides can serve as templates for the design of selective GPCR ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof-of-concept for the development of cyclotide-based peptide ligands.[3] In summary, combining structure-activity analysis and peptide chemistry, we are aiming to generate selective, potent and stable peptide ligands that are potentially oral bioavailable and may be useful for the treatment of a wide range of challenging, but yet untreated diseases. 1. Gruber et al. Curr Pharm Des 2010; 16:3071–88 2. Gruber & Muttenthaler PLoS One 2012; 7(3):e32559 3. Koehbach et al. PNAS 2013; 110:21183–88

394 EXTRACTION AND PURIFICATION OF SIALIC ACID FROM SUBMANDIBULAR MUCINE OF CATTLE Nkeonye O., Sallau B., Ameh D. Background: Sialic acid has many biological functions and is an important component of many pharmaceutical products. Due to its high cost, there is a need to find a cheaper source for its production. The


121 aim of this research was to extract, purify, quantify and characterize sialic acid from the submandibular mucine of cattle. Methods: The submandibular gland of cattle was obtained from an abattoir, homogenized at 4°C using phosphate buffer saline at pH 7.2, incubated with phospholipase and lipase at different temperatures and time intervals then hydrolyzed using 0.1 M H2SO4. The homogenate was then purified by eluting it through an ion exchange column using NaCl concentrations of 0.0–0.5 M. Fourier Transform Infrared spectroscopy was then carried out and the result compared with that of standard sialic acid obtained from Sigma ran concurrently. Results: Incubation with phospholipase led to an increase in quantity of sialic acids released. Acid hydrolysis of the homogenized submandibular glands led to a 4.5 fold increase in sialic acid released. The extracted sialic acid was eluted during ion exchange chromatography using 0.2 and 0.3 M concentrations of NaCl. The FTIR analysis revealed that the extracted sialic acid was as pure as the standard sialic acid. Conclusion: The results showed that the submandibular mucine of cattle is rich in sialic acid. Enzymes can be used to increase the quantity of extracted sialic acid which can find application in pharmaceutical research and drug development.

395 FLAVONOLS - POTENTIAL NEW TREATMENTS FOR OCULAR NEOVASCULARIZATION CAUSING LOSS OF VISION: TARGETTING NADPH OXIDASE SIGNALING Dusting G., Chan E., van Wijngaarden P., Hakami N., Peshavariya H. The proliferation of new blood vessels in the retina is a leading cause of vision impairment. Excessive neovascularization also compromises vision after corneal injury and surgery. NADPH oxidase (Nox) is involved in cell signaling for retinal neovascularization: we have shown the Nox2 isoform contributes to oxygen-induced retinopathy (OIR) in mice. We have now investigated the involvement of Nox4 isoform in both OIR and corneal neovascularization, and show that a synthetic flavonol acts as a selective inhibitor of Nox4 expression, and thereby reduces aberrant ocular neovascularization. Methods: In HUVEC Nox isoforms and adhesion molecule expressions were evaluated by PCR. HUVEC proliferation and tubulogenesis were determined in a Matrigel assay. For OIR, neonatal wildtype and Nox4-/- mice aged 7 days (P7) were exposed to 5 days hyperoxia (75% O2), followed by room air. Eyes were harvested on P8 and P17 for quantitation of retinal vaso-obliteration and neovascularization, respectively. Retinal expression of Nox4 and VEGF-A were measured by PCR and superoxide was detected by dihydroethidium staining of fresh frozen sections. Corneal injury was produced in mice by an alkali burn and assessed after 14 days. Results: Inhibition of Nox4 in vitro using either RNAi or a flavonol (2HF, 10-50 lM, shown to inhibit Nox4 expression specifically) markedly reduced HUVEC proliferation and tubulogenesis. 2HF also abolished cytokine-induced ICAM-1 and VCAM-1 expression. In wild-type mice, OIR was characterized by neovascularization at P17, which was associated with increases in Nox4 (12-fold) and Nox2 (4-fold) as well as VEGF (2.5-fold) gene expression. At P17 the inner retina also showed superoxide generation and accumulation of macrophages and microglia. Nox4-/- mice exhibited markedly less retinal neovascularization (7.90.7%, n=13) but similar elevations of VEGF (2-fold) as wildtype (13.60.8% and 2.5-fold, respectively; n=14). In contrast, in Nox2-/mice VEGF did not increase. Analogous to Nox4-/-, in OIR studies daily treatment with 2HF (1 mg/kg/day, ip) also reduced retinal neovascularization (by 30%) compared to vehicle-treated, wildtype control mice. Conclusions: Clearly Nox4 facilitates retinal neovascularization in this mouse model, causing vision loss. Therapies targeting Nox4 could be of value to reduce aberrant retinal neovascularization in retinopathy of prematurity, diabetes, proliferative macular degeneration and corneal neovascularisation after injury.

396 GENETIC DIVERSITY IN BLACK SOUTH AFRICANS FROM SOWETO May A., Hazelhurst S., Li Y., Norris S., Govind N., Tikly M., Hon C., Johnson K., Hartmann N., Staedtler F., Ramsay M. Due to the unparalleled genetic diversity of its people, Africa is attracting growing research attention. Several African populations have been assessed in global initiatives such as the International HapMap and 1000 Genomes Projects. Notably excluded, however, is the southern Africa region, which is inhabited predominantly by southeastern Bantu-speakers, suffering under the dual burden of infectious and noncommunicable diseases. Limited reference data for these individuals hampers medical research and prevents thorough understanding of the underlying population substructure. Here, we present the most detailed exploration, to date, of genetic diversity in 94 unrelated southeastern Bantu-speaking South Africans, resident in urban Soweto (Johannesburg). Participants were typed for approximately 4.3 million SNPs using the Illumina Omni5 beadchip. PCA and ADMIXTURE plots were used to compare the observed variation with that seen in selected populations worldwide. Results indicated that Sowetans, and other southeastern Bantu-speakers, are a clearly distinct group from other African populations previously investigated, reflecting a unique genetic history with small, but significant contributions from diverse sources. To assess the suitability of our sample as representative of Sowetans, we compared our results to participants in a larger rheumatoid arthritis case-control study. The control group showed good clustering with our sample, but among the cases were individuals who demonstrated notable admixture. Sowetan population structure thus appears unique compared to other black Africans, which may have clinical implications. Drug discovery, development and prescription may need more careful consideration within the South African context, in order to adequately cater for the diverse range of genetic backgrounds present. Our data represent a suitable reference set for southeastern Bantu-speakers, on par with a HapMap type reference population, and constitute a prelude to the Southern African Human Genome Programme.

397 GHANAIAN MEDICINAL PLANTS - A SOURCE OF WOUND HEALING AGENTS Ekuadzi E., Dickson R., Fleischer T., Pistorius D., Oberer L. Background: This work examined three indigenous Ghanaian plants; Margaritaria discoidea (Baill.) M€ull Arg (Euphorbiaceae), Gouania longipetala Hemsl. (Rhamnaceae), and Glyphaea brevis (Spreng) Monachino (Tiliaceae), with antibacterial, antioxidant and anti-inflammatory properties, for use in the treatment of wounds and infectious diseases. Methods: The 70% ethanol extracts of the plants were assessed for their in vitro antibacterial effects using micro dilution tests. Antioxidant effects were determined using the free radical scavenging assay, lipid peroxidation assay, total phenol content and antioxidant capacity. Carrageenan induced oedema model in chicks was used to assess the anti-inflammatory effects. Using various separation techniques, 12 isolates were obtained. Their identities were established using spectroscopic methods. Results: The extracts showed considerable antibacterial, antioxidant and anti-inflammatory effects. One new flavonoid glycoside, along with three known flavonoid glycosides, two securinega alkaloids and one triterpene were isolated from M. discoidea. One monoacyl glyceride, a deoxypreussomerin and a lasiodiplodin were isolated from G. longipetala. Epicatechin and its dimer were isolated from G.brevis. The new flavonoid glycoside was elucidated as hydroxygenkwanin 8C-[a-rhamnopyranosyl (1?6)]-b-glucopyranoside [1]. Some isolated compounds demonstrated free radical scavenging and antibacterial activities. Conclusions: Microbial infection and oxidative stress are implicated in inhibiting or delaying the wound healing process. Therefore any


122 agent capable of preventing this process may facilitate wound healing. These results provide some scientific justification for the traditional use(s) of these plants.

398 GPCRDB: AN INFORMATION SYSTEM FOR G PROTEINCOUPLED RECEPTORS Gloriam D.

4. B. Vroling et al. Nucleic Acids Res., 39 (1): D309-19, Jan. 2011. 5. V. Isberg et al. Nucleic Acids Res., 42 (D1): D422-5, Jan. 2014. 6. A. Pawson et al. Nucleic Acids Res., 42 (D1): D1098-D1106, Jan 2014.

399 GRAPEFRUIT JUICE PREVENTED THE RISE IN LIVER ENZYMES AFTER PARACETAMOL OVERDOSE IN RATS Baleni R., Walubo A., Bekker Z.

Background: For the past 20 years, the GPCRDB (www.gpcr.org/ 7tm) has been a popular resource for G protein-coupled receptor (GPCR)-related data and obtained more than 1000 citatations [1-5]. The GPCRDB contains experimental data on sequences, structures, ligand-binding constants, mutations and oligomers, as well as many different types of computationally derived data, such as multiple sequence alignments and homology models. Methods: Our current implementation has focused on increasing the user-friendliness broadening the target audience to all GPCR researchers. The web-based toolbox is developed using standard web technologies such as PHP, MySQL, JavaScript and SVG, and only requires a modern web browser to run. Results: We have developed novel tools to analyze and visualize GPCR data. The tools are highly complementary to IUPHARs ‘Guide to Pharmacology’ [6], allowing for multi-receptor comparison of sequences and structures. Novelties include: • Cross-linking to IUPHARs ‘Guide to Pharmacology’ [6] • Crystal structure browser with annotations • Interactive helix box plots and snake plots with custom coloring and download • Receptor similarities for sub-sites/domains, e.g. binding or dimerization sites, using generic numbering schemes • Similarity searches based on user-selected receptor subsequences • Phylogenetic trees generated based on any sub-alignment and set of receptors • Sequence conservation statistics and alignment amino acid properties, e.g. hydrophobicity, hydrogen bond donor/acceptor capability or size • Structure-based sequence alignments and updated homology models Conclusions: The GPCRDB continues to facilitate GPCR research by providing reference alignments, easy-to-use tools and diagrams for visualization. The latest release includes a powerful, yet user-friendly computational toolbox aimed at the broadest possible audience within the GPCR research community, and his highly complementary to the excellent pharmacological data provided by IUPHARs “Guide to Pharmacology” [6]. 1. F. Horn et al. Nucleic Acids Res., 26 (1): 275-279, Jan. 1998. 2. F. Horn et al. Nucleic Acids Res., 31 (1): 294-297, Jan. 2003. 3. F. Horn et al. Nucleic Acids Res., 29 (1): 346-9, Jan. 2001.

Background: Paracetamol (PARA) is a widely used analgesic and antipyretic agent. While it is generally safe for use at recommended doses, acute overdose of PARA can cause fatal liver damage. Despite the understanding that some cytochrome P450 isoforms are responsible for activation of PARA to the hepatotoxic metabolite, N-acetyl-p-benzoquinone-imine (NAPQI), the use of enzyme inhibitors for prevention and/or treatment of PARA hepatotoxicity is still not well researched. Therefore, grapefruit juice (GFJ), a well known enzyme inhibitor, was investigated for the prevention of hepatotoxicity after PARA overdose in rats. Methods: The study was approved by the animal ethics committee, which also imposed dosage limitations for this experiement. Twelve groups of 5 Sprague Dawley rats each were treated with single oral dose of either saline, PARA only 1000 mg/kg, PARA + GFJ low dose (2 ml) and PARA + GFJ high dose (3 ml). The remaining six groups were treated with bergamottin, a GPJ derivative and known enzyme inhibitor, instead. Thereafter, 5 rats from each group were sacrificed after 24, 48 and 72 h and, on each occasion, blood samples were collected for determination of liver &renal function, FBC and PARA concentration. A piece of liver was sent for histopathalogy. Results: By 48 h the liver enzymes in the PARA only group were significantly (P < 0.05) higher than in the GFJ + PARA and bergamottin + PARA groups. The concentrations, median (range), were: alkaline phosphate (ALP) 319 [71–328] u/L and alanine transaminase (ALT) 56 [50–59] u/L for PARA only, vs. ALP 44 [39–58] u/L, ALT 40 [36–49] u/L for GFJ, ALP 34 [24-401] u/L, ALT 46 [41-49] for bergamottin, and ALP 96 [75–238] u/L and ALT 38 [37–43] u/L for the control group. Conclusion: GFJ and bergamottin prevented PARA induced increase in liver enzymes after paracetamol overdose. Although the increase in liver enzymes did not meet the criteria for clinical hepatotoxicity, the results are promising, and call for use of a higher dose.

400 HARNESSING CARBOHYDRATE ACTIVE ENZYMES AS DRUG TARGETS AND TOOLS FOR DEVELOPING GLYCOTHERAPEUTICS Osanjo G., Aluvaala E., Wadegu M., Bulimo W., Mulaa F. Background: Glycans and the enzymes that modify them, the carbohydrate active enzymes (Cazymes) are key actors in biomolecular communication and in disease pathogenesis.Essential biological recognition information encoded by glycans includes the human ABO blood group determinants which mediate transfusion rejection reactions, cellular trafficking during inflammatory responses and cancer cells metastases, infection by viruses such as the Influenza A virus and promotionof neurogenesis. Neuraminidases and alpha-L-fucosidases are Cazymes that catalyse the removal of terminal sialic acid and fucose residues from glycans respectively. Objectives: We explored the structure and function relationships of Cazymes and their glycan ligands using the neuraminidase and alphaL-fucosidase (TmFuc) from Influenza A virus and Thermotoga maritima respectively. Methodology: The Influenza A H3N2 viral neuraminidase was obtained by generating cDNA using a reverse transcriptase, followed by gene cloning and expression in Escherichia coli. Alpha-L-fucosi-


123 dase (TmFuc) was cloned from the hyperthermophile Thermotoga maritima. Directed molecular evolution approach was used to engineer transglycosidase activity of TmFuc. Transferase activity of the Cazymes were assessed by nuclear magnetic resonance and capillary electrophoresis. Results: Non-recombinant TmFuc catalysed synthesis of candidate oligosaccharides by transfer of a fucosyl residue from fucoside donors to galactoside acceptors with alpha-(1-2) regioselectivity at low yields (7%). Similar transglycosidase activity was not observed with the Influenza A virus neuraminidase. Following a cycle of mutagenesis and in vitro recombination of Tmfuc, it was shown that the best mutant exhibited a 32-fold increase in the transferase/hydrolytic kinetic ratio. Discussion and conclusion: Molecular modeling suggested that the mutations: T264A, Y267F L322P of Tmfuc increased transglycosylation by causing a reorientation of the amino-acids that are in direct contact with the substrates resulting in a better docking energy. Such recombinants with high transglycosidase activity may constitute novel enzymatic tools for synthesis of fuco-oligosaccharides. While the Influenza A virus neuraminidase did not show trans-sialidase activity, the methodology developed could be used to evaluate susceptibility of Influenza A virus to neuraminidase inhibitors, the current first line medicines for management of influenza A virus infections. 401 HEME OXYGENASE PHARMACOLOGY: DESIGN OF INHIBITORS AND ACTIVATORS Nakatsu K., Jason V., Vukomanovic D., Hum M., McLaughlin B., Rahman M., Szarek W., Jia Z., Brien J. Heme oxygenase (HO) is responsible for the generation carbon monoxide (CO, the second of three gasotransmitters), ferrous iron and biliverdin, which is rapidly converted to bilirubin. CO and biliverdin/ bilirubin are now accepted to possess cytoprotective properties by virtue of their anti-inflammatory, anti-apoptotic and anti-oxidant effects. Much of the early progress in HO studies was built on pharmacological tools that inhibited HO activity (i.e. metalloporphyrins, such as tin protoporphyrin) or induced one of the isozymes (HO-1). Our laboratory has focussed on the design of second generation inhibitors of HO, and to enhance selectivity they have been designed on skeletons that differ from the porphyrins and heme. Our QC-xx series of drugs inhibited HO with little effect on soluble guanylate cyclase or nitric oxide synthase. Some QC-xx drugs were selective for HO-1, with little inhibitory effect on HO-2, while others inhibited both enzymes. Many but not all QC-xx drugs inhibited CypP450 activity. More recently we have reported on the discovery of drugs that selectively inhibit HO-2, with little effect on HO-1. Several of these have IC50 values for HO-2 below 10 micromolar, and for HO-1 over 100 micromolar. We have also discovered compounds that activate HO. Some of our activators increase Vmax of HO-2 up to 30-fold, and without effect on HO-1. Other activators increase the activity of both HO-2 and HO-1. These pharmacological tools should be useful to explore HO function and may have practical applications.

402 HEPATO-RENAL TOXICITY EFFECTS OF B-SUCCESS (A NIGERIAN HERBAL REMEDY) ON ALBINO RATS Agbasi P., Orisakwe O. A toxicological assessment of B-success (BS) herbal remedy was carried out on liver, heart and kidney of adult albino rats. Twenty apparently healthy adult albino rats were randomly selected for the study. They were divided into four groups of five rats each. The first group received no drugs and serves as control. The second group received twenty five percent (25%) of the LD50 of the drug. The third group was given 50% and the fourth group received 75% of LD50 of BS respectively. All the animals were given free access to water and feed

for the entire period of the study which lasted for 90 days. The alanin transaminase (ALT), aspartate transaminase (AST) and urea of 50% and 75% dose groups were significantly increased compared to control. While only the creatinine value of 75% dose group was significantly increased compared to control P < 0.05. Also, the values of creatinine, ALT and AST of rats in group 4(75%LD50) were dose dependently increased P < 0.05 compared to lower dose treatment groups of 2 (25% LD50) and 3 (50%LD50). The total bilirubin of groups 2, 3 and 4 were not significantly increased P > 0.05 compared to control. The value of total cholesterol, high density lipoprotein (HDL) cholesterol of groups 2, 3 and 4 were significantly increased P < 0.05 compared to control. The low density lipoprotein (LDL) cholesterol values of groups 3 and 4 were dose dependently decreased P < 0.05 compared to control. Overall, this study implies that BS is toxic to liver and kidney and protective to heart at the doses studied.

403 HEPATOPROTECTIVE EFFECTS OF GARLIC AND ONION IN NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) IN RATS Ebeid F., Seif el-Din S., Sabra A., Hammam O., El-Lakkany N. Background: Non-alcoholic fatty liver disease (NAFLD) includes a broad spectrum of fat-induced liver injury, ranging from mild steatosis to cirrhosis and liver failure. This study investigates the hepatoprotective properties of garlic and onion in NAFLD rat model. Methods: Ninety male Sprague-Dawley rats were randomly divided into nine groups; normal (I), NAFLD induced with high fat diet (HFD; II), NAFLD switched to regular diet (RD; III), NAFLD-HFD or NAFLD-RD treated with garlic (IV, V), onion (VI, VII) or the combined garlic+onion (VIII, IX) respectively. A NAFLD rat model was established by feeding the animals with a high-fat diet for 12 weeks. These animals were then treated with garlic or/and onion or vehicle for 8 weeks (weeks 13–20) and then killed to obtain serum samples and liver tissues. Liver histology, lipids, parameters of oxidative stress, TNF-alpha and TGF-beta were measured. Results: The liver in NAFLD-HFD showed typical steatosis, accompanied with mild to moderate lobular inflammatory cell infiltration. Serum levels of ALT, AST, ALP, leptin, cholesterol, triglycerides, TNF-alpha, TGF-beta and hepatic MDA were significantly increased (P < 0.05) compared with normal group. This was accompanied with reduction of hepatic GSH, GR, GPx, GST, SOD and serum adiponectin. These changes were to a less degree in NAFLD-RD group. Combined administration of garlic+onion produced a significant decrease in liver steatosis, serum liver enzymes, oxidative markers and lipid peroxidation. Moreover, NAFLD-induced inflammation was also mitigated via reduction of TNF-alpha and TGF-beta. These results were better in the group IX vs. group VIII. Conclusions: Garlic or onion alone could be considered as a potent food supplement in the prevention of fatty liver disease. Combined administration of both supplements gave better results than each one alone.

404 HYPOGLYCAEMIC POTENTIAL OF PYTHON REGIUS OIL Okere S. The hypoglycermic potentials of python regius oil was studied by treating alloxan induced diabetic rats with different concentrations of the oil for the period of 14 days. It was observed that there was significant difference from day 1 too day 14 as compared to day 1 to day 7 thus the therapeutic effect of the oil will be best observed on prolonged use. The oil caused a significant decrease in blood glucose level from 221.921.02 to 131.470.96 mg/dl, in serum cholesterol level from 184.342.38 to 133.213.85 mg/dl, in level of serum bilirubin from 10.3161.27 p/100 ml, to 6.6251.27jj/100 ml, as well as in the level


124 of liver enzymes ALT and AST from 32.000.78 UI to 17.001.44 UI for ALT and from 31.001.92 UI to 17.000.14 UI for AST. These findings establishes the fact that python oil has hypoglycaemic potentials thus it can be recommended for use in the treatment of diabetes

405 IDENTIFICATION AND PHARMACOLOGICAL EVALUATION OF A NOVEL POSITIVE ALLOSTERIC MODULATOR OF ALPHA 7 NICOTINIC RECEPTOR CHANNEL Wang K., Tang J., Luo L., Sun Q. Background: The alpha-7 nicotinic acetylcholine (ACh) receptors are pentameric ligand-gated ion channels that are distributed in the brain where its activation yields neuronal excitation. Pharmacological studies indicate that activation of alpha7 channels improves cognitive performance, including deficits in attention, learning, working memory, and cognitive flexibility. Since a primary effect of the prolonged presence of an agonist is to produce desensitization at nAChRs, allosteric augmentation of nicotinic alpha-7 receptor function is considered to be a potential therapeutic strategy for Alzheimer’s disease and schizophrenia. We recently designed and synthesized a novel series of type I positive allosteric modulator (PAM) of alpha7 nicotinic acetylcholine receptor (nAChR), and tested their pharmacological effects both in vitro and in vivo. Methods: For functional expression, Xenopus laevis oocytes were injected with approximately 20 ng of human a7 nAChR cRNA or alpha4beta2 cRNA (1:1 ratio). For two-electrode voltage clamp recordings at room temperature (20°C), oocytes were held at -90 mV and currents were recorded 2–4 days after cRNA injection. ACh was applied to the recording chamber for 10 s at 3 ml/min. Test compound was applied for 120 s before ACh application, allowing for sufficient preincubation. Results: Electrophysiological screening was carried out by preincubation of oocytes expressing human alpha7 nAChR with test compound and followed by application of 100 mM ACh that evokes 30% of the maximum ACh response (Imax). Test compounds were inactive in the absence of direct agonists. A representative compound TSW486 identified from novel series resulted in a 3 to 4-fold increase in peak current amplitude with an EC50 of 3.2 mM, compared with the control. To further evaluate the compound effect, concentration-response curves for ACh were generated in the presence and absence of 10 mM TSW486 compound. TSW486 shifted the EC50 from 288 mM to 137 mM. The maximal efficacy of ACh was increased about 2-fold. The selectivity of TSW486 was also tested against alpha4beta2. Conclusions: The TSW486 compound presents a modulator activity at alpha7 nAChR, causing an increase of the peak amplitude of AChevoked alpha 7 currents in dose-dependent manner and a decrease of the EC50 of ACh.

406 IDENTIFICATION OF MODULATORS FOR SUCNR1 BY SCREENING OF A SOSA LIBRARY WITH A BIOLUMINESCENT CAMP ASSAY Gilissen J., Dupuis N., Derj A., Soni A., Dilly S., Pirotte B., Hanson J. Background: Succinic acid (SA), a metabolic component that takes part in the citric acid cycle, has been described as the cognate agonist for the orphan receptor SUCNR1 (GPR91). This receptor belongs to the G Protein-Coupled Receptor family (GPCR) that play an essential role in regulating many physiological functions and represent 30% of targets for currently marketed drugs. Several studies on KO models suggested different roles through the activation of SUCNR1. Nevertheless, the characterization of the pharmacology and physiology of SUCNR1 is limited by the lack of pharmacological tools. Methods: In order to identify ligands modulating SUCNR1 Gai activity, we adapted a specific cAMP level measurement assay based on a modified luciferase fused with cAMP binding domain (Glosensorâ

Promega). Upon cAMP binding, conformational changes induce luminescent signal. This assay allows end-point and real time measurements. We designed a protocol compatible with the screening of 96well plates distributed molecules libraries. We screened with our assay the Sigma LOPAC library (composed of 1280 active compounds that might have an activity on new targets at high concentration) on HEK293 expressing SUCNR1. In parallel, we docked a part of the ZINC database ‘lead-like’ molecules against SUCNR1 built by homology modeling from the b2adrenergic receptor (SA binding site described by He et al.). Results: We performed a primary agonist screening on 1280 compounds of a SOSA library at two different concentrations (100– 10 lM) with a cAMP assay (Z0 =0.4–0.6). For the primary screening, we set up a criterion of activity higher than 20% compared to WT cells. One hundred and fourteen compounds fitting these parameters were subjected to a secondary screening performed in triplicates. Results analysis provided 16 putative modulators of SUCNR1 Gai activity. We followed a similar strategy in another screening to find candidates with an antagonist profile. The virtual screening has sorted 30 compounds with substantial affinity that are under thorough pharmacological investigations to confirm activity. Conclusions: We set up a real-time luminometric cAMP assay for SUCNR1. We selected a dozen putative modulators of SUCNR1 that will be deeply evaluated. Furthermore, we obtained 30 potentially active compounds from a virtual screening. Their pharmacology will be confirmed in functional assays.

407 IDENTIFICATION OF PHOSPHOPROTEINS REGULATED BY SERINE/THREONINE PROTEIN PHOSPHATASE INHIBITION IN HUMAN HEPATOCELLULAR CARCINOMA CELLS Ward M., Botting C., Lucitt M., Spiers P. Serine/Threonine Protein Phosphatases (PsP) serve as an exquisite switch in controlling several key oncogenic and kinase signalling pathways involved in cell proliferation, apoptosis, migration, and invasion (Shi, 2009). While much is known about kinase regulation of oncogenic pathways, little is known about the role PsP play. In this study we use a phosphoproteomic approach to investigate the effect PSP inhibition plays in altering protein class function in hepatocellular carcinoma cell line Hep3B. Human hepatocellular carcinoma cells were treated with 40 nM okadaic acid in serum free conditions for 24 h. Phosphoproteins were isolated using a Phosphoprotein Enrichment &Purification Kit (Clontech, USA), ran on 10% SDS-PAGE gel and stained with coomassie blue (0.25%). Protein bands were excised, digested in-gel with trypsin and analysed by nLC-ESI-MS/MS. The MS/MS data files generated were analysed using Mascot 2.1 software. Three of more matching peptides and a significant probability score (P < 0.001) were required for a secure identity assignment. All MASCOT search results were inspected manually to ensure correct matches by bioinformatics. Classification of identified proteins was completed using PANTHER Gene OntologyTM to categorise all identified proteins based on their protein class, pathway and biological function. Cytoscape software was used to visualise the serine/threonine interactome of identified proteins. Three hundred and fifty-two phosphoproteins were identified in Hep3B cells by mass spectrometry under basal conditions. Following exposure to okadaic acid, 180 new phosphoproteins were identified plus the loss of 5 basal expressed proteins. These new phosphoproteins were associated with nucleic acid binding (24%), cytoskeleton (10.4%), transcription factors (6.8%), signalling (2.1%), cellular junction (2.1%), cell adhesion (2.6%), and enzyme modulation (8.9%). Pro-metastatic proteins such as PAK2, AKT2, PLCB3, CTNNA1, Hypoxia up-regulated protein 1 (HYOU1), RUVL1, HSPA14 and ROCK2 were all detected following phosphatase inhibition. The phosphoprotein interactome showed that many of the new phosphoproteins interacted with by the different serine/threonine phosphatases. Our results indicate that Ser/Thr phosphatases inhibition leads to the activation of protein networks involved in tumour growth, metabolism,


125 cellular communication and invasion. As many new anti-cancer drugs target kinase and phosphatase controlled pathways, our data highlights the complexity and diversity of cellular functions protein phosphatases play in the cell.

408 IISOLATION AND CHARACTERIZATION OF THE ANTIDIARRHEAL CONSTITUENTS OF WALTHERIA INDICA L. (STERCULIACEAE) LEAVES Ayinde B., Uti I., Owolabi O., Adhikari A., Amjad H., Iqbal C. Background: In ethnomedicine, Waltheria indica is claimed to be used in treating dysentery and diarrhea. Methods: This work examined the effects of the leaf methanol extract on gastrointestinal motility using charcoal meal transit and castor oil induced methods in mice and rats respectively. The extract (20–80 mg/ mL) was also evaluated on acetylcholine (Ach) - induced ileum and latter partitioned into chloroform and aqueous fractions with each fraction tested on the ileum. Separation of the aqueous fraction using vacuum liquid chromatography with increasing concentrations of ethyl acetate in dichloromethane followed by methanol and thin layer chromatography on silical gel GF254 led to the isolation of two flavonol compounds (1 and 2) which were also tested on the isolated ileum at 2.5–20 mg/mL. The compounds were then subjected to spectroscopic analysis to determine their structures. Results: In the control animals, the charcoal moved up to 71.83 % of the intestine, whereas the movement was reduced to 42.87% in animals treated with 400 mg/kg of the extract compared to 39.53% in animals treated with Atropine (0.1 mg/kg). In castor oil induced diarrhea, the onset and frequency of stooling in control rats were 101.0 min and 5 .20.9 wet stools, respectively after castor oil administration. The extract (100 mg/kg) delayed the onset of stooling by 62.33 2.33 min and reduced the number of stools to 1.2 0.6, but atropine treated animals did not produce any stool. The extract significantly inhibited the ileal contractions produced by ACh and the aqueous fraction showed higher effects than the methanol extract. Relaxation of the ileum by 40 mg/ml of the fraction was higher than that produced by 80 mg/ml of the extract. Chromatographic separations further enhanced the relaxant effects of the isolated compounds. Compounds 1 and 2 showed similar Rf of 0.46 in CH3Cl- CH3OH (3.5:1.5). While compound 1 at a concentration of 5 mg/ml completely abolished the ileum contraction produced by 80 lg/ml of ACh, compound 2 blocked the effect of the ACh at 20 mg/ml. Conclusion: The results of this work justify the ethnomedicinal application of W. indica leaf in the management of diarrhea.

409 IL-6R BLOCKADE AS A THERAPEUTIC APPROACH TO OVERCOME CISPLATIN RESISTANCE IN HEAD AND NECK CANCER Nör C., Zhang Z., Warner K., Bernardi L., Visioli F., Helman J., Roesler R., N€ or J. Background: Head and neck squamous cell carcinoma (HNSCC), one of the most common types of cancer, exhibits an unacceptably poor patient survival. The addition of chemotherapy to surgery and radiation led to an improvement in local disease control and organ preservation however, the overall survival of patients with HNSCC has not changed significantly. This has been attributed to chemoresistance and incidence of distant metastasis. Recent evidence has unveiled a sub-population of highly tumorigenic, multipotent cells capable of self-renewal in HNSCC. The cancer stem cells (CSC) proliferate slowly and might be involved in resistance to conventional chemotherapy. We have shown that cancer stem cells are found in perivascular niches and rely on endothelial cell-secreted factors (particularly IL-6) for survival and

self-renewal in HNSCC. We hypothesized that the therapeutic blockade of IL-6 may benefit cancer patients. Methods: A human HNSCC tumor was dissociated and cells submitted to flow sorting. Orospheres, (non-adherent spheroids), were generated in ultra-low attachment plates after treatment with 0–2 lM cisplatin and/or rhIL-6. HNSCC cell lines (UM-SCC-22A, UM-SCC-22B) were treated with cisplatin and/or rhIL-6, IL-6 antibody or IL-6 receptor inhibitor (Tocizilumab) and the expression of stem cell markers was evaluated by western blot. Human dermal microvascular endothelial cells (HDMEC) were silenced for IL-6 and co-implanted with UM-SCC-22B tumor cells in immunodeficient mice for tumor growth evaluation. Results: IL-6 potentiated cisplatin-induced orosphere formation when primary human HNSCC cells were sorted for ALDHhighCD44high immediately after surgery (P = 0.026). The cytotoxic drug cisplatin alone increases the expression of stem cells markers Bmi-1 and CD44. Interleukin-6 potentiated this effect. Notably, cisplatin-induced Bmi-1 was inhibited by IL-6R blockade in HNSCC cells. Tumor volumes were significantly smaller in mice implanted with the sh-IL-6 endothelial cells (P = 0.039). Conclusions: IL-6R antibody can revert the induction of Bmi-1 expression after cisplatin treatment. The knockdown of IL-6 in the tumor vasculature suppresses tumor growth. The IL-6 receptor should be further evaluated as a new molecular target for the development of adjuvant therapies.

410 IN VITRO ACTIVITY OF A NEW SERIES OF SYNTHETIC COMPOUNDS FOR ANTITUBERCULOSIS DRUG DEVELOPMENT Ngwane A., Styger G., Pietersen R., Kendrekar P., Wiesner L., Baker B., van Helden P., Wiid I. Background: Development of new, effective, safe and affordable tuberculosis (TB) antibiotics has been identified as an important priority for global TB control. In this study we report the potency of two analogs (NP091 and PK141) from a series of synthetic compounds. These compounds were initially screened for antimalarial studies and were furthermore screened against Mycobacterium tuberculosis (Mtb). Methods: The BACTEC 460 system was used for minimum inhibitory concentration (MIC) determination in both (Mtb) H37Rv and multi-drug resistant (MDR) and extensive drug-resistant (XDR) clinical Mtb isolates. Time-kill studies were conducted using agar proportional method and colony forming units (CFUs) were determined. Cytotoxicity against THP-1 cell-line was determined by the MTT assay. Furthermore ex vivo studies were carried in mouse bone-marrow derived macrophages infected with Mtb H37Rv and snapshot in vivo pharmacokinetics studies were also conducted in mice. Results: These compounds (NP091 and PK141) have been identified as “leads” with an MIC of 2.5 and 10 lM, respectively, against Mtb H37Rv. They have also been shown to be active against (MDR) and (XDR) Mtb clinical isolates. This suggests that these compounds may have a different mechanism to the first-line TB drugs and may not have cross resistance. Cytotoxicity data has shown that NP091 is highly selective compared to PK141 for Mtb in relation to the THP-1cell-line with selective indexes (SI) of 14 and 5, respectively. An SI of at least 10 is sufficient enough for a compound to be considered as a “lead” for TB drug development. In vivo pharmacodynamics and pharmacokinetics studies revealed poor bioavailability of both compounds. Conclusions: The above results suggest that NP091 has the potential to be further investigated as a TB drug lead. Knowledge concerning its mechanism of action is needed to identify new Mtb drug targets and to guide the synthesis and structure activity relationship (SAR) studies aimed at identifying a potential optimised TB antibiotic.


126 411 IN VITRO ASSESSMENT OF THE ANTI-DIABETIC ACTIVITY OF SCLEROCARYA BIRREA AND ZIZIPHUS MUCRONATA da Costa Mousinho N., Van Tonder J., Steenkamp V. Background: Diabetes mellitus is a growing threat to human health, and herbal remedies offer the potential for alternative treatment strategies that avoid the undesirable side-effects associated with the commonly available drugs. The aim of this study was to determine the in vitro anti-diabetic and antioxidant activity of aqueous and methanolic extracts of Sclerocarya birrea (A. Rich.) Hochst. (Anacardiaceae) and Ziziphus mucronata Willd. (Rhamnaceae), which are traditionally used to treat diabetes. Methods: The effects of the crude extracts on a-amylase and a-glucosidase was determined using enzymatic assays. Antioxidant activity was carried out by determining the ability of the extracts to scavenge the 2,2-diphenyl-1-picryl hydrazyl (DPPH) and 2,20 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals. The effect of the crude extracts on insulin secretion in cultured RIN-5F pancreatic bcells was investigated using ELISA. The effect of the crude extracts on glucose uptake on HepG2 human hepatocarcinoma cells was assessed using fluorescence. Results: The aqueous and methanolic extracts of S. birrea inhibited a-amylase activity. At the highest concentration tested, inhibition was 83.1% and 87.9% for methanolic and aqueous extracts, respectively. Crude extracts of both S. birrea and Z. mucronata, displayed inhibitory activity against a-glucosidase. The methanolic extract of Z. mucronata displayed the strongest inhibitory activity (15.2%) Crude extracts of S. birrea displayed the strongest free radical scavenging ability. Although no significant increase in insulin secretion from cultured RIN-5F cells was noted, the crude extracts increased glucose uptake in HepG2 cells. Conclusions: These findings provide evidence that S. birrea and Z. mucronata possess antioxidative and anti-diabetic properties.

412 IN VITRO ASSESSMENT OF THE ANTICANCER ACTIVITY OF EIGHT AFRICAN HERBAL REMEDIES IN A PANEL OF CANCEROUS CELL LINES Hurrell T., Steenkamp V., Cordier W. Background: Cancer is a progressive, heterogeneous disease which as a result of biological diversity results in drug resistance. As such, novel treatment options, such as herbal remedies, are being sought. Plants contain diverse phytochemical properties which have been known to display anticancer activity. However, numerous plants remain to be screened for anticancer activity. The aim of this project was to assess anticancer activity of eight African plant extracts to identify those with potential to be developed further for use in cancer treatment. Methods: Hot water and methanol extracts (10% w/v) were prepared through standard brewing and maceration techniques, respectively. Cytotoxicity was assessed 72 h post-exposure in HepG2 hepatocarcinoma, Caco-2 colon carcinoma, SK-Br-3, MDA-MB-231 and MCF-7 breast carcinoma as well as EA.hy926 primary endothelial hybrid cell lines using the sulphorhodamine B assay. Cell cycle analysis was conducted in MCF-7 cells, 24 h post-exposure using flow cytometric techniques. Results: Ten of the sixteen extracts displayed IC50s ≤ 30 lg/ml which included both extracts of Acokanthera oppositifolia root-bark, Boophane disticha bulbs, Mundulea sericea roots, Solanum aculeastrum fruits in addition to the methanol extract of Tabernaemontana elegans roots and Scadoxus puniceus bulbs. The hot water extract of A. oppositifolia (1.29–13.86 lg/ml) and methanol extract of T. elegans (0.35– 3.57 lg/ml) demonstrated the most prominent cytotoxicity. The hot water extract of S. puniceus (IC50 >100 lg/ml), as well as both extracts of Moringa oleifera leaves (IC50 > 100 lg/ml) and Ziziphus mucronata roots (IC50 >70.98 lg/ml) demonstrated the lowest cytotoxicity. SK-Br-3 and MCF-7 cells were the most susceptible to the

extracts. Similar cytotoxicity in EA.hy926 was evident in the range of anticancer activity indicating primary cytotoxicity. Preliminary analysis of the hot water extract of A. oppositifolia and methanolic extract of T. elegans suggests G2 and G1 phase cell cycle accumulation, respectively at the IC50. Conclusion: Anticancer activity was especially apparent in extracts from the Apocynaceae family (A. oppositifolia and T. elegans) through induction of cell cycle arrest. Although primary cytotoxicity might hinder further development for anticancer applications, research to determine to which extent is warranted.

413 IN VIVO ASSESSMENT OF THE ANTIMALARIAL ACTIVITY OF CASSIA SINGUEANA AND CYMBOPOGON CITRUTUS Ior L. The problem of increasing drug resistance in malaria therapy has made its treatment a major source of concern globally. This has necessitated the need to explore traditional medicines in the search for new/potential anti-malarials for both prophylaxis and chemotherapy. Cassia singueana and Cymbopogon citrutus are widely spread in northern Nigeria and claimed to possess multiple therapeutic properties, including anti-malarial activity. Cassia singueana bark was extracted with 50% ethanol,while the root and leaf of Cymbopogon citrutus was extracted with cold water. Phytochemical analysis and oral acute toxicity of the extracts were evaluated. The Suppressive and Prophylactic anti-plasmodial activities against chloroquine sensitive strain of Plasmodium berghei berghei in mice were evaluated. Pathological effects associated with malaria infections; pyrexia and weight loss or poor weight gain similarly assessed. Results showed the presence of carbohydrates, alkaloids, tanins, flavonoids, cardiac glycosides, saponins and steroids in Cassia singueana, while Cymbopogon citrutus showed the presence of alkaloids, carbohydrates, tannins, flavonoids, steroids and saponins. The oral median lethal dose of both extracts was greater than 5000 mg/kg body weight. The extract Cassia singueana at (400–600 mg/kg) exerted significant (P < 0.05) chemosuppressive effects between 72.7% and 90.5% and prophylactic effects between 79% and 83% against the Plasmodium berghei berghei. Similarly, Cymbopogon citrutus (200–800 mg/kg) produced a significant chemosuppressive effects between 20.83% and 80.56% for the leaf extract and 55.38–77.78% for the root extracts. This result showed that the plants have anti-plasmodial property that can be explored for the management of malaria.

414 IN VIVO PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION OF NOVEL ANTIMALARIAL AMODIAQUINE ANALOGUES Strydom N., Ongarora D., Chibale K., Smith P., Wiesner L. Background: Drug discovery requires not only an active compound, but a desirable drug that must excel in a number of diverse in vivo criteria. A series of amodiaquine analogues were synthesised with the aim of preventing the formation of known toxic amodiaquine reactive metabolites. Three compounds showed desirable in vitro antiplasmodial activity. Analysis of the pharmacokinetic and pharmacodynamic properties was also carried out in order to establish the lead potential of these analogues. Aim: To evaluate the drug metabolism and pharmacokinetic profiles of novel antimalarial compounds and further evaluation of their efficacy in rodent models. Methods: Using murine models and liquid chromatography-mass spectrometry the blood concentrations of these compounds were measured at specific time points after oral and intravenous administration. This data was modelled using non compartmental analysis to obtain


127 bioavailability results, and a pharmacokinetic profile of the compounds. Pharmacodynamic evaluation used a Plasmodium berghei GFP infected murine model. Infection was measured by flow cytometry with treated and untreated groups compared. Results: All three compounds showed excellent in vivo results with high rates of inhibition of the malaria parasite. These results show promising pharmacokinetic and pharmacodynamic properties. Conclusion: A liquid chromatography-mass spectrometry method was used to evaluate the pharmacokinetic properties of three antimalarial compounds in selected murine models. Understanding the in vivo pharmacokinetic profile of these compounds in combination with their in vivo efficacy is important in order to progress these drugs along the drug discovery value chain.

415 IN-VIVO AND IN-VITRO EVALUATION OF THE ANTIINFLAMMMATORY AND IMMUNOMODULATORY EFFECTS OF SCHWENKIA AMERICANA L. IN EXPERIMENTAL ANIMALS Nwabunike I.A., Mbaoji F.N., Onyeto C.A., Ezike A.C., Okoye T.C., Nworu C.S., Okoli C.O., Akah P.A. Objectives: The antiinflammatory and immunomodulatory activities of the methanol:methylene chloride extract of the aerial parts of Schwenckia americana L. (Solanaceae) (SAE) was studied and the mechanisms of antiinflammatory effects were evaluated. Methods: Bioactivity-guided studies on SAE and its fractions was done using the egg albumin-induced rat paw edema as activity guide. Further screening for antiinflammatory activity was accomplished using the xylene-induced ear edema, formaldehyde induced arthritis, and cotton pellet-induced granuloma formation. The putative antiinflammatory mechanisms were studied using acetic acid-induced vascular permeability, carrageenan-induced neutrophil migration, and heatand hypotonic solution- induced hemolysis. The immunomodulatory effects of SAE was evaluated using the neutrophil adhesion test and carbon clearance assay in rats. Results: Bioactivity-guided studies on SAE and fractions revealed significant (P < 0.05) inhibition of rat pedal edema development with an increasing magnitude upon fractionation of SAE in the order considering the area under curve, Fraction A(1.55)> dichloromethane fraction (2.01)> SAE(2.33) compared to acetylsalicylate (2.14). SAE also significantly (P < 0.05) inhibited acetic acid-induced vascular permeability and topical edema development in the mouse ear. Granuloma tissue formation and neutrophils count in rats, and heat and hypotonic solution- induced hemolysis of ox erythrocytes were also inhibited. SAE also caused a slight increase in phagocytic activity and neutrophil recruitment and adhesion. Phytochemical analysis of SAE and fractions revealed the presence of terpenoids and steroids in abundant proportions especially in Fraction A. Conclusion: SAE possessed good antiinflammmatory and some immunostimulatory potentials. The anti-inflammatory mechanism of S. americana may partly be through membrane stabilization, and inhibition of vascular permeability, neutrophil migration, cellular infiltration and proliferation. The terpenoids and steroids abundant in the most active fraction as seen from the phytochemistry may be the phytoconstituents responsible for the observed pharmacological effects.

416 IN-VIVO ANTI-INFLAMMATORY AND ANTI-ASTHMATIC STUDY OF EFFECT OF PARTIALLY PURIFIED EXTRACTS FROM THE LEAVES OF LABISIA PUMILA Okechukwu P., Ekeuku S. This present study investigated the anti-asthmatic activity of Labisia pumila leaf extract using histamine, serotonin and bradykinin induced

paw edema and histamine and acetylcholine induced bronchoconstriction. 1% histamine, serotonin and bradykinin (0.1 ml/rat) was dissolved with 0.9% (w/v) saline and subcutaneously injected to induce edema formation. Also, 1% histamine and acetylcholine was dissolved in saline and was used to induce bronchoconstriction in guinea pigs. Oral treatment of partially purified fractions A-E (100 mg/kg), and indomethacin (10 mg/kg) inhibited significantly (P < 0.001) the formation of the histamine, serotonin and bradykinin-induced paw edema measured in the fifth hour. The highest inhibition was observed in fraction A and E which were 5.07% (0.674 0.002 mm) and 5.63% (0.670 0.004 mm) for histamine, 4.24% (0.678 0.002 mm) and 5.65% (0.668 0.002 mm) for serotonin and 9.59% (0.622 0.002 mm) and 8.72% (0.628 0.002 mm), compared to the negative control (0.710 0.003 mm), (0.688 0.002 mm) and (0.708 0.002 mm) respectively. In histamine and acetylcholine-induced bronchoconstriction, partially purified fractions A-E (100 mg/kg), as well as pyrelamine maleate salt (1 mg/kg) gave significant protection against bronchoconstriction (P < 0.001) at the fourth hour. The highest protection was observed in fraction A and C 55% (123 0.000 s) and 58% (123 0.000 s) for acetylcholine-induced bronchoconstriction and 48.17% (100.333 12.454 s) and 45.70% (85.333 8.667 s) respectively, compared to the negative control (45 0.000 s) and (39.333 0.667 s) respectively. For the 14 days treatment in histamine and acetylcholine-induced bronchoconstriction, partially purified fractions A (100 mg/kg), as well as pyrelamine maleate salt (1 mg/kg) gave significant protection against bronchoconstriction (P < 0.001) at the 24th hour. Fraction A showed protection of 68.84% (134.980 5.025 s) for histamine and 51.21% (110.260 1.750 s) for acetylcholine compared to the negative control (35.600 1.140 s) and (34.180 1.385 s) respectively. The present study confirmed that the extracts exhibited anti-asthmatic activity by its anti-inflammatory and bronchodilatory action.

417 INHIBITION OF TRPV1 ION CHANNELS CONTRIBUTES TO THE ANTINOCICEPTIVE EFFECT OF BUNODOSINE 391 ON CAPSAICIN-EVOKED THERMAL HYPERLGESIA Ferreia Júnior W., Bressan E., Gomes Campos Nascimento L., Junqueira Zaharenko A., Picolo G., Reeh P.W., Cury Y. Background: Bunodosine (BDS) 391 is a low molecular weight and non-peptidic compound purified from the Brazilian sea anemone Bunodosoma cangicum venom. We have previously shown that BDS induces antinociceptive effect on carrageenan- and PGE2-induced mechanical hyperalgesia, mediated by activation of 5HT3 receptors. The aim of the present work was to characterize the effect of BDS 391 on capsaicin-evoked thermal hyperalgesia and the involvement of TRPV1 ion channels in this effect. Methods: Male Wistar rats were used for the behavioral studies. Cultured DRG neurons from C57BL/6 mice and human (h) TRPV1expressing HEK293t cells were used for Ca2+ imaging. Behavioral experiments were previously approved by CEAUIB (Butantan Institute). Handling of mice used for cell culture/calcium imaging was approved by the Erlangen local district government (Germany). Capsaicin-evoked thermal hyperalgesia was induced by hind paw intraplantar (i.pl.) injection of capsaicin (1 nmol/50 lL). The hyperalgesia was evaluated 15, 30 and 60 min after capsaicin and determined by the latency (in s) of the hind paw withdrawal to heat stimulation. BDS (0.015, 0.15, 1.5 and 150 lM/50 lL) or saline (control) were administered i.pl. 15 min before capsaicin. The possible effect of BDS on TRPV1 ion channels was investigated in vitro, through Ca2+ imaging studies. The capsaicin-evoked Ca2+ influx in DRG neurons or hTRPV1-expressing HEK293t cells was quantified before and after treatment of the cells with BDS 391. Results: Intraplantar capsaicin injection induces significant thermal hyperalgesia characterized by the decrease in latency withdrawal to heat stimulation. BDS 391, in low doses and dose-dependently inhibits capsaicin-evoked hyperalgesia, indicating a potent antinociceptive effect on thermal hyperalgesia. Since capsaicin-induced hyperalgesia is


128 mediated by TRPV1 activation, we investigated if the antinociceptive effect of BDS could result from a modulatory action of this compound in these channels. BDS 391 inhibits capsaicin-evoked Ca2+ influx in both, DRG neurons and hTRPV1-expressing HEK293t cells, indicating a modulatory action on TRPV1 activity. Conclusion: The present study demonstrates the antinociceptive effect of BDS 391 on thermal hyperalgesia, by a mechanism that could involve, at least in part, inhibition of TRPV1 channels. 418 INHIBITORY EFFECTS OF CARVACROL ON SEROTONIN INDUCED CONTRACTONS OF ISOLATED BOVINE CORONARY ARTERY Aydin Y., Aydin Y., Aydin S. Carvacrol is one of the volatile oxygenated monoterpenes found in nature which was reported to have hypotensive and smooth muscle relaxant effects. Cardiovascular diseases are one of the main cause of morbity and mortality of modern civilizations. The aim of this study was to investigate the effects of carvacrol on serotonin induced contractions of coronary artery rings. Isolated bovine coronary artery rings of ramus circumflexus obtained from local slaughterhouse were used in this study. Endothelium intact and endothelium denuded rings of the coronary arteries (n=7) were tested in isolated organ baths with commercially obtained serotonin and carvacrol. Cumulative concentration response curves for serotonin (108–10-4 M) on carvacrol pretreated (108–5 9 104 M) bovine coronary arteries were obtained. Data were statistically evaluated using one way variance analysis followed by Tukey’s HSD for multiple comparison (P < 0.05) and ED50 values were calculated using R packages. Carvacrol was observed to inhibit contractions in endothelium contact and endothelium denuded rings at 10-4 and 5 9 10-4 M doses and inhibitory effect of carvacrol on the endothelium intact arteries were higher than endothelium denuded preparations. ED50 values of endothelium intact coronary artery rings were higher than endothelium denuded rings indicating the role of endothelium on the relaxant actions of carvacrol. Carvacrol was shown to interact with TRP channels (Xu et al, 2006) and the role of calcium channels was reported in the mechanism of action of hypotensive effects of carvacrol (Aydin et al, 2007). In this study we report the inhibitory actions of carvacrol on serotonin induced contractions and the role of endothelium on isolated bovine coronary artery. Aydin Y, Kutlay O, Ari S, Duman S, Uzuner K, Aydin S. (2007) Hypotensive effects of carvacrol on the blood pressure of normotensive rats. Planta Med., 73: 1365–1371. Xu H, Delling M, Jun JC, Clapham DE.(2006) Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat. Neurosci., 9: 628–635.

coverX Corporation (Freeman, CA, USA). For RDT 3 dose levels of teriparatide in either formulation were daily administered, for 28 days, to rats (Rattus norvegicus, n= 72), strain WKAH/hok (36 male), evaluating histopathological findings at necropsy, clinical chemistry, local tolerance, immunogenicity and systemic exposure (with the highest dose); 12 rats received saline, as control. For RB, 24 healthy volunteers (10 women) received either formulation of teriparatide (20 lg SC), in a cross-over design: plasma teriparatide was measured with the kit PTH(1-34) High Sensitivity EIA, (American Laboratory Products Co., Salem, NH, EEUU) and results analyzed with WinNonLin software. The safety evaluation included the search for anti-teriparatide antibodies by in-house ELISA. Results: RC50 (lM) values for Osteofortil and Forteo in the arrestin assay were 0.011355 vs 0.013582; in the cAMP assay, 4.80E-05 vs 6.92E-05 and in the internalization assay, 0.0081566 vs 0.0079345, respectively. Teriparatide increased body weigth in males, and slightly alkaline phosphatase, total proteins, CK and calcium, without difference between products. No differences were found in the immunotoxicology evaluation. Local tolerance and antibody induction was similar. In systemic exposure, elimination was very rapid (no teraparatide was detected at 4 h after injection). In the RB study in humans, Osteofortilâ/Forteoâ ratios (as percentual mean and CI90%) were: Cmax 94.58%; 85.29–104.87, area under the curve (AUC)0-4 h 105.5%; 97.77–113.84 and AUC0-∞ 104.40% 96.97–112.39. These ratios comply with Argentina’s regulation for bioequivalence. Conclusions: primary pharmacodynamics, RDT and RB of Osteofortilâ and Forteoâ are similar

420 INNOVATIVE PPARd AGONISTS BY FRAGMENT BASED DRUG DISCOVERY APPROACH Silva P., Maltarollo V., Ganesan A., Dale J., Emery F. The Fragment based drug discovery (FBDD) approach is a strategy that has emerged as an additional and contrasting to drug discovery in recent years. This approach involves constructing fragments, small molecules with low molecular weight, which are potent ligands for the target.1 Currently, a class of receptors directly related to the regulation of lipid metabolism has attracted interest of pharmaceutical industries, these class is known as peroxisome proliferator-activated receptors (PPARs).2 Considering the PPARs promising targets for drug discovery process, this work proposed the development of innovative PPARd agonists by the emerging strategy of FBDD.

419 INITIAL DEVELOPMENT OF A NEW TERIPATIDE FORMULATION Farias J., Farias J., Keller G., Parodi F., Papouchado M., Silva M., Bello R., Lucini A., Di Girolamo G., Diez R. Background: Teriparatide is an analogue of human parathyroid hormone, used to treat severe osteoporosis, through activation of PTH1R, which results in increased bone regeneration. To develop a new pharmaceutical product containing teriparatide, EMA guidelines were used to define critical steps. In this communication, results corresponding to comparison in primary pharmacodynamics (in vitro), repeat-dose toxicity (RDT, in rats) and relative bioavailability (RB, in humans) between the product under development (Osteofortilâ, Biosidus S.A., Buenos Aires, Argentina) and the reference (innovator) product (Forteoâ, EliLilly, Indianapolis, IN, EEUU) are presented. Methods: Both compounds were profiled with 3 PTHR1 Biosensor Assays as agonist (arrestin, cAMP and receptor internalization) at Dis© 2014 The Authors Basic & Clinical Pharmacology & Toxicology © 2014 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 115 (Suppl. 1), 1–374

129 A compounds library was proposed by merging FBDD, based in the substitution of the bicyclic scaffold of compounds with known activity as PPARd agonist2,3 by the fragments A, naphthyridinole and B, pyrazolopyridinole, wich were described as promising fragments to drug discovery by Pitt (Figure 1).4 The synthesis of the fragments A was realized by pyridine chemistry, and B was achieved through the cyclisation of a N-acyl-N-nitroso intermediate (Scheme 1). Both synthesis are robust and scalable, and the compound are highly soluble in water. Once prepared the fragments, were conducted a similarity study by Tanimoto coefficient. The results indicated similarity of over 90%, which makes possible to propose the hypothesis that the structural modification of the bicyclic fragments PPARd agonists known in the literature by fragments A and B will not affect the affinity of new compounds for biological target, enabling the structural innovation and discovery of new bioactive compounds. This work describes the first step of a FBDD approach, the obtaining of fragments A and B. These fragments representing promising scaffolds to drug discovery as well as to substitute bicyclic core of PPARd agonist by merging FBDD approach. The next step is the synthesis of PPAR agonists, and then the fragment elaboration based in Docking and biochemical assays.

421 INSULIN-INDEPENDENT GLUCOSE UPTAKE IN SKELETAL MUSCLE FOLLOWING b2-ADRENOCEPTOR ACTIVATION IS MEDIATED BY MTORC2 AND GLUT4 Summers R., Sato M., Dehvari N., Oberg A., Dallner O., Sandstrom A., Olsen J., Csikasz R., Hutchinson D., Bengtsson T. Background: Although activation of b2-adrenoceptors (ARs) in vitro increases glucose uptake in skeletal muscle, the signalling pathways involved are unclear. In vivo, sympathetic effects on glucose homeostasis are influenced by glucose from the liver, insulin from the pancreas, as well as glucose uptake into peripheral tissues such as white fat, brown fat and muscle. Methods: In L6 myotubes glucose uptake was measured using3H 2 deoxy-glucose, protein phosphorylation by western blotting and GLUT4 translocation by immunohistochemistry. Results: Both insulin- and isoprenaline-stimulated glucose uptake in L6 cells was inhibited by LY294002, suggesting a role for PI-3-kinase, but LY also inhibits other members of the PI-3-kinase/PIKK family. Akt is downstream of PI-3-kinase, but was clearly not phosphorylated at T308 or S473 following b2-AR stimulation, whereas insulin increased Akt phosphorylation at both sites. The Akt inhibitor X inhibited insulin-stimulated glucose uptake and Akt phosphorylation but not isoprenaline-mediated glucose uptake. AS160 was also phosphorylated following insulin but not isoprenaline treatment. Thus b2-AR-stimulated glucose uptake does not utilize major components of the insulin pathway. Cell-permeable cAMP analogues and cAMP increased glucose uptake, whereas an impermeable analogue had no effect. The selective mTOR inhibitors Torin-1 and KU0063794 reduced isoprenaline-mediated glucose uptake. Isoprenaline caused phosphorylation of mTOR at S2481, and insulin at both S2448 and S2481. Akt inhibition decreased insulin-induced phosphorylation of mTOR at S2448 but not isoprenaline-induced phosphorylation of mTOR at S2481. The mTORC1 inhibitor rapamycin (30 min) had no effect on isoprenaline and 8-Br-cAMP-stimulated glucose uptake whereas 48 h exposure inhibits mTORC2 assembly and inhibited isoprenaline-stimulated glucose uptake, suggesting involvement of mTORC2. siRNAs for Rictor abolished isoprenaline-stimulated glucose-uptake whereas Raptor siRNA or control siRNAs had no effect. GLUT4 translocation examined by immunohistochemistry in non-permeabilized L6 cells was observed following b2-AR stimulation as well as in native L6 myoblasts or myotubes, cells transiently transfected with GLUT4mycGFP or stably transfected with GLUT4myc. GLUT4 translocation in response to isoprenaline was abolished by mTOR inhibition. Conclusions: mTORC2 is the key regulator of b2-AR-mediated glucose uptake in skeletal muscle. b-AR-mediated glucose uptake is there-

fore dependent on mTORC2, changes in actin reorganization and on GLUT4 translocation.

422 INTEGRATIVE ANALYSIS ON NEUROPHARMACOLOGICAL EFFECTS OF HONEY Akanmu M., Hatanaka F., Tamada K., Takumi T. Honey (Hachimitsu in Japanese) is a natural sweet substance that bees produce by transforming flower nectar or other sweet secretions of plants. It is reported to have health promoting properties. The beneficial effects of honey have been attributed to its contents. The composition of these constituents and their effectiveness may be due to geographical locations and the sources of plant nectars. Therefore, we investigated the antioxidant, circadian rhythm, locomotive activity of different honey samples from three geographical locations of Nigeria and Japan: (ID: Idanre; EW: Ewu; JG: Jigawa; KD: Kaduna; IF: Ife; UD: Umudike; JA: Japanese). In vitro model was used in the determination of the antioxidant property. The circadian rhythm experiment was determined using Rat-1 fibroblast cells. Locomotor activity was determined after 24 h administration of honey at 5%v/v concentration in drinking water. Recording was done for 24 h in male C57BL6J Jms Slc mice. The effect of honey samples on corticosterone level in plasma and brain monoamines from the following parts of the brain: Olfactory bulb, prefrontal cortex, hippocampus, hypothalamus, pons and medulla, mid brain and cerebellum in immobilized stress mice were determined in young male Jcl:ICR mice Result obtained showed that all the honey samples have antioxidant activity in this order (EW>KD>ID>JG>UD>JA>IF). Most of the honey samples have slight effects on the circadian rhythm at 10 or 5% V /v final concentration. However, EW honey sample showed the most significant effect on the circadian rhythm on Rat-1 cells at 1 or 0.5 % V /v concentration. The results obtained in immobilization stressinduced mice showed that honey samples potentiated the corticosterone level. The results showed that honey samples have differential effects on the brain monoamines and the metabolites depending on the part of the brain. Stress-induced increase in serotonin level was significantly and differentially reversed by honey administration (EW, ID, JA) in prefrontal cortex and hypothalamus regions of the brain. The study on locomotor activity (24-h analysis) showed that honey samples decreased the locomotor activity in mice. In conclusion, the study showed that honey has antioxidant property, central inhibitory effects and also has significant effects on brain monoamines.

423 L-ASCORBIC ACID- AND L-ASCORBIC ACID 2-GLUCOSIDE ACCELERATE IN VIVO LIVER REGENERATION AND LOWER SERUM ALANINE AMINOTRANSAMINASE ACTIVITY IN 70% PARTIALLY HEPATECTOMIZED RATS Kimura M., Moteki H., Ogihara M. The effects of L-ascorbic acid and its stable analogue L-ascorbic acid 2-glucoside on the restoration of liver mass and recovery of liver function after 70% partial hepatectomy (PH) were compared with other natural vitamin C analogues in rats in vivo. L-ascorbic acid (100 mg/kg/ day, i.p.)- and L-ascorbic acid 2-glucoside (50 mg/kg/day, i.p.)-treated rats showed an approximately 1.3-fold increase in the ratio of liver weight (LW) to body weight (BW), when compared to saline (as control)-, L-dehydroascorbic acid (150 mg/kg/day, i.p.) - and D-isoascorbic acid (150 mg/kg/day, i.p.)-administrated rats on day 3 after PH. Accordingly, 5-bromo-2’-deoxyuridine (BrdU)-labeling index in the regenerating liver was significantly higher in L-ascorbic acid (100 mg/ kg/day, i.p.)- and L-ascorbic acid 2-glucoside (50 mg/kg/day, i.p.)-treated rats compared with saline-treated control rats on days 0.5 and 1. LDehydroascorbic acid (150 mg/kg/day, i.p.)- and D-isoascorbic acid


130 (150 mg/kg/day, i.p.)-treated rats did not significantly increase either LW/BW ratio or BrdU labeling index. Dose-dependent stimulation of liver regeneration by L-ascorbic acid and L-ascorbic acid 2-glucoside (1–150 mg/kg/day, i.p.) at day 3 after PH was completely inhibited by co-administration of monoclonal anti-insulin-like growth factor (IGF)-I receptor antibody (10 lg/kg/day i.p.). In control rats, liver-related serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activites were rapidly elevated on day 1, and then decreased to near pre-operative levels on day 5 following PH. L-Ascorbic acid (100 mg/kg/day, i.p.) and L-ascorbic acid 2-glucoside (50 mg/kg/day, i.p.) significantly lowered the serum ALT on day 1 after PH compared with saline-, L-dehydroascorbic acid (150 mg/kg/day, i.p.)- and D-isoascorbic acid (150 mg/kg/day, i.p.)-administered rats. These results demonstrate that L-ascorbic acid and L-ascorbic acid 2-glucoside significantly promote the regeneration of normal liver mass and function, as well as recovery from the liver damage induced by surgical resection via stimulation of IGF-I receptor in rats.

424 LIFETRAIN: TOWARDS A EUROPEAN FRAMEWORK FOR CONTINUING PROFESSIONAL DEVELOPMENT IN BIOMEDICAL SCIENCES Hardman M., Brooksbank C., Janko C., Linden H. Background: The medicines research and development process has recently undergone considerable change and will continue to change: biomedical professionals are now expected to be much more agile than previously, moving and collaborating between disciplines, sectors and geographical locations. This necessitates that they continually develop and maintain the required competencies to work most effectively. The Innovative Medicines Initiative (IMI) is Europe’s largest publicprivate partnership which aims at improving the research environment in all sciences involved in medicines research (www.imi.europa.eu). The IMI Education and Training projects have developed LifeTrain, an emerging pan-European framework for continuing professional development in the biomedical sciences. Methods: LifeTrain’s approach focuses on working collaboratively to develop competency profiles for the different roles required in medicines research and development. It has been developed with four major stakeholder groups - professional/scientific bodies, course providers, employers and individual professionals. LifeTrain is coordinated by the EMTRAIN project (www.emtrain.eu), on behalf of all the IMI funded Education and Training projects. Results: The LifeTrain framework is comprised of four sets of agreed principles; one for each of the four stakeholders groups, and has a growing list of signatories who have agreed to the principles of the framework and to work towards their implementation. Responsibility for learning lies with the individual professional, but is supported by employers, professional/scientific bodies and course providers working together to provide an appropriate environment for learning. Within the LifeTrain framework, several stakeholder groups, including the IMI projects Eu2P (www.eu2p.eu), PharmaTrain (www.pharmatrain. eu) and SafeSciMET (www.safescimet.eu) as well as professional bodies like EPHAR (www.ephar.org) are currently developing certification processes to recognise that bearers excel in standards of education, skills, experience and professional standing in their respective disciplines. Conclusion: With an increasing number of IMI LifeTrain signatories the current approaches to CPD in the biomedical sciences will dramatically change. Individual professionals will be guided by clearly defined competency profiles. They will develop and maintain their personal competency portfolios recognised by both professional/scientific bodies and employers. This stimulates mutual recognition of competencies to facilitate mobility: across disciplines; between academia, industry and Regulatory Authorities; and across geographical boundaries.

425 LONGITUDINAL AND SUBGROUP ANALYSIS OF PRECLINICAL PUBLICATION PATTERNS OF NEWLY LAUNCHED DRUGS K€oster U., Nolte I., Michel M. Background: Preclinical data are important for the mechanistic understanding of newly launched drugs but, in contrast to clinical data, there are no guidelines which preclinical data need to be communicated at what time to the scientific community. Methods: Recently, we have analysed the publication pattern of preclinical data for >120 drugs approved in 1991-2011. For these drugs >1400 original articles have been published before or up to one year after approval. We have now expanded those findings by longitudinal and subgroups analysis. Results: The number of published articles per compound ranged between 0 and 90 and did not exhibit a clear trend over time (mean/median per approval year: 1991: 19/13, 1996: 14/9, 2001: 9/3, 2006: 9/5, 2007: 8/2, 2011: 8/5). We have also analysed the following parameters for longitudinal trends: use of in vivo studies, studies in pathophysiological settings, studies involving transfected cells or genetically modified organisms, and studies using human material. Moreover, subgroups analysis was performed to analyse publication patterns by drug type (small molecule, peptide, antibody, other biological), by therapeutic area, by first-in-class vs. follow-up, by location of originating company (US, Europe, Asia), by type of company (big pharma, small-to-medium enterprise, biotech), and by specific sponsoring company (for those with at least 5 approved compounds within our database). Discussion: Our data illustrate how general trends in the research environment (e.g. attempts to reduce animal use, availability of molecular techniques and greater focus on drug effects in pathophysiological settings and in human tissues) have affected publication patterns of preclinical data for newly launched drugs within a 20-year period.

426 LOW MOLECULAR WEIGHT FUCOIDAN AMELIORATES TUBULOINTERSTITIAL FIBROSIS IN DIABETIC NEPHROPATHY VIA INHIBITION OF EPITHELIALMESENCHYMAL TRANSITION AND FIBROTIC PROCESSES Zhou H., Yang B. Renal fibrosis provides an excellent treatment target for chronic kidney disease (CKD) including diabetic nephropathy (DN), since a large variety of pathophysiologically different diseases converge finally into this single process. Activation of TGF-ß-mediated pathways may play a key role in the pathogenesis of tubulointerstital fibrosis (TIF), suggesting that modulation of this pathway may prevent DN. The aim of this study was to determine whether LMWF can reduce harmful TGFß-mediated cell responses in DN. In vitro, 10 ng/ml TGF-ß induced EMT and activated downstream pathways in human proximal tubular (HK2)cells. The treatment of LMWF significantly reversed EMT and dose-dependently inhibited accumulation of extracellular matrix proteins in response to TGF-ß. In type 1 or type 2 diabetic rat model, we also observed inducing diabetes resulted in TGF-ß signaling activation and increased expression of TGF-ß downstream signals. We found that LMWF treatment may improve renal dysfunction and fibrogenesis in two vivo diabetic rats. Thus, in this study we demonstrated that LMWF reduce harmful TGF-ß-mediated cell responses in renal interstitial fibrosis, suggesting the potential benefit of LMWF to halt the progression of DN. And target blockade of TGF-ß/Smad and TGF-ß/ MAPKs signaling by LMWF may provide a new therapeutic potential for tubulointerstitial fibrosis in DN in type 1 or 2 diabetes.


131 427 MANGIFERIN: NATURAL GLUCOSILXANTHONE WITH ANTI-ANGIOGENIC ACTIVITY Delgado-Hernandez R., Hernandez-Balmaseda I., Rodeiro I., Vanden Berghe W. Angiogenesis, the development of new blood capillaries and vessels, is required in physiological processes but is also involved in pathological conditions such as tumor growth and proliferation. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. Mangiferin is the main chemical ingredient of Mangifera indica L. stem bark extract and this extract is used in Cuba for the ethnomedical treatment of cancer. A number of biological activities of mangiferin have been suggested, including anti-inflammatory immunomodulatory and antitumoral abilities. The present study evaluated the effects of mangiferin treatment on migration assay in endothelial and murine melanoma cells stimulated with bFGF during 24 hour. Mangiferin were assessed in the Chorioallantoic membrane (CAM) angiogenesis assay. Mangiferin (100ug/ mL), present in the culture medium in combination with bFGF, significantly exhibited an inhibitory effect on cell migration in vitro. Moreover, 50 ug/disk of mangiferin also significantly prevented bFGF induced angiogenesis in the CAM angiogenesis assay. In addition, in a second experimental designed on CAM, mangiferin (50 ug/eggs) showed protective effect on infiltration of tumoral breast cancer cell line (MDAMB231-GFP) stimulated with bFGF in the CAM eggs. Altogether, these results reveal that Mangiferin hold promise for development of new antitumor treatments via interference with angiogenesis in cancer progression.

428 MECHANISM STUDY OF SYNERGISTIC ANTITUMOR EFFECT OF BERBERINE AND DOXORUBICIN ON BREAST CANCER CELLS BASED UPON GENOMIC SCREENING Liu W., Song X., Gu W., Cao Y., Yan L., Jiang Y., Cao Aim: The present study was designed to investigate the mechanism of Berberine and Doxorubicin’s synergistic antitumor effect on breast cancer cells MDA-MB-231 based upon genomic screening. Methods: To provide a detailed understanding of the mechanism of synergy effect of Berberine and Doxorubicin, we performed genomewide expression profiling of MDA-MB-231 cells, treated 0, 4, 16 and 24 h with Berberine/Doxorubicin, using Agilent human lncRNA+mRNA Array v2.0. Differentially expressed genes (DEGs) analysis, gene oncology (GO) analysis and pathway analysis were done using bioinformatics techniques. Transcriptional expression changes were verified and investigated by qRT-PCR and Western blot analysis. Results: Pathway analysis of DEGs showed MAPK pathway held high enrichment in both mRNA DEGs and lncRNA DEGs. Gene oncology (GO) analysis identified that many of the target genes in MAPK pathway were involved in regulation of nucleotide binding, ATP binding and protein binding. The results of qRT-PCR analysis confirmed the microarray data. Furthermore, to investigate the detailed mechanism, western blot analysis was done with the results showing that ERK and p-ERK protein expressions were down-regulated in Berberine single use group and the synergy group. Conclusion: Preliminary study showed the synergy group of Berberine and Doxorubicin differs with the single drug use group in gene expression profiles. ERK/MAPK pathway was affected significantly in the synergy group and Berberine single use group which was a possible mechanism for Berberine and Doxorubicin’s synergistic antitumor effect on breast cancer cells.

429 MECHANISMS UNDERLYING THE VASODILATORY EFFECTS OF CYSTAMINE IN SMALL MESENTERIC ARTERIES Simonsen U., Pedersen M., Mogensen S., Mulvany M. Background: The tissue transglutaminase (t-TG, transglutaminase 2) inhibitor cystamine has been shown to be neuroprotective and to attenuate structural vascular alterations characteristic for essential hypertension. Vasodilatation may contribute to these effects. The present study hypothesized that cystamine evokes small vessel relaxation by blocking smooth muscle calcium entry and/or inhibition of Rho kinase. Methods: Contraction studies, fura-2 measurements of intracellular Ca2+ ([Ca2+]i) and Western blot determination of phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain (MLC2) were used to study the effects of cystamine in rat mesenteric small arteries. Results: RT-PCR revealed the presence of transglutaminase 1, 2, and 3 in the heart, and also transglutaminase 4 in testes, but only transglutaminase 2 was expressed in mesenteric and small femoral arteries.Cystamine (100 lM) rightward shifted and successively depressed concentration-response curves for phenylephrine, 5-hydroxytrypatmine (5-HT) and and the thromboxane analogue, U46619, while 1 mM cystamine was required to suppress concentration-response curves for extracellular K+. In phenylephrine-contracted arteries, cystamine caused concentration-dependent relaxations and reduced [Ca2+]i, while in potassium-contracted arteries, cystamine induced less relaxation and without changing [Ca2+]i. In contrast to inhibitors of calcium-activated and ATP-sensitive K channels, the inhibitor of voltage-gated potassium KV7 channels, XE991 inhibited relaxations induced by low cystamine concentrations. Incubation with a suicide substrate for transglutaminase, cadaverine, inhibited relaxations induced by cystamine and by a selective inhibitor of transglutaminase 2. Cystamine relaxations were also inhibited by an inhibitor of phospholipase C. Phenylephrine increased myosin phosphatase targeting subunit 1 (MYPT1)-Thr855 and myosin light chain 2 phosphorylation. Cystamine and the Rho kinase inhibitor Y27632 (1 lM) reduced basal MYPT1-Thr855 phosphorylation, but only Y27632 reduced phenylephrine-induced MYPT1-Thr855 phosphorylation. MLC2 phosphorylation was unaltered in the presence of cystamine. Conclusions: Our findings suggest that cystamine by inhibition of receptor-coupled transglutaminase leads to opening of KV7 channels and reduction of intracellular calcium as well as to force suppression which is followed by vasodilatation in rat small mesenteric. The vasodilator effect may contribute to the inhibitory effects of cystamine on the vascular remodelling characteristic for essential hypertension.

430 METHOXYPOLYETHYLENE GLYCOLS (MOPEG’S): A NOVEL TREATMENT PARADIGM FOR GLAUCOMA Samudre S. Background: Recent emphasis on glaucoma treatment has revolved around the development of agents that might have neuroprotective properties as well as their ability to reduce intraocular pressure (IOP). MoPEG’s are novel compounds that have the potential to mitigate both scenarios. The current study determines MoPEG’s ability to reduce IOP as well as protect retinal cells. Methods: For neuroprotection studies: NMDA (2 ll, 100 mM) was injected into both eyes of 6 SD rats that were pretreated 4 days with topical vehicle, 100 mM MoPEG (t.i.d.), followed by 2 weeks of treatment. For chronic IOP experiments, groups of 7 SD rats that had surgically generated ocular hypertension and were treated for 6 months t.i.d. with vehicle, WIN 55-212-2 or MoPEG. Full field ERGs (ffERGs) and histology was used to examine retinal function in both cases, together with IOP, McDonald Shadduck and slit lamp exams and clinical chemistries.


132 Results: In the NMDA experiments, MoPEG mitigated NMDA induced retinal damage and were significantly effective compared to vehicle alone (P < 0.003). Untreated NMDA ffERG a-b wave activity was significantly reduced to 8516 lV from a baseline of 2955 lV (P < 0.001), with significant improvements to 16013 lV (MoPEG) (P < 0.05). In the IOP experiment, treatment with WIN or MoPEG significantly suppressed (P < 0.05) the IOP elevation created by the surgery. The prolonged elevation in IOP compromised retinal status as ffERG response in the vehicle treated ligated eyes was 476% of the unligated contralateral control eyes. Treatment with MoPEG mitigated this decline in retinal status to 709%, (P < 0.0001, n=7/group). There was no decline in retinal status after treatment with MoPEG. Clinical chemistries remained within normal limits. There were no untoward systemic effects caused by any of the agents. Conclusions: Topical treatment with MoPEG safely reduced IOP and protected retinal cells in both acute and chronic rat models with pathological elements that may be seen in glaucoma, without adverse systemic effects.

431 MITOCHONDRIA-DEPENDENT APOPTOGENIC ACTIVITY OF THE AQUEOUS ROOT EXTRACT OF CROTON MEMBRANACEUS AGAINST HUMAN BENIGN PROSTATIC CELLS Afriyie D., Asare G., Bugyei K. Background: Benign prostatic hyperplasia (BPH) and its related cancer is a disease of high public health impact, observed mostly in the elderly above 50 years. Though its etiology and pathophysiology remains obscure, imbalance of molecular mechanisms of proliferation and apoptosis appears to underpin development of BPH and its cancer. Proven efficacy, with minimal side effects of phytotherapeutics compared to conventional drugs used for BPH and its cancer, has resulted in their use worldwide. Aqueous root extract of Croton membranaceus (CM) is widely used for the management of BPH and its cancers in Ghana. Previous studies showed it had potential anti-atherogenic and cardiac protective activity, significantly reduced serum prostate specific antigen and prostate epithelium significantly, and is non toxic. However, its mechanism of action remains to be elucidated. Hence, in this study, the effects of CM on cell viability and apoptosis in human BPH-1 cells were investigated. Methods: Effect of various doses of CM (0, 1, 3 and 5 mg/ml) on viability and morphology of BPH-1cells for 24, 48 and 72 h was determined by MMT assay and phase contrast microscopy respectively. Apoptosis-inducing effects of CM after 48 h at the cellular level were investigated with Hoescht 33258, JC-1 dye staining, and flow cytometry analysis. RT-PCR and Western blot were further used to confirm its apoptotic effects at the molecular level. Results: Treatment with CM (0, 1, 3 and 5 mg/ml) significantly (P < 0.05 at 24 h, and P < 0.001 at 48 and 72 h) and dose-dependently inhibited the proliferation of BPH-1 cells. CM caused significant dose-dependent alteration in BPH-1 cell morphology and reduction in cell density. CM dose-dependently induced brightly stained chromatin nuclear, significant DNA fragmentation (P < 0.01) with presence of G0/G1 subdupliod cells, and loss of mitochondria membrane potential (P < 0.01) in the treated cells compared to control after 48 h. CM significantly upregulated mRNA and protein levels of Bax, but no significant change was observed in mRNA and protein levels of Bcl2. Conclusion: The study revealed that, mitochondria dependent apoptosis induction is one of the mechanisms CM antiproliferative activity in BPH, and possibly its cancers.

432 MODIFIED PROTOCOL FOR EXCISION WOUND HEALING Ayande P. Introduction: Wounds are generally classified according to degree of insult into superficial wounds; injury to the epidermis, and deep-seated wounds; injury to the epidermal, dermal, and subcutaneous layer. A wound is described as acute if it heals under 8 weeks, and chronic when it lasts much longer.The healing of skin wounds progresses through sequential and overlapping phases of inflammation, granular tissue formation, re-epithelialisation and remodelling. Each phase of healing is directed by a complex coordination and interaction of several cell types contained within the wound, including inflammatory cells such as neutrophils, macrophages, lymphocytes, and platelets. Current Protocol: The excision wound model comes across as one convenient wound model often adopted to assess molecular, cellular, and tissue movements in rodents. It represents an animal model that provides access to investigate complex tissue movements associated with repair such as haemorrhage, granulation tissue formation, re-epithelialisation, and angiogenic processes. Circular wounds have a larger and even ratio of total wound area to wound edge which is symmetrical to the wound centre and repair of injured skin areas undergo coordinated cellular movements to restore epidermal, dermal, and subcutaneous tissue structures as described by Frank and K€ampfer (2003). Modified Protocol: This protocol however, reveals significant observations at variants with the proposition of Frank and K€ampfer; that animals inflicted with multiple circular wounds will cope well with the injuries. It is noted that, bleeding could be profuse in the wounding process leading to ischemia in the vicinity of wounds. That inflammation, infectious pathologies, excisional pain and necrosis increases with increasing number of wounds, and that single large circular dorsal wounds be preferred to small-multiple circular wounds. This excision wound method provides for the creation of larger wounds with varying diameters but lessens the physiological stress associated with inflammation, prevents cross-contamination by wound exudates and the likelihood of frequent infliction of pain due to handling. Conclusion: Single large circular dorsal wounds are preferred to small-multiple circular wounds.

433 MOLECULAR DESIGN STRATEGY FOR ENHANCED ESTROGENIC ACTIVITY OF DIARYLHEPTANOIDS USING CARBORANE CLUSTER DERIVATIZATION: SKELETAL SCAFFOLD OR BULKY PROSTHETIC APPENDAGE Lawal B., Priere L. Background: Diarylheptanoids (DAHs) are known to exhibit significant estrogen receptor (ER) activity and as such identified as candidates for safe and effective phytoestrogen replacement for conventional post-menopausal estrogen supplement. Significant numbers of breast cancer cases also depend on estrogen receptor agonist binding thereby worsening pre-existing cases. Downstream consequence of ligand binding to estrogen receptors not only depends on the affinity of the ligand to the receptors, but also to the steric effect of bound ligand on the conformation and spatial orientation of helix 12 of the estrogen receptor binding domain. Boron-containing clusters however are known to exhibit considerable chemical stability in addition to versatile physicochemical properties. Derivatisation of endocrine disruptors with carborane clusters e.g dicarba-closo-dodecaborane combining its hydrophobic and chemical properties with unique structural features will afford flexibility in the design of ER ligands for enhanced binding affinity coupled with and selective agonistic and antagonistic properties. We therefore investigated the possibility of employing carborane derivatives as either ‘skeletal backbone’ or prosthetic attachment in DAHs to achieve functional selectivity using in-silico molecular modeling and docking experiments.


133 Methods: Carboranes derivatives of DAHs were generated using HyperChem, using Allow Ions option and geometry optimization with the semi-empirical AM1 method. The obtained geometries of the carborane derivatives were compared with those obtained from ab initio calculations at the Hatree-Fock/6-31G* level. Because HyperChem does not provide any platform for docking, and since Autodock does not provide parameters for boron atoms, all boron atoms are converted to carbon atoms after optimization and subsequently converted to pdbqt file that Autodock can handle. Docking was performed using Autodock vina and conformational poses of ligands are scored and compared. Results: As designed, it was found that depending on the in-silico molecular design of the carborane derivatives of DAHs, it was possible to fine-tune the properties of the resulting molecules to obtain desired agonistic or antagonistic action on the estrogen receptors. Conclusions: Carborane derivatives of DAHs improved the ligand efficiency on estrogen receptors and provides a possible strategy for design of highly efficient estrogen receptor ligands with either agonistic or antagonistic properties.

434 MULTIPARAMETRIC EVALUATION OF THE CYTOPROTECTIVE EFFECT OF THE MANGIFERA INDICA L. STEM BARK EXTRACT AND MANGIFERIN IN HEP-G2 CELLS

i.n.a of the VSV LD50 in young male mice, three populations (P1, P2, P3), are identified in the olfactory bulb based on CD45 and CD11b expression. We are now characterizing the dynamics of these populations during the course of the CNS encephalitis. Methods: Immunocytochemistry, Flow cytometry and FluorescentActivated Cell Sorting (FACS) analysis of vesicular stomatitis virus (VSV) infected Cd11c/eyfp transgenic and wild type C57/b6 mice, as well as OT1 and OT2 transgenic mice were used in this research. Results: Following i.n.a of VSV, P1 contains activated microglia, while P2 &P3, express either a CD11c+ F4/80+ Gr-1+ and Ly-6Chi (P2) or a CD11chi CD115neg F4/80neg Gr-1neg and Ly-6Chi (P3) with a NK1.1+ IKDC sub-population. P2 and P3 can present antigen to T lymphocytes, as well as induce a TH1 directed cytokine profile in proliferating CD4 T cells. Among the Nk1.1+ cells, two interferon producing killer dendritic cell (IKDC) phenotypes have been characterized, indicating novel immune mediators to cope with the VSV encephalitis. As the disease progresses in the young mice, the ratio/phenotypes of P1, P2 and P3 changes. In adult mice, VSV encephalitis is resolved, whereas in aged mice, the time course/pathology of the illness is markedly different from young mice. Aged mice die later than the young mice and the bDCs display an increase in stress granule markers. Conclusion: Our ongoing research of neuroendocrine interactions with immune cells in the steady state, and following models of brain insult, is instrumental in deciphering the mechanisms of susceptibility and resistance to various CNS insults throughout life.

Rodeiro I., Tolosa L., Donato M., Herrera J., Castell J., GomezLech on M., Delgado-Hernandez R. Mango (Mangifera indica L.) stem bark standardized extract (MSBE) is a product with biological properties. It contains a variety of polyphenols and a xanthone (mangiferin), the major component (up to 20%). Clinical use of this extract determines the necessity of evaluating its safety in humans. This study was undertaken to investigate the potential cytoprotective hepatic effects of MSBE and mangiferin against tert-butyl hydroperoxide or amiodarone-induced toxicity, compounds reported to produce oxidative effects and mitochondrial damage, by using a high content screening multiparametric assay that measures nuclear morphology, cell viability, intracellular calcium concentration and ROS production in HepG2 cells. MSBE and mangiferin produced no toxicity up to 500 lg/ml. A marked recovery of cell viability reduction induced by model toxicants was observed in the cells pre-exposed to MSBE or magniferin (5–100 lg/ml). This effect could be attributed to both the partial restoration of the intracellular calcium levels and the quenching of ROS generated in cells after herbals exposure. The cytoprotective effect of MSBE is due, at least in part, to mangiferin, but other constituents contribute to its effects. In addition, we explored the possible interaction of both products on the P-glycoprotein (P-gp), one of the ABC transporters responsible for resistance of tumours. Our results show MSBE and mangiferin above 100 lg/ml inhibit the activity of P-gp in HepG2 cells.

435 MULTIPLE FUNCTIONS FOR CELLS OF DENDRITIC CELL LINEAGE IN THE STEADY STATE AND VIRALLY INFECTED BRAIN AND PITUITARY Bulloch K., Gagnidze K., Gal-Toth J., Glennon E., Trochtenberg A., Chung Y., Kaunzner U. Introduction: Dendritic cells (DC), the antigen presenting cells of the immune system, have been identified within the CNS. These cells, termed brain DCs (bDC), are characterized by their morphology, DC phenotypes, and their responses to brain trauma, intracerebral ventricular inoculation (i.c.v.) of interferon gamma and intranasal application (i.n.a.) of vesicular stomatitis virus (VSV). Anatomic studies of young and adult mice reveal bDC in discrete regions of the steady-state brain and pituitary, whereas in old mice, bDC accumulate in the region associated with cognitive decline during normal brain aging. At day 4, post

436 NEUROPHARMACOLOGICAL PROFILE OF THE ETHANOLIC EXTRACT OF PERSEA AMERICANA MILL (LAURACEAE) IN MICE Oyemitan I., Ojo E. Persea americana is a common medicinal plant used ethnomedicinally in the management of several diseases in many South-Western States of Nigeria where it is used to treat malaria, hypertension, rheumatism, febrile convulsions and diabetes among other uses. Presently, there is paucity of report on the central activity of the ethanolic extract of the dried seed which has been suggested for potency; hence this study. Fresh ripened fruits of P. americana were purchased from the local market and their seeds removed, sliced into small pieces, dried under sun-light and grounded into coarse particles before extracted with 70% ethanol and the filtrates concentrated in vacuo. Acute toxicity (LD50) profile was determined orally (p.o.) and intraperitoneally (i.p.) in mice of mixed sex (20–25 g). The neuropharmacological evaluation was done by testing the extract at doses of 250, 500 and 100 mg/kg, i.p. Behavioural effects on rearing and locomotion was determined by open field; anxiolytic activity was tested on hole board and elevated plus maze (epm); sedative effect was assessed by ketamine-induced hypnosis and the parameters recorded include sleep latency (SL) and total sleeping time (TST); and finally, anticonvulsant effect was evaluated by pentlylene tetrazole-induced (PTZ), strychinine-induced and maximal electric shock (MES)-induced convulsion models. Results were analyzed and compared to standard drugs as positive controls and normal saline as negative control. The LD50 of the extract in mice was determined to be ≥5000 mg/kg, p.o., and 2250 mg/kg, i.p. The extract at (250–1000 mg/kg) dosedependently caused significant (P < 0.01-0.001) reduction in rearing and locomotor activity; caused increase in number of head dips and time spent in the open arms of the epm; shortened SL and increased TST induced by ketamine (100 mg/kg, i.p.). The extract also dosedependently blocked PTZ-induced convulsion, blocked hind limb tonic extension on the MES but ineffective against strychnine-induced convulsion. It is concluded that ethanolic extract of the dried seed of Persia americana demonstrated significant depressant effects on the CNS and exhibited anxiolytic, sedative and anticonvulsant effects in mice, thus providing pharmacological basis for the ethnomedicinal uses of the plant in addition to serving as a lead in drug discovery.


134 437 NEUROPROTECTION EFFECT OF KAI-XIN-SAN THROUGH ERK AND PI3K PATHWAYS IN CHRONIC MILD STRESSINDUCED DEPRESSIVE RATS AND HIPPOCAMPAL NEURONS Dong X., Wang T., Yu B., Mu L., Hu Y., Liu P. Kai-Xin-San (KXS) is a famous traditional Chinese medicine used for the treatment of emotional disease. Previously, we have found that KXS demonstrated significant antidepressant behavior and promoted the BDNF and phosphorylation of cAMP response element binding protein (CREB). In the present study, we investigated the antidepressant-like effect of KXS by observing its influence on pCREB and its upstream signal pathway. Solitary rising combined with the chronic unpredictable mild stress (CMS) was used to establish the rat model of depression. The rats were given KXS for 3 weeks, the behavior change and the following items in hippocampus and prefrontal cortex were detected simultaneously: CREB, pCREB, BDNF, TrkB, ERK, pERK, PI3K, Akt, GSK-3b, pGSK-3b; primary hippocampal neurons were cultured, and the effects of TFSA on cell viability, BDNF release, pTrkB,TrkB, ERK, pERK were observed. We observed the rugulation of TFSA on TrKB/ERK/BDNF signal pathway in Primary hippocampal neuronal cultures.Our results showed that treatment with the KXS significantly improved the behavior and simultaneously increased the pCREB, BDNF and TrkB levels in the hippocampus and prefrontal cortex tissues of depressive rats. In future studies revealed that KXS could significantly increase the protein expression of ERK, pERK, PI3K, Akt, GSK-3b and pGSK-3b in the hippocampus and prefrontal cortex of the depressed rats; and TFSA increased the cell viability, BDNF release, and pTrkB,TrkB, ERK, pERK expression in primary hippocampal neurons at the same time. We believe that antidepressant effect of KXS is concerned with the increase of pCREB and its upstream signal pathways: TrkB/ERK/CREB and TrkB/PI3K/ CREB.

438 NEUROPROTECTIVE POTENTIAL OF LERCANIDIPINE IN MIDDLE CEREBRAL ARTERY OCCLUSION MODEL OF FOCAL CEREBRAL ISCHEMIA IN RATS Gupta S., Gupta Y. Background: Worldwide, stroke is a second leading cause of death above 60 years of age, and is the fifth leading cause of death in people aged between 15 and 59 year old. At present, recombinant tissue-plasminogen activator (rt-PA) is the only thrombolytic drug approved for the treatment of acute ischemic stroke. Risk factors and recurrence further necessitates prophylactic approach for stroke. Till date calcium channel blockers (CCBs) such as nimodipine, isradipine have failed to show efficacy in previous clinical trials. The objective of the present study was thus to assess the neuroprotective potential of lercanidipine, a third generation 1,4-dihydropyridine (DHPs) L-type CCB with additional ability to cross blood-brain-barrier, anti-oxidant, anti-inflammatory and vasodilatory property in an experimental model of stroke in rats. Methods: Male Wistar rats (220–250 g) were subjected to 2 h of middle cerebral artery occlusion (MCAo) followed by 24 h reperfusion to induce transient focal cerebral ischemia. Non-hypotensive dose of lercanidipine was selected based on non-invasive tail cuff method. Pretreatment protocol included lercanidipine (0.25, 0.5 and 1 mg/kg, i.p.) or vehicle administration 1 h before MCAo. Neurological deficit score, motor deficit paradigms and cerebral infarction using 2,3,5-triphenyl tetrazolium chloride (TTC) staining were performed 24 h after MCAo. Based on pre-treatment effect, lercanidipine was administered 1 h postMCAo at an effective dose of 0.5 mg/kg, i.p. The parameters assessed were cerebral blood flow (CBF) by laser doppler flowmetry and cerebral infarction. Results: Significant improvement in neurological deficit score, motor impairment and infarct volume reduction was observed with lercanidi-

pine at 0.5 and 1 mg/kg, i.p. (P < 0.05) when treated 1 h before MCAo. There was an increase in CBF (85.89% of baseline) with lercanidipine at 0.5 mg/kg, i.p., 1 h post-MCAo group (P < 0.001) within 2 h after reperfusion as compared to vehicle-treated MCAo control group (64.61% of baseline). Cerebral infarct volume was also reduced significantly to almost 50% in lercanidipine 0.5 mg/kg, i.p., post-MCAo group (P < 0.05) as compared to MCAo-control group. Conclusions: Present study paves the way for calcium channel blocker with anti-oxidant and anti-inflammatory properties as a novel therapeutic strategy for stroke.

439 NEUROPROTECTIVE POTENTIAL OF SELECTED MEDICINAL PLANTS AGAINST ROTENONE-INDUCED APOPTOSIS IN SH-SY5Y NEUROBLASTOMA CELLS Seoposengwe K., Van Tonder J., Steenkamp V. Introduction: Parkinson’s disease (PD) is the second most common chronic neurodegenerative disease. Currently, there is no promising PD cure and this has resulted in extensive research into complementary and alternative medicines. The aim of this study was to investigate the effect of methanol and ethyl acetate extracts of Lanneaschweinfurthii (Engl. Engl) (Anacardiaceae), Zanthoxylumcapense (Thunb. Harv) (Rutaceae), Scadoxuspuniceus ((L.)Friis&Nordal) (Amaryllidaceae) and Crinum bulbispermum (Burm. f.) Milne-Redh.&Schweick) (Amaryllidaceae) on rotenone-induced toxicity in SH-SY5Y neuroblastoma cells. Methods: Cytotoxicity of plant extracts and rotenone was assessed using the sulforhodamine B (SRB) assay. Fluorometry was used to measure intracellular redox state (reactive oxygen species (ROS) and intracellular glutathione content), mitochondrial membrane potential (MMP) and caspase-3 activity, as a marker of apoptotic cell death. Results: When comparing SRB cytotoxicity results obtained from cells induced with 10 nM of rotenone to the rotenone positive control, both methanol and ethyl acetate extracts of Z. capense and S. puniceus induced a significant increase in cell viability at both 3.125 lg/ml and 6.25 lg/ml concentrations. C. bulbispermum did not induce any increase in cell viability at concentrations tested, whereas L. schweinfurthii induced a non-significant increase in cell viability at 6.25 lg/ ml. Regarding SRB cytotoxicity results obtained from cells induced with 50 nM of rotenone, none of the extracts induced an increase in cell viability when compared to rotenone. All extracts significantly decreased caspase-3 activity when compared to rotenone. When comparing methanol extracts to minocycline, both S. puniceus and C. bulbispermum significantly decreased caspase-3 activity in cells at 25 lg/ml. Comparing ethyl acetate extracts to staurosporine all extracts significantly reduced caspase-3 activity except for L. schweinfurthii that did not have any effect at concentrations tested, and Z. capense, S. puniceus and C. bulbispermum that did not have any effect at the lowest concentration 3.125 lg/ml. Conclusions: These findings provide evidence that Z. capense and S. puniceus possess potential neuroprotective effects. Moreover, all four extracts were shown to possess anti-apoptotic effects with S. puniceus, C. bulbispermum and Z. capense showing strong anti-apoptotic effects.

440 NOVEL IMIDAZO [1, 2-A] PYRIDINES AS POTENTIAL ANTILEUKAEMIC AGENTS Ismail Z., de Koning C., Harmse L., Dam J. Background: Leukaemia is the most prevalent cancer amongst children in South Africa. Despite recent advances in chemotherapy, challenges such as primary drug resistance and toxicity warrant the search for alternative agents to treat leukaemia. Against this background, novel imidazopyridines were synthesised and evaluated for their ability to inhibit the K562 leukaemia cell line in vitro.


135 Methods: Cells were maintained in RPMI-1640 media in a humidified incubator with a 5% CO2 atmosphere. Test compounds were synthesized by recombinatorial chemistry and are arbitrarily referred to as JD-compounds. Cell viability was determined by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) cytotoxicity assay. The half maximal inhibitory concentration (IC50) was obtained for compounds that caused more than an 80% decrease in cell viability when exposed to 200 lM of test compound for 48 h. Phase-contrast and fluorescence microscopy, with the fluorochromes acridine orange and ethidium bromide, were used to assess whether the morphology of treated cells display features of apoptosis or necrosis. Results: The results identified 9 test compounds that were able to reduce growth in K562 cells by at least 80%. The IC50 values of the four most active test compounds, JD-52, JD-74, JD-88 and JD-122 were 10.682.3, 12.822.4, 1.870.9 and 17.584.5 lM, respectively. The IC50 values of the other test compounds, JD-60, JD-75, JD-80, JD-119 and JD-137 were 34.023.8, 25.674.5, 63.784.1, 23.512.4 and 26.151.2 lM, respectively. Phase-contrast and fluorescent microscopy images displayed typical apoptotic features like cell shrinkage and cell surface blebbing for both JD-88 and JD-122 treated cells. JD-122 treated cells displayed condensed nuclei stained with ethidium bromide indicating the loss of membrane integrity. Untreated control cells showed intact cell membranes, nuclei and lysosomes as determined by acridine orange and ethidium bromide. Conclusion: This investigation identified four novel compounds (JD52, 74, 88 and 122) with promising cytotoxic activity against the K562 cell line. Preliminary investigations indicate that these compounds are able to induce apoptotic cell death. Further studies will be performed to confirm the mode of cell death on the K562 cell line and will be expanded to include other leukaemic cell lines and animal models.

441 O-PRENYLATED COUMARINS: POTENT HUMAN 15LIPOXYGENASE-1 INHIBITORS Sadeghian H., Jabbari A. Introduction: Human 15-lipoxygenase-1 (15-HLO-1) is recently welldocumented as a target for reducing the biosynthesis of eoxines, one of the known pro-inflammatory mediators. It has been showed that the 15-HLO-1 pathway can generate the pro-inflammatory eoxines in eosinophils, mast cells, and nasal polyps from allergic subjects, indicating that inhibition of 15-LO-1 might be an attractive target for treatment of inflammatory respiratory disorders such as asthma, rhinitis, and chronic obstructive pulmonary disease (COPD) in humans. Also the critical role of the 15-HLO-1 metabolite, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), in the progression of prostate cancers and inhibition of 15-LO-1 activity for apoptosis induction in PC3 cells has been demonstrated. Material and methods: All of the O-prenylated coumarins were synthesized via reaction of farnesyl or geranyl bromide with desired monohydroxycoumarins. 15-HLO-1 was prepared from harvested PC3 cells and its activity was determined by measuring the 13-HODE production rate. The inhibitory potency of the coumarins against HLO-1 was performed by using of the kinetic procedure: measurement of the enzyme activity within 60–120 s after addition of the substrate to the enzyme-inhibitor mixture at 235 nm. Bonding affinity of the designed molecular structures toward modeled 15-HLO-1 was studied by using of AutoDockTools (ADT) software. Results and discussion: Amongst the synthetic analogs, 6-farnesyloxycoumarin showed the most potent inhibitory activity (IC50 = 1.3 lM) while the lowest activity was observed for 3-farnesyloxycoumarin. Complementary studies on inhibitory mechanism showed that the mentioned compounds inhibit lipoxygenase activity by competitive mechanism. For each farnesyl and geranyl derivatives, it was found that there is a satisfactory relationship between IC50 values and the lowest calculated inhibitory constants (Ki) of the docked conformers.

Conclusion: It was found that for farnesyl and geranyl derivatives the role of coumarin substitution site in HLO-1 inhibition is very predominant. SAR studies showed that hydrogen bonding, CH/p, anion-p and S-O=C interactions with FeIII-OH, Leu408, Glu357 and Met419 were the distinct intermolecular interactions which can lead to important role of the coumarin substitution site in HLO-1 inhibitory potency, respectively.

442 OPTIMIZED GRAPHIC AND STATISTICAL EVALUATION OF TELEMETRY-DERIVED DATA SETS FROM CARDIOVASCULAR SAFETY PHARMACOLOGY STUDIES IN DOG, MINIPIG AND CYNOMOLGUS MONKEY Markert M., Guth B., Trautmann T., Graf K., Krause F., Meyer W., Ege A. Background: Safety testing of drugs includes preclinical toxicological and safety pharmacological studies. Safety pharmacology investigates effects on physiological function as opposed to histopathology. Druginduced effects on the cardiovascular (CV) system are of particular importance and can be investigated in conscious, unrestrained animals using telemetry-based technology. This allows for continuous monitoring of CV parameters, but produces very large data sets that are challenging for data analysis. Methods: Animals (dog, minipig, primate) instrumented with a fullimplant telemetry- based device (ITS, Konigsberg Instruments) allowing measurement of aortic pressure, left ventricular pressure, ECG and body temperature were used in randomized, cross-over design studies with three oral verum doses or vehicle. The data acquisition was beatto-beat (Notocord). To allow for visualization and statistical analysis, the data was binned as 10 min median values and stored in an Oracle database. Spotfireâ was used for visualization and the statistical analysis was performed using SASâ. Results: We obtained excellent telemetric signal quality through optimized antennae placement and therefore found stable and highly reproducible hemodynamic parameters in all species. Between 120,000 beats (dogs) and 400,000 beats (cynomolgus) collected over 24 h were analyzed. Access to the stored digital data was fast using SAS and Spotfire which also resulted in a user-friendly graphic illustration and statistical analysis within a short time after completing data acquisition. Graphical analysis of the ECG was also able to detect small, timedependent waveform changes. Statistical evaluation demonstrated a statistical power sufficient to detect small changes in the cardiovascular parameters with a modest number of animals (N=4). Conclusions: Large data sets generated in safety pharmacology CV studies present a challenge for data reduction and evaluation. The novel approach described offers convenient data handling and a flexible graphic illustration of the data to enhance and speed study evaluation.

443 OXIDATIVE INJURY UNDERLIE A HOST OF HUMAN DISEASES: PROTECTIVE POTENTIALS OF A GINGERED POLY-HERBAL FORMULATION Idakwoji P., Ajayi O. Background: Free radicals cause oxidative injury, which now appears to be the fundamental mechanism underlying a number of human pathologies including atherosclerosis, rheumatoid arthritis, inflammation, autoimmune pathologies and digestive system disorders. Anti-oxidants scavenge free radicals thereby protecting cells against their damaging effects. This study investigated the polyphenolic content and anti-oxidant potentials of partially purified fractions of a gingered polyherbal formulation.


136 Methods: The Methanolic extract of a gingered poly-herbal formulation (made up of equal proportions of Camellia sinensis, Nauclea latifolia, Phyllantus spp, Khaya senegalensis and Zingiber officinale) was partially purified on a sephadex LH20 column using methanol/ethanol mixtures. Ten (10) fractions obtained were examined for total polyphenols, flavonoids and antioxidant properties using Folin-Ciocalteu, Colorimetric assay and 1,1-diphenyl 1,2- picrylhydrazine (DPPH) radical scavenging methods respectively. Observations: Fractions 5 and 9 showed highest total polyphenol content of 8000.00 and 7800.00 mg respectively while fractions 1 and 7 showed lowest polyphenol content of 2530.60 and 1800.30 mg respectively. Fractions 5 and 9 also showed the highest flavonoid content of 5560.56 and 4690.36 mg respectively while fractions 1 and 7 showed lowest flavonoid content of 2060.97 and 1520.38 mg respectively. A positive correlation was observed between total polyphenol content and antioxidant activity as the fractions with highest polyphenolic content showed the highest antioxidant activities. Conclusion: With the high polyphenolic content and antioxidant activities exhibited by some fractions, this polyherbal formulation may be useful in the treatment and management of oxidative stress related diseases.

444 PACHYMIC ACID DERIVED FROM PORIA COCOS ENHANCES PENTOBARBITAL-INDUCED SLEEPING BEHAVIORS VIA GABAERGIC SYSTEMS IN MICE Oh K., Shah V., Han J., Choi J., Hong J. Most sedative-hypnotics used in insomnia treatment target the g-aminobutyric acid (GABA)A receptors. Triterpenes comprise one of the most interesting groups of natural products due to their diverse pharmacological activities. This study investigated possible mechanisms underlying the sedative-hypnotic effect of pachymic Acid (PA), the predominant triterpenoids in Poria cocos, through behavioral experiments. In this study, we investigated PA as novel compound for sleep aid reagent via GABAAergic systems and Cl channel activation. We explored the behavioral experiments in mice by PA (1, 3 and 5 mg/ kg), respectively. Oral administration of PA markedly suppressed locomotion activity in mice. This compound also confirmed prolonged sleeping time along with reduced sleep latency, and showed synergic effects with muscimol (0.2 mg/kg), in potentiating sleep onset and enhancing sleep time induced by pentobarbital, both at the hypnotic (40 mg/kg) and sub-hypnotic (28 mg/kg) dose. Additionally, it was found that with an increase in PA concentration (mM), it showed elevated levels of intracellular chloride influx levels in primary cultured hypothalamic neuron cells. Moreover, western blot quantitative results showed PA with increased amount of protein level expression of GAD65/67 over a broader range of doses. The expression studies showed that PA treatment increased of a- and b-subunits protein levels, and decreased in amount of c-subunit protein levels in GABAA receptors. The present report provides evidence for the hypnotic effects of PA with enhanced pentobarbital-induced sleeping behaviors possibly via GABAAergic mechanism and Cl channel activation. Taken together it is proposed that this experiment can provide basic evidence that PA may sought for novel class of compound for sleep disturbed subjects with insomnia.

445 PAIN SENSITIVITY FOLLOWING CHRONIC CONSUMPTION OF FRESH AND NATRON - TREATED PALM OIL DIET IN MICE Nneli R.

ingredients in South East Asia and tropical Africa and lower oxidation status or level of rancidity (Gapor et al, 1989). In Nigeria, palm oil is treated with natron in the preparation of a local delicacy, ‘jollof tapioca’ (‘abacha’) to reduce the viscosity. The objective of this study was to determine the effects of fresh palm oil (FPO) and natron - treated palm oil (NTPO) on pain sensitivity in mice. Methods: Twenty seven male Swiss white mice randomly selected were divided into three groups - A (control fed normal ration); B (fed fresh palm oil (FPO) diet); C (fed natron - treated palm oil (NTPO) diet). The feeding experiments lasted for 14 weeks and all animal had free access to water. Thereafter the pain study was done using the hot plate method and the formalin test as modified by McNamara et al, (2007). The pain parameters were observed in early and late phases respectively, then 30, 50, 70, 90 min post - treatment as well as behavioural parameters - hind paw lick, attention, rearing, grooming were observed. Also, the maximal inhibitory effect using aspirin as control. Statistical analysis was done using ANOVA and P < 0.001 was considered significant. Results: The results showed that grooming was higher in the NTPO than FPO and control (P < 0.001) but lower in the FPO compared to the control in the early phase. In the late phase, duration of hind paw lick andattention was significantly higher in the NTPO than control. Maximal inhibitory effects of FPO and NTPO (5 mg/kg) and aspirin (10 mg/kg) as positive control were highest after 50 min post - treatment (P < 0.001). The frequency of rearing in the FPO was higher compared to NTPO group in the formalin test. Conclusion: Chronic consumption of NTPO diet increased pain sensitivity, while FPO diet decreased it thus corroborating the local usage of FPO as a pain reducing substance.

446 PCA, A SCAVENGER OF DICARBONYL COMPOUNDS, PREVENTS METHYLGLYOXAL-INDUCED INJURY IN HUMAN LENS EPITHELIAL CELLS Wang Y., Li W., Pu X., Du G. Background: Methylglyoxal (MGO), a glucose derived dicarbonyl intermediate, is a major precursor of advanced glycation end products (AGEs) which have been linked to the development of diabetic complications. PCA, a phenolic acid compound, is found in the roots of Salvia miltiorrhiza. This study was to investigate the effect of PCA against MGO-induced toxicity in human lens epithelial cells. Methods: Cell viability was assessed by WST-1. Mitochondria redox activity was assayed by resazurin. Mitochondrial membrane potential was assayed by JC-1 staining. The DNA integrity was evaluated by the analysis of the DNA oxidation biomarker 8-OHdG using immunofluorescence assay. AGEs formation was monitored by fluorescence assay. RAGE protein expression was assayed by Western blotting. Results: PCA significantly reduced AGEs formation and alleviated MGO-induced mitochondrial dysfunction and apoptosis in SRA01/04 cells. Furthermore, PCA was capable of inhibiting MGO-mediated AGEs formation and blocking receptor of AGEs expression in SRA01/ 04 cells. Conclusion: The results suggest PCA could be useful in attenuation of MGO-induced cell damage and the possible mechanism is involved in modulating AGEs-RAGE axis in human lens epithelial cells, which suggests that PCA has a potential protective effect on diabetic cataract.

447 PHARMACEUTICAL RESEARCH, POST GRADUATE TRAINING AND SERVICES AT THE NORTH-WEST UNIVERSITY Tredoux K., Du Plessis J., Bonechans B., Grobler A.

Background: Palm oil is edible plant oil derived from the pulp of fruit of oil palm (Reeves et al, 1979). It contains several saturated and unsaturated fats (Poku, 2002). Palm oil is a very common cooking

Pharmacen, a Centre of Excellence at the Potchefstroom Campus of the North-West University, sets the trend for cutting edge Pharmaceuti-


137 cal research and innovation. We deliver postgraduate students and post-doctoral fellows and contribute to provide solutions for the dynamic health care needs of the people of South Africa and globally. Our mission is excellence in research and the accumulation of new scientific knowledge. We have world-class research facilities and technologies where we focus on optimal drug delivery and translational neuroscience and therapeutics. The DST/NWU Preclinical Drug Development Platform forms part of a national initiative by the Dept of Science &Technology to establish preclinical drug development in South Africa. The following studies are performed in small animals at the facilities of the North-West University: ADME, safety pharmacology including plethysmography, cardiovascular monitoring using telemetry and open field mobility. Specialized services include xenografts, transgenic mouse studies and blood, plasma and urine bioanalysis. The facilities are world class with continuous environmental monitoring and control of temperature, humidity, light and pressure. The Centre for Pharmaceutical and Biomedical Services provides and manages various pharmaceutical and biomedical services by means of independent business units. These units, The Research Institute for Industrial Pharmacy incorporating the Centre for Quality Assurance of Medicines, the Clinical Pharmacokinetic Laboratory and the Cosmetics Efficacy Laboratory, provide analytical laboratory-, training- and consultation services to various national and international pharmaceutical and cosmetic companies, procurement organisations, medicine regulatory authorities, private medical practitioners, pathology practices and the Department of Health.

448 PHARMACODYNAMICS OF SCHISANDRIN B- AND FRUCTUS SCHISANDRAE OIL-INDUCED HYPERTRIGLYCERIDEMIA, HEPATOMEGALY, AND LIVER DAMAGE Pan S., Zhang Y., Zhou S., Yu Q., Wang X., Sun N., Zhu P., Yu Z., Ko K. Background: Fructus Schisandrae (FS) is widely used as a tonic and adaptogenic in China and Russia. Schisandrin B (Sch B) is a dibenzocyclooctadiene compound isolated from FS. Our previous studies have showed that Sch B can induce hypertriglyceridemia and hepatomegaly. Here, the pharmacodynamics of Sch B and FS oil on serum/ hepatic triglyceride (TG) and liver weight/function were investigated. Methods: FS used in the present study was identified as Schisandra chinensis (Turcz.) Baill. (BeiWuWeiZi in Chinese). Oil was extracted from FS seeds using petroleum ether. Adult male ICR mice were orally given a single-dose Sch B 0.125–2 g/kg or FS oil 0.3–5 g/kg. Serum/hepatic lipids and liver weight/function were assayed from 6 to 120 h post-dosing. TG was determined with GPO-PAP method. Alanine aminotransferase (ALT) was measured by automatic Biochemistry Analyzer. Hepatic malondialdehyde (MDA) and protein level using TBA and G-250 dye method, respectively. Results: Sch B and FS oil treatment increased serum TG levels (up to 427% and 123%, respectively) at 6–72 h post-dosing, with a value of Emax 5.50 and 4.60 mM and KD 0.43 and 2.84 g/kg being estimated, respectively. Hepatic TG contents were increased by 40–158% and 35–85% at 6–120 h after Sch B and FS oil treatment, respectively. The values of Emax and KD of Sch B/FS oil increasing hepatic TG contents were 22.94/15.02 lmol/g and 0.78/3.03 g/kg, respectively. Hepatic index was increased by 16–60% (Emax=11.01, KD=0.68 g/kg) and 8–32% (Emax=9.88, KD=4.47 g/kg) at 12–120 h after administration of Sch B and FS oil, respectively. Serum ALT activity was elevated (approximately 60%) at 48–96 h after Sch B, but not FS oil, treatment. Sch B and FS oil treatment increased hepatic MDA by 70 and 22% at 24–96 h post-dosing, respectively. Conclusions: The potency and duration of hypertriglyceridemia, hepatomegaly, and liver injury caused by Sch B is more than FS oil at high doses. Sch B, but not FS oil, elevated serum ALT activity. Sch B has more affinity to tissues and Emax than FS oil.

449 PHARMACOKINETIC EVALUATION OF AN ANTIMALARIAL COMPOUND, NP-102, IN MICE Gibhard L., Pravin K., Abbay E., Taylor D., Wilhelm-Mouton A., Swart K., van der Westhuizen J., Smith P., Wiesner L. Background: Malaria remains a great burden on humanity. Humans are infected largely by five parasite species, of which P. falciparum eliciting the most severe infections. Although significant advances have been made in the prevention and treatment of malaria, malaria control is now prejudiced by an increasing tolerance of the parasite to one or more drugs within artemisinin combination therapies (ACTs), therefore an urgent need exists for development of novel and improved therapies. The University of the Free State chemistry department previously synthesised an anti-malarial compound, NP-046. In vitro studies illustrated an enhanced efficacy for both chloroquine-sensitive and -resistant P. falciparum strains. NP-046 has been subjected to a comprehensive pharmacokinetic study in mice but showed a very low oral bioavailability of approximately 3%. Efforts to enhance the bioavailability of NP-046 have resulted in the synthesis of a number of structural analogues including NP-102. Similar to NP-046, NP-102 illustrated activity against intra-erythrocytic stages of both chloroquinesensitive and -resistant strains of P. falciparum. We describe here the pharmacokinetic experiments conducted on compound NP-102. Methods: Pharmacokinetic studies were conducted for NP-102 in C57/BL6 mice. NP-102 was administered at a dose of 5 mg/kg intravenously or 15 mg/kg orally. Blood samples were collected by means of tail bleeding at pre-determined time intervals. Drug concentrations were determined with a LC/MS/MS method. Results: The Cmax obtained following oral administration of NP-102 was 0.7 0.3 lM, the time to peak NP-102 concentration was 0.5 h and the mean AUC0-inf value 147 min.lM. The mean oral bioavailability for NP-102 was 20% and the apparent elimination half-life was 6.1 h. Discussion: The study indicated an improvement in the Cmax and AUC0-inf as well as a relatively good bioavailability of NP-102 when compared to parent analogues in this series. In addition to the results of the pharmacokinetic study, the drug also conformed to acceptable in vitro efficacy and general cytotoxicity criteria and will be subjected to further in vivo efficacy studies.

450 PHARMACOLOGICAL ACTIVITIES OF MOROCCAN ESSENTIAL OILS Badiaa L. The history of medicinal and aromatic plants is associated with the development of civilizations in which aromatic and medicinal plants had an important role. Herbal-derived compounds are the basis for large proportion of the commercial medications used today in developing countries for treatment of several diseases. An essential oil (EO) is internationally defined as the product obtained by hydro-, steam- or dry-distillation of a plant or of some of its parts, or by a suitable mechanical process. For food and beverage consumption, essential oils products used are on the Generally Recognized as Safe list approved by the Food and Drug Administration (FDA). For medical purposes essential oils need to fulfill national and international Pharmacopoeia recommendations. Physical standards of essential oils are also specified by the Association Francßaise de Normalisation (AFNOR) as well as the International Organization for Standardization (ISO). The use of essential oils in the pharmaceutical, agricultural and nutritional fields are due to their antimicrobial, antiviral, antifungal, insecticidal, antioxidant and anti-inflammatory activities. In this presentation, the biological activities of the essential oils were compared with those of the main components in order to enlighten if the activities of these compounds reflect the pharmacological activities . The chemical variability may contribute to the existence of EO chemotypes which may


138 partly explain the contrasting properties of the essential oils isolated from the same species. Also in vivo and in vitro animals studies reveals a therapeutic target for cardiovascular pathologies. The most EO was effective against lipid peroxidation, in scavenging NO free radicals, showed the best chelating power. Not all of the major compounds of the EO were responsible for the whole activity of the EOs. The EOs antioxidant and anti-inflammatory activities was plant species dependent and not always attributable to the EOs main components. Nevertheless, the EOs anti-proliferative activities were more related to their main volatile components.

451 PHARMACOLOGICAL BASIS FOR THE MEDICINAL USE OF CHAMOMILE IN HYPERTENSION Mehmood M. Background and objective of study: Medicinal herbs have been used through dawn of the history to treat various diseases, such as cardiovascular disorders and are serving as best alternative remedies to combat ever-increasing disease burden in the society. Matricariachamomilla commonly known as ‘Chamomile’ is well known for its medicinal use in hypertension. This study provides pharmacological basis to the medicinal use of M. chamomilla in cardiovascular disorders. Methods: In vitro experiments on isolated rat aorta were carried out. The spasmolytic effect of different concentrations of crude extract of Matricariachamomilla (Mc.Cr) was studied on phenylephrine (P.E 1 lM) and high K+ (80 mM)-induced contractions in rat aortic tissues using isolated tissue bath set up coupled with PowerLab data acquisition system. Results: The extract of Matricariachamomilla showed both vasoconstrictive and dose dependent vasodilatory effects on isolated rat aorta. It showed both vasodilatation and vasoconstriction in aortic tissue. On basal tone, Mc.Cr showed phentolamine sensitive vasocontractile effect, while against induced contraction of high K+ (80 mM) and P.E, the plant extract exhibited dose-dependent relaxation. When tested in Ca+2 free Kreb’s solution for its effect on P.E-evoked contractile peaks, Mc.Cr caused attenuation at 1–5 mg/ml showing its inhibitory effect on the release of Ca2+ from intracellular stores. Conclusion: These results indicate that Matricariachamomilla extract possesses vasomodulatory constituents possibly mediating through effect through Ca2+antagonistic and a-adrenergic receptor modulating activities, thus providing a rationale to its medicinal use in hypertension. However, further studies are required to identify the possible chemical constituents responsible for its therapeutic use in hypertension.

452 PHARMACOLOGICAL EVALUATION OF CUBAN MEDICINAL PLANTS ON THERAPEUTIC TARGETS OF TYPE 2 DIABETES Sánchez Calero J., Young L.C., Marrero Faz E., Harvey A. Background: The aim of the study was to determine the antidiabetic efficacy and the underlying mechanism, of seven medicinal Cuban plants: Allophylus cominia (L.) Sw., Ocimum tenuiflorum, Persea americana, Sechium edule green and white varieties, Momordica charantia and Jatropha aethiopica, some of them reported in Cuban ethnomedicine. Methods: The aqueous extracts from these plants and their fractions were evaluated on type 2 diabetes therapeutic targets: protein tyrosine phosphatase 1B (PTP1B), dipeptidyl peptidase-IV (DPPIV), glucose uptake in 3T3-L1 adipocytes, plasma glucose levels in a neonatal model of diabetic rats induced by Streptozotocin and antioxidant effects.

Results: The results revealed that only aqueous extracts from A. cominia, O. tenuiflorum and P. americana inhibited the enzymatic activity of PTP1B in an extract concentration dependent manner, resulting more active the more polar fractions from A cominia and P americana extracts and fraction 2 from O tenuiflorum extract. All the extracts and fractions showed a slight inhibition of enzymatic activity of DPPIV, A. cominia extract inhibited the enzymatic activity of this protease in a concentration dependent manner, only fraction 1 from P. americana extract showed a moderate inhibitory effect. A cominia, O tenuiflorum and P. americana extracts exhibited a moderate enhance of glucose uptake in 3T3-L1 adipocytes, resulting more active fractions 6 and 10 from A cominia extract and fraction 10 from P americana extract. The same three plants extracts exhibited a potent scavenger effect on DPPH radicals, while J. aethiopica extract showed a moderate inhibition in the generation of this free radical, a great variety of fractions from these plants extracts showed antioxidant activity in this assay. All extracts showed antioxidant properties in L929 fibroblasts exposed to oxidative stress, resulting many fractions active in this assay. The oral administration daily of A cominia aqueous extract at 0.5 g/kg during 21 days to type 2 diabetic rats recovered the normal glycemic values demonstrating the efficacy of A cominia as antidiabetic. Conclusions: The present research demonstrated that aqueous extracts from A cominia, P americana and O tenuiflorum and some of its fractions (AcF6, AcF10 and PaF10), are promising candidates for the development of antidiabetic phytopharmaceuticals.

453 PHARMACOLOGICAL LEVELS OF WITHAFERIN A (WITHANIA SOMNIFERA) SUPPRESS INVASION CONCOMITANTLY WITH EPIGENETIC SILENCING OF PROMETASTATIC GENE EXPRESSION IN TRIPLE NEGATIVE BREAST CANCER CELLS Vanden Berghe W. Today, interfering with metastatic spread remains one of the main challenges in cancer therapy. Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of metastatic cancer types. In the present study, epitheliallike MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells were exposed to pharmacologically relevant concentrations of WA or its closely related analogue withanone (WN). Interestingly, only WA, but not WN treatment demonstrated attenuation of multiple cancer hallmarks in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells. Further, Ingenuity Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flowcytometry and realtime cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Metastasis development includes several discrete steps highly susceptible to epigenetic regulation, all of which occur in a context of a tumour-promoting microenvironment, both by internal and external cues. We found that essential components of metastasis development, including urokinase plasminogen activator, ADAM8 metallopeptidase, and tumour promoting cytokine TNFSF12 are regulated epigenetically by DNA methylation in breast cancer as revealed by 450k Illumina BeadChipArray, MCIp, EpiTyper MassArray and CpG pyrosequencing. Moreover, WA decreases breast cancer invasion by increasing methylation of these genes concomitantly with gene repression. In con-


139 clusion, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer.

454 PHARMACOLOGICAL PROPERTIES OF PEGYLATED HYALURONIDASE - THE NEW DRUG FOR REGENERATIVE MEDICINE Zhdanov V., Dygai A., Zyuz’kov G., Artamonov A. Background: We have previously shown the ability to modify the functions of stem cells (SC) with use of hyaluronidase. This enzyme splits the hyaluronic acid of extracellular matrix on polymers, activating the processes of cell division and differentiation, and also it causes SC migration into the blood. The aim of this work was to study the specific activity of immobilized hyaluronidase (ImGD) on the model of experimental hepatitis, as well as investigation of drug pharmacokinetic and safety. Methods: In the experiments rabbits, Wistar rats and mice CBA/ CaLac were used. Immobilization of enzyme molecules was carried out on polyethylene oxide with use of directional flow of accelerated electrons. Chronic hepatitis (CH) was simulated by administration of CCl4 for 3 weeks. Then ImGD (which doesn’t disintegrate in organism in contrast to the native enzyme) was injected intragastric five times. On the 21st-40th day of the experiment the biochemical, morphological, clonal studies and immunoassay were carried out. Pharmacokinetics was studied using a fluorimetric detection of ImGD, the toxicity with standard techniques. Results: ImGD possesses significant anti-holestatic, anti-inflammatory and anti-sclerotic action. These effects are associated with activation of bone marrow SC proliferation, their mobilization and directed homing into the affected liver. A decrease in SDF-1 -factor produced by the bone marrow microenvironment cells and an increase in the secretion of this chemokine by liver cells, both being influenced by ImGD, determine the progenitor cells migration in the liver at the conditions of CH. ImGD is rapidly absorbed from the gastrointestinal tract, the concentration in blood plasma reaches its maximum in 2 or 3 h. The drug is not cumulated in the body, it is excreted in the feces and in the urine during the first day. In a therapeutic dose the drug doesn’t cause the death or any changes in the internal organs and systems, and also it has no specific toxicity. Conclusions: The results indicate that ImGD possesses a high hepatoprotective activity associated with the activation of bone marrow SC and their migration in the target organ. The drug is not toxic and is rapidly metabolized in the body.

455 PHARMACOLOGICAL STUDIES ON A STANDARDIZED EGYPTIAN RICE BRAN EXTRACT Khayyal M., Abdel-Aziz H., Mohsen R., Helal A., M€uller W., Eckert G. Rice is the primary food source for over 70% of the world. Once milled, rice bran quickly becomes rancid and is unfit for human consumption and is either dumped or used as animal fodder. Using modern technology, we have been able to stabilize rice bran immediately after milling. An extract was prepared using n-hexane and analyzed phytochemically to contain policosanol, gamma oryzanol and tocotrienol as well as a high level of macronutrients. Both gamma oryzanol and tocotrienol have been shown to possess potent anti-oxidant activity. The extract was standardized to contain 2% gamma oryzanol. The standardized extract was studied pharmacologically to investigate its potential therapeutic usefulness. In doses of 30–100 mg/kg, it effectively reduced the blood pressure in L-NAME induced hypertension in rats in a dose-dependent manner without appreciably affecting the heart rate. In models of both acute and chronic inflammation, it was

comparable to diclofenac in reducing inflammation and the levels of associated markers, such as serum TNFa and myeloperoxidase activity. In vitro studies showed the extract to have hypocholesterolemic properties and to inhibit lipid peroxidation. In cell cultures of INS-1 cells, it had a dose-dependent insulinotropic effect probably mediated by gamma oryzanol and policosanol and showed promise as an antidiabetic agent. Recently, we showed it to protect against mitochondrial dysfunction in guinea-pig brains, suggesting that it might be useful against Alzheimer disease. Acute and subchronic toxicity studies in rats have shown that the extract is safe and free from mutagenic effects. The wide spectrum of activity and safety profile of the standardized Egyptian rice bran extract have now paved the way towards introducing it in formulations to be used as a useful nutraceutical agent.

456 PHELA REVERSED CYCLOPHOSPHAMIDE-INDUCED SUPPRESSION OF IGG AND IGM IN A RAT MODEL Lekhooa M., Walubo A., Du Plessis J., Matsabisa G. Background: Phela is a herbal medicine product under development for use as an immune booster in immune-compromised individuals. Unfortunately, the lack of diseased models by which to evaluate the mechanism and efficacy of products purported to be immune boosters has hampered the development of these products. Here, using Phela as test compound, a rat model of cyclophosphamide-induced immune suppression was used to investigate the mechanism of immunomodulation by Phela Methods: Sprague dawley rats were used and approval from animal ethics committee was obtained. Two groups of 15 rats were pre-treated with cyclophosphamide (CP: 100 mg/kg) once weekly to induce immune suppression. On the eighth day the control group continued on the CP-only treatment. The test group was treated with Phela (15.4 mg/kg) daily and once weekly dose of CP. Five rats were sacrificed after 7, 14 and 21 days of treatment in each group. Blood was analyzed for liver, renal and haematology functions and the CD4 and CD8 counts were analyzed by flow cytometry. IgG, IgM and IL-2 measurements were done by ELISA. The kidney, liver, spleen, thymus, were weighed and examined for any pathology. Body weight was recorded before and after the study. Results: Phela minimized thymus weight loss and prevented body weight reduction (%: mean sd) after 14 and 21 days [Phela-group: (+215) and (+158) vs. Control-group (+108) and (+63)]. Furthermore, Phela overcame CD4 and CD8 count suppression from 14 days (P < 0.08) and significantly after 21 days (P < 0.05). IgG concentration was significantly increased in Phela-treated group over 7 days (P = 0.01) and 14 days (P = 0.008) and returned to baseline after 21 days. IgM levels remained high through-out the study though not statistically significant. After 21 days Phela overcame neutrophils and lymphocytes suppression, (910^9/l: SDmean) [Phela-group: (1.050.27) and (3.661.24) vs. Control-group: (0.630.23) and (2.400.31]. Conclusion: Phela prevented progression of the immunesuppression in rats treated with cyclophosphamide as indicated by increasing IgG and IgM levels. This implies that Phela may stimulate antibody production in patients with a compromised immune system and that Phela’s mechanism of immunomodulation is partly humoral.

457 PHENOTYPE ANALYSIS AS PLANT SELECTION CRITERIA IN DRUG DISCOVERY FROM NATURAL PRODUCTS FOR SYNDROMIC DISEASES 1: A CASE STUDY OF METABOLIC SYNDROME Bello S., Chika A. Background: In natural product research, plant selection methods include ethno-botanical surveys or simple folkloric tales, but these are


140 not very predictive. It is conceivable that secondary metabolites in plants are linked by genomics and proteomics to observable phenotypes like plant height, venation, color of leaves, flowers among others. Mathematical models of these phenotypes using database of plants whose extracts are known to have activities and plants whose extracts are known be inactive against targeted diseases may be used to develop a predictive rule or formula that may be used to select new plants with potential activity against such diseases. This hypothesis was tested, with Diabetes mellitus and Metabolic syndrome as targets. Method: Database of plants that have been found to have activity or no-activity against Diabetes and /or components of the metabolic syndrome was developed by systematic Internet search using criteria developed through Delphi protocol. Receiver operator characteristic curve analysis (ROC) was used to check the predictive utility of continuous variables (height of plant, length and width of leaves) for pharmacological activity, while diagnostic analysis (Likelihood ratio) was used to evaluate the predictive utility of categorical variables (Venation, Flowering, Color) using pre-specified cutoff values. Predictive phenotypes were combined into a single model, which was then use to select 5 plants (for each disease target) from a cultivar. Five other plants were also randomly selected for comparison. Aqueous extract of all plants were screen in wet-lab experiment using Alloxan induced diabetic rats, Salt induced hypertensive rats, Diet Induces Obesity mice. Result: A total of 495 plants were retrieved and used for modelling. Only height of plant (less than 1 m) and venation (Compound) were found to be predictive of pharmacological activity. On screening, four of five plants (80%) were correctly predicted to have antidiabetic activity; all five plants (100%) were correctly predicted as anti-obesity but none (0%) was correctly predicted as antihypertensive. Random selection performed 10%, 0% and 0% in the same domains respectively. Conclusion: Mathematical modelling could an effective predictor of pharmacological activity in plants but model refinement with more criteria may be required.

458 PHENOTYPE ANALYSIS AS PLANT SELECTION CRITERIA IN DRUG DISCOVERY FROM NATURAL PRODUCTS FOR SYNDROMIC DISEASES 2: METHANOLIC LEAF EXTRACT OF LEPTADENIA HASTATA PERS. (DECNE) [LH] FOR METABOLIC SYNDROME Bello S., Chika A. Background: In a previous screening study, Leptadenia hastata Pers. (Decne) was the best performing plant, of those selected by mathematical models, as active against components of metabolic syndrome. LH was further evaluated in this study. Method: After acute oral toxicity in male Wister rats, the effects of oral LH on Diet induced obese (DIO) female C57BL6/JolaHsd mice, male B6.V-Lep (ob)/J mice were evaluated at doses of 250 mg/kg bid (most effective dose from previous screening) and compared to 10 ml/ kg bid vehicle treated and 5 mg/kg bid Sibutramine treated controls. Effects on weight gain, Glucose tolerance test profile; Serum total cholesterol, non-esterified fatty acid, HDL cholesterol, Triglycerides, plasma leptin, insulin and 24 h energy expenditure were determined. Furthermore, the effect of LH on Liver glycogen and triglyceride and RNA expression profile of metabolic enzymes of Brown adipose tissues were determined using standard methods and protocols. Result: The LD50 of LH was above 5000 mg/kg with no signs of acute toxicity. In DIO mice LH caused 18.7% reduction in body weight vs. 7.7% reduction by Sibutramine compared to control. The percentage reduction in body weight by LH on Ob/OB mice was smaller (10%). Furthermore, LH reduced food intake, increased energy expenditure, reduced percentage fat mass relative to body weight, increased insulin sensitivity, and decreased hepatic glycogenolysis. Also treatment with LH resulted in down regulation of pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), glucose-6phosphatase (G6Pase) and phosphoglucomutases (1, 2 and 3).

Conclusion: LH, selected by mathematical modelling, has important activity against components of the metabolic syndrome and outperformed controls.

459 PHOSPHODIESTERASE-4D DEFICIENCY REVERSES AMYLOID-b42-INDUCED MEMORY DEFICITS AND PRODUCES ANTIDEPRESSANT-LIKE EFFECT IN MICE Xu J., Zhang C., Cheng Y. Background: Phosphodiesterase 4 (PDE4) is a member of a family of cAMP-catabolizing enzymes in distinct mammalian tissues. PDE4 has been implicated in various clinical conditions, including depression and memory deficits. The PDE4D family is comprised of eleven different isoforms, we investigated the effect of long-form PDE4D knockdown on amyloid-b1-42 (Ab42) -induced memory deficits in mice and evaluated the antidepressant effect in C57/BL mice subjected to chronic unpredictable mild stress (CUMS). Methods: The long-form PDE4D was knocked down by lentiviral RNA construct containing a specific microRNA hairpin structure. Morris water maze (MWM) and novelty object recognition tests were used to examine whether long-form PDE4D knockdown reversed memory impairment caused by Ab42 in mice. The antidepressant-like activity was evaluated using forced swim test (FST), tail suspension test (TST) and sucrose preference test. Western blotting analysis was used to assess protein levels of phosphorylated cAMP response element-binding protein (pCREB), brain-derived neurotrophic factor (BDNF), interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), and nuclear factor-jB (NF-jB) to explore the neurochemical mechanisms. Results: Microinfusions of lentiviruses successfully resulted in reduced expression of PDE4D4 and 4D5 proteins, knockdown of PDE4D reversed Ab42-induced cAMP decline and memory deficits in mice. Downregulation of PDE4D4 and 4D5 increased pCREB and BDNF and reduced IL-1b, TNF-a, and NF-jB in the hippocampus of Ab42-challenged mice. PDE4D knockdown also reversed stressinduced cAMP decline and exerted antidepressant-like effects. PDE4D deficiency caused increased pCREB in the hippocampus of stressmice. Conclusions: These results suggest that long-form PDE4D knockdown may offer a promising treatment for memory loss associated with Alzheimer’s disease and produce a therapeutic effect for depression disorder.

460 PHYTOCHEMICAL COMPOSITION AND ABORTIFACIENT POTENTIAL OF METHANOL EXTRACT OF DENNETTIA TRIPETALA BAKER F. LEAF (ANNONACEAE) IN PREGNANT ALBINO RATS Odoh U. The objective of this study was to carry out phytochemical screening of Dennettia tripetala leaves and ascertain its abortifacient potential in pregnant albino rats. The pregnant rats were treated on days 10–18 at 8 h intervals. Those in group A (positive control) were orally administered 2.85 mg/kg body weight of mifepristone, while those in group B and C were orally treated with 250 and 500 mg/kg body weight of the extract. Group D received 0.5 ml of 3% tween 80. Quantitative and qualitative phytochemical analysis were also performed on the leaf extracts. Phytochemical screening of the extract showed positive results for alkaloids (1.18%), tannins (3.18%), glycosides (0.612%), saponins(0.041%) and flavonoids (0.256%). For the LD50, no death was recorded with the high dose of 5000 mg/kg. The extract significantly reduced (P < 0.05) the number of life foetus, weight and survival ratio of the foetus, numbers of implantations, Corpora lutea and implantation index. The number of dead foetus, number and percentage of rats that aborted, resorption index, pre- and post-implantation


141 losses increased significantly. The effect of the plant extract was comparable with mifepristone. Respiratory distress, salivation, diarrhoea, changes in the appearance of hair as well as maternal mortality was not observed at any time during the exposure period. This study has provided evidence to the age-long claim of D. tripetala leaves in ‘washing the uterus’. The abortifacient properties were most pronounced at 500 mg/kg body weight of the extract. Overall, the extract may be used as an abortifacient and therefore not safe for consumption as oral remedy during pregnancy.

461 PHYTOCHEMICAL CONSTITUTENTS AND HYPOGLYCEMIC PROPERTY OF COLA ACUMINATA SEED ON ALLOXAN-INDUCED DIABETIC RATS Sangodele J., Okere S. The seeds of Cola acuminata were investigated for their anti-diabetic properties. The extracts were obtained by maceration in cold water. Alloxan was used to induce diabetes in the rats at 180 mg/kg body weight and diabetes was confirmed after 48 h. The Phytochemical analyses of the plants showed the presence of tannin, cardiac-gylcoside, saponin and alkaloids, but anthraquinone, steriod, terpenoid were observed to be absent. Cola acuminata was also observed to cause a reduction in blood glucose level from 5990.667 mg/dl to 591.202 mg/dl. The extract showed a significant (P < 0.05) decrease of about 90.15% of blood glucose level in alloxan induced diabetic rats. Cola acuminata showed a higher level of potency when compared to a known anti-diabetic drug; Glanil. Glanil was observed to cause a decrease of 26.04% which is quite low compared with the cola specie hence, Cola acuminata extract was observed to be more potent.

462 PHYTOCHEMICAL, ANALGESIC AND ANTIINFLAMMATORY STUDIES OF THE CRUDE ETHANOLIC EXTRACT OF THE LEAF OF GLOBIMETULAR BROUNII VAN TIEGHEM (FAMILY: LORANTHACEAE) Atiku I., Umar P.U., Musa A., Yahaya S.M., Abdullahi S.M., Hanwa U.U., Lawal E.A., Abdurrahman H. Globimetula braunii is a bushy parasitic plant growing on Terminalia catappa, which has a variety of use in African traditional medicine. The leaves of the plant was subjected to preliminary phytochemical analysis, Acute Toxicity and analgesic activity using Hot plate and acetic acid induced pain method in mice, while the anti-inflammatory activity was tested using Carrageenan-induced paw oedema in rats. The Phytochemical analysis of the ethanolic leaf extract of Globimetulabraunii revealed the presence of Carbohydrate, Steriods/Triterpenes, Tannins, Saponin, Alkaloids and Flavonoids; The LD50 was found to be > 5000 mg/kg which shows that the plant extract is relatively safe. The results from the Hot-plate method showed that the extract was statistically significant at (P < 0.02 and P < 0.01), this shows that there is increase in the latency of pain in the extract compare to standard drug pentazocine (20 mg/kg), while the Acetic acid induced method the extract was statistically significant at (P < 0.02) with a percentage of inhibition of 65.5%, 65.2%, and 67.7% at the doses of 12.5, 25, and 50 mg/kg and compared with the standard drug piroxicam (10 mg/kg) with a percentage inhibition of 54.3%. The extract was also found to significantly (P < 0.05) inhibite oedema at all doses: 250, 500 and 1000 mg/kg.This shows that the plant contains phytochemical constituent with analgesic and anti-inflammatory activities. Comparisn with the host plant shows that the two plants have most of their phyto chemical constituents in common and their analgesic and anti-inflammatory activities are comparable.

463 PRECLINICAL CHARACTERIZATION OF THRX-113587: A NOVEL, POTENT AND SELECTIVE INHIBITOR FOR THE NOREPINEPHRINE TRANSPORTER Shen F., Smith J., Tsuruda P., Stangeland E., McNamara A., Cremers T., Martin W. Background: Norepinephrine (NE) and serotonin (5-HT) modulate CNS neurotransmission and, as such, are implicated in a variety of physiological and pathophysiological processes. Monoamine reuptake inhibitors exhibit a broad range of selectivity profiles and are commonly categorized by their relative activity in vitro at NE and 5-HT transporters (NET and SERT, respectively). Here, we describe the profile of THRX113587, designed to be selective for NET, and compare it to atomoxetine, the only FDA-approved selective NE reuptake inhibitor. Methods: Transporter selectivity was determined using in vitro, ex vivo and in vivo methodologies; radiolabeled neurotransmitter uptake in vitro, CNS transporter occupancy ex vivo, and CNS neurotransmitter microdialysis and behavior (rat formalin and serotonin syndrome models; RFM and RSS) in vivo. Results: THRX-113587 potently inhibited NET (IC50 = 0.4 nM) and exhibited 160-fold selectivity for NET over SERT. At 10 mg/kg (IP), it achieved 70% NET occupancy, negligible SERT occupancy and selectively increased NE concentrations (> 5-fold over baseline) without affecting 5-HT. Maximal and selective engagement of NET was associated with 80% NET and SERT occupancy at 30 mg/kg. Even the minimally efficacious dose of 10 mg/kg yielded >80% NET and 68% SERT occupancy. Consistent with ex vivo occupancy results, atomoxetine (10 mg/kg IP) increased both 5-HT and NE. However, elevations in NE were of a greater magnitude than those observed for 5-HT. THRX-113587 failed to augment 5-HT-induced behaviors in the RSS model, whereas atomoxetine did so at only 3-fold higher doses than required for antinociceptive efficacy. Conclusions: The data from these complementary approaches demonstrate that THRX-113587 is a potent and selective NET inhibitor. By comparing the pharmacological profile of this novel NET-selective inhibitor to that of atomoxetine we found that inhibition of NE reuptake alone yields sub-maximal efficacy in the RFM, and that dual engagement of NET and SERT may be required to achieve complete antinocieption in this rodent pain model.

464 PREDICTION OF IN VIVO BIOAVAILABILITY OF SELECTED FLAVONOLS FROM SUTHERLANDIA FRUTESCENS Mbamalu O., Syce J., Samsodien H. Background: Sutherlandia frutescensis a herbal medicinal product widely used in South Africa. It has gained renewed interest due to its purported activity (supported by in vitro studies) against HIV. Preparations are underway for a clinical trial to assess such activity. Previous studies identified flavonol glycosides as appropriate markers for products containing this plant. These may not be bioavailable as reported in past studies using animals. In vivo bioavailability may be higher for the flavonol aglycones and their metabolites than for the glycosides. The objective of this study was to assess (using prototype flavonol glycosides and aglycones of Sutherlandia frutescens) the likelihood of flavonol absorption after oral administration in humans. Methods: Structural models of flavonol glycosides and aglycones were tested for drug likeness using MOE (Molecular Operating Environment), a chemoinformatics and computational resource for drug discovery. In vivo bioavailability was assessed using descriptors proposed independently by Lipinski, and Veber.


142 Results: With respect to Lipinski’s criteria, the number of hydrogen bond donors, number of hydrogen bond acceptors, molecular mass and octanol-water partition coefficient were 10, 15, 610.5 Da and -1.1, respectively for the prototype flavonol glycoside; 4, 5, 286.2 Da and 2.3, respectively for quercetin; 5, 6, 302.2 Da and 2.0, respectively for kaempferol. With respect to the Veber criteria, the number of rotatable bonds and the polar surface area were 6 and 196.1 A, respectively for the prototype flavonol glycoside; 1 and 96.9 A, respectively for quercetin and 1 and 115.0 A, respectively for kaempferol. Conclusions: Flavonol aglycones, but not the prototype flavonol glycoside, met Lipinski’s and Veber’s criteria for in vivo bioavailability. This indicates that the flavonol glycosides may not be bioavailable and may likely not be suitable blood markers for assay of Sutherlandia frutescens after oral administration in humans. Caution in this interpretation is however required since natural products often do not comply with Lipinski’s rule.

465 PRELIMINARY SCREENING OF ANTI-NOCICEPTIVE ACTIVITIES OF THE ETHANOL LEAF EXTRACT OF PAULLINIA PINNATA LINN (SAPINDACEAE) IN LABORATORY ANIMALS

We have currently synthesised two conjugates - a combination of derivative chlorin e6 with isoniazid and N-acetyl-3-indolinones and we are expecting the results of the in-vitro biological tests soon.

467 PRINCIPAL COMPONENT ANALYSIS OF 17 CYTOKINES AND CHEMOKINES SUGGESTS THAT AUTOLOGOUS ENDOTHELIAL CELL: PBMC CO-CULTURES DELINEATE SEVERE AND MILD CYTOKINE STORM CAUSING BIOLOGICS: A NEW ASSAY FOR CYTOKINE STORM DETECTION AND RESEARCH Reed D., Kirkby N., Mohamed N., Galloway-Phillipps N., Paschalaki K., Starke R., Randi A., Hansel T., Mitchell J. The Northwick Park drug trial disaster in 2006, involving the CD28 superagonist TGN1412, defined an urgent unmet need for improved human tissue bioassays to predict cytokine storm responses to biological drugs. TGN1412 did not cause a response in animals and does not activate monocultures of human peripheral blood mononuclear cells (PBMCs) or human umbilical vein endothelial cells (HUVEC). However co-cultures of human PBMCs and HUVEC do respond to TGN1412 with a cytokine

Mohammed I., Abubakar Mohammed M., Umaru L. The ethanol leaf extract of paullinia pinnata at tested doses of 50, 100 and 200 mg/kg was evaluated for its analgesic activities on acetic acid-induced abdominal writhing and thermal induced test in mice. The extract produces a dose dependent inhibition of abdominal constriction in mice (70.4, 80 and 82%) in the acetic acid-induced abdominal constriction model, and all the values were significant (P < 0.001) compared with the negative control (normal saline) group. The extract increased the reaction time of mice to thermal stimulation. Significant (P < 0.05) activity was observed (62.8, 60.99 and 63.6%) with the extract after 30 min post administration (50, 100 and 200 mg/kg) of all the tested doses. Morphine increased reaction time of mice to thermal stimulation by 93%. Preliminary phytochemical screening of the extract revealed the presence of flavonoids, tannins, saponins and steroids. The intraperitoneal LD50 was found to be 692.82 mg/kg. The results of this study indicate the presence of biologically active substance which may be beneficial in the treatment of pain.

466 PREPARING OF NOVEL CHIMERIC COMPOUNDS BASED ON CONJUGATION OF ANTI-TUMOR AND ANTI-TB DRUGS Fedoseev P., Grin M., Nikitin A. Photodynamic therapy is a well-known technique for the treatment of cancer that has saved many lives. In photodynamic therapy, the three key components in most cases use a photosensitizer, light sourse and tissue oxygen. The combination of these three components causes chemical destruction of tumor tissue by the action of produced singlet oxygen. The photosensitizer accumulates predominantly in tumor tissues, and after the accumulation, it is only necessary to irradiate the desired target with light of a specific wavelength . As a photosensitizer commonly used natural and synthetic porphyrins demonstrating strong photochemical effect. Modern development of photosensitizers for photodynamic therapy aims at improving accumulation in tissues (tropism) and increasing the effective wavelength of the irradiated light when the compound is active. So far is used compounds with the ratio of tumor tissue compound accumulation / normal tissue compound accumulation - up to 30. But we have the compounds with the ratio up to 180. In our work, we decided to use a combination of a photosensitizer with other drugs that have performed well in the treatment of tuberculosis - isoniazid and indole derivatives . We expect a new and unique biological effect of so-called chimeric compounds - chemical conjugate anticancer and anti-TB drugs, which can show increased anti-cancer / TB effect, or a third, unknown effect .

Fig. 1. Scores plot (left) and loadings plot (right) for principal component analysis of BOEC and PBMC monocultures and same donor BOEC:PBMC co-cultures treated with the TGN-like anti-CD28; ANC28 (1/5D10; 10 lg/ml), Herceptin (10 lg/ml), Campath (10 lg/ ml), Herceptin (10 lg/ml), Avastin (10 lg/ml) or Arzerra (10 lg/ml) for 24 h and the following cytokines and chemokines were measured and included in the analysis; GM-CSF, IFNc, IL-2, IL-6, CXCL8, TNFa, IL-1b, IL-10, IL-12p70, Eotaxin, Eotaxin 3, IP10, MCP-1, MCP-4, MDC, MIP-lb, TARC. Values were entered for each variable for all treatment conditions which were the average of n = 5 donors.


143 storm response, typified by key cytokines including CXCL8, TNFa, GM-CSF, IFNc and IL-2. Whilst effective, these types of assays use cells from different donors and this heterologous cell mixing mighty limit detection, compromise the signal and cause immune responses. We have previously reported that endothelial cells derived from adult progenitors (blood outgrowth endothelial cells) (Paschalaki et al. 2013 Stem Cell) can be used to create autologous co-cultures of endothelial cells and PBMCs (Reed et al. 2013 Toxicology Letters, Interlaken, Switzerland. S164) and these assays respond to the, so-called TGN-like, antiCD28 superagonist ANC28 to release CXCL8. Here we have measured release of a panel of 17 cytokines and chemokines in autologous BOEC: PBMC co-cultures stimulated with ANC28 (akin to TGN1412 which causes severe cytokine storm), Campath (mild cytokine storm) or Herceptin, Avastin and Arzerra, which do not cause cytokine storm. For TGN1412, as for new biological drugs, it is critical to assess any generic as well as drug specific pattern of cytokine release and to analyse this in an unbiased manner in order to confirm safety and/or efficacy. Here we used unsupervised principal component analysis of cytokines and chemokines released from mono and co-cultures of autologous BOECs and PBMCs treated with therapeutic antibodies or ANC28 in a proof of concept study (Figure 1). Data from ANC28 stimulated co-cultures formed a distinct cluster, separated from other antibody treated co-cultures and from all mono culture conditions. The loadings plot indicated positive correlations between all cytokines and chemokines measured. We suggest that this kind of analysis and the use of autologous co-cultures will prove useful in the future testing of new biological drugs for their propensity to cause cytokine storm responses.

468 PRO- AND ANTIOXIDANT EFFECTS AND CYTOPROTECTIVE POTENTIALS OF NINE EDIBLE VEGETABLES IN SOUTHWEST NIGERIA Omisore O. Antioxidant and cytoprotective activities of boiled, cold, and methanolic extracts of nine edible vegetables in Southwest Nigeria were evaluated in the 1,1-diphenyl-2-picrylhydrazyl free radical assay and haemagglutination assay in bovine erythrocytes respectively. Crassocephalum rubens showed the highest antioxidant activity (56.5%), Solanum americanum and Vernonia amygdalina exhibited moderate antioxidant activity (26.0–37.5%) while Celosia argentea and Talinum triangulare were pro-oxidants. The studies on the cytoprotective effect showed that all the plant extracts demonstrated a very low haemagglutination titre value between 0.32 and 5.56 except S. americanum methanolic extract which had a titre of 50.0. These results indicated correlation between the antioxidant properties and the haemagglutination values of these plant extracts.

469 PROTECTIVE ROLE OF AQUEUOS LEAF EXRACT OF VERNONIA AMYGDALINA IN CYCLOPHOSPHAMIDE INDUCED UROTOXICITY Ikeh C., Ikeh P., Ezike A., Ajibesin K., Akah P. Cyclophosphamide (CP) is one of the most widely used alkylating anticancer agents. Urotoxicity is one of its major adverse effects. The present study investigated the potentials of Vernonia amygdalina (VA) aqueous leaf extract in protecting CP-induced urotoxicity in rats using sodium-2mecarptoethane sulfonate (MESNA) as a positive control. Urotoxicity was induced with CP 200 mg/kg. Biochemical parameters (Glutathione, Supraoxide dismutase, Catalase and Malondialdehyde assays), and histopathological examination of the urinary bladder/kidney sections were evaluated in the VA-treated and control groups. Results showed that the extract of V. amygdalina significantly protected (P < 0.01) the urothelium as evidenced in the biochemical and histopathological parameters evaluated. This protection was comparable to that produced by MESNA.

Histopathological analysis further revealed that the urinary bladder architecture of V. amygdalina treated groups remained intact when compared to CP group. The result of the present study clearly revealed that the aqueous leaf extract of V. amygdalina can prevent urotoxicity induced by CP, and, thus can be beneficial as therapeutic adjuvant in the management of CP and other oxazaphosphorine toxicities.

470 PSYCHOPHARMACOLOGICAL EFFECTS OF STANDARDIZED METHANOL STEM BARK EXTRACT OF CROSSOPTERYX FEBRIFUGA Tijani A., Alhaji Adamu M., Samuel O. Background: Crossopteryx febrifuga preparations are widely used in African Traditional Medicine practice for management of malaria, fever and painful inflammatory disorders. Objective: The aim of the study was to provide additional safety pharmacology data through investigation of possible central nervous system effects of Crossopteryx febrifuga (CF) stem bark extract in mice. Methods: The methanol stem bark extract of Crossopteryx febrifuga was standardized using Reversed Phase High Performance Chromatography (RHPLC). The effects of stem bark extract of Crossopteryx febrifuga (50, 100 and 200 mg/kg body weight) and diazepam (1 mg/kg) were tested on anxiety-like behaviour on Elevated Plus Maze (EPM), Zero Maze and hole board. Its effect on amphetamine and apomorphine-induced hyper locomotion was studied using the open field apparatus while nootropic effect of extract was evaluated using Ymaze and Elevated Plus Maze (EPM). The effect of the extract on motor co -ordination was assessed using rota rod apparatus. Results: The standardized CF extract (50–200 mg/kg, p.o.) and diazepam (1 mg/kg, i.p.) significantly (P < 0.05) increased the time spent in open -arm of the EPM and significantly (P < 0.05) decreased number of head dips on holeboard. The extract, chlorpromazine and haloperidol produced significantly (P < 0.001) reduced amphetamine- and apomorphine-induced hyperlocomotion in time and dose-dependent manner. The extract significantly (P < 0.05) increased Spontaneous Alternation Behaviour (SAB) when compared to vehicle and diazepam treated mice. The extract significantly decreased post treatment escape latency when compared to effect of vehicle and diazepam but did not significantly affect motor coordination of the mice in rota-rod performance. Conclusion: The results suggest that the Crossopteryx febrifuga standardized extract contains psychoactive substances with possible anxiolytic-like, antipsychotic and nootropic effects.

471 REGULATION OF CYTOKINE SIGNALING AND AIRWAY INFLAMMATION BY ACTIVATION OF G-PROTEIN SIGNALING (AGS) 3 AND G-PROTEIN REGULATORY MOTIF PEPTIDES Song K., Cha H., Ock M. Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections are critical consequences for mortality and morbidity in human beings. Many of these diseases are strongly involved to mucus overproduction and mucous hypersecretion. Mucus overproduction and airway obstruction are common features in airway mucosal inflammation. The mechanism by which SDF1a induces MUC1 overproduction, however, has not been fully explored. The aims of this study were two-fold; firstly, to examine the Activator of G-protein signaling (AGS) 3-dependent mechanism by which SDF1a reduces MUC1 gene expression and airway inflammation, and secondly, to identify specific molecules which could suppress SDF1ainduced airway inflammation at a G-protein coupled receptor level. Here we suggest that SDF1a induces MUC1 gene expression via CXCR4 receptor. Interestingly, SDF1a signaling induced protein-pro-


144 tein interaction between MUC1 and CXCR4 to regulate physiological phenomena. In addition, we showed that AGS3 plays as a suppressor for SDF1a-induced MUC1 and TNFa gene expressions by regulating with Gai, whereas it could not control MUC1-mediated IL-6 gene expression. More interestingly, G-protein Regulatory (GPR) motif in AGS3 bound to Gai and decreased MUC1 gene expression, whereas increased TNFa gene expression. In addition, GPR mutation (DDQR?DDAR) increased MUC1 and TGFb gene expression, but decreased TNFa and IL-6 gene expressions. In addition, mutant GPR peptide inhibited CXCR4-induced dendritic extension compared with consensus GPR peptide on 2D and 3D matrix culture. Mutant GPR peptide inhibited also significant morphologic changes and inflammatory cell infiltration after SDF1a exposure in mouse lungs. In addition, synthezied mutant GPR peptide also inhibited inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and lungs. These results suggest that GPR motif may be essential for regulating MUC1/Muc1 and cytokines in inflammatory microenvironment and mutant GPR peptide play as suppressive compound to decrease respiratory inflammation in the airway.

clophosphan, prednisolone, haloperidol, phenazepam, diclofenac etc.) decrease the toxicity of this drugs and tend to enhance, in a number of instances, its pharmacological efficacy. Further studies of RA dilutions of different antibodies showed that they exhibit consistent ‘pro-antigen-like’ (co-antigenic) effect: modify antigen activity without inhibiting it. A number of inherent characteristics have been established for such agents that enable the integration of the latter to modern pharmacology: specificity, absence of tolerance to RA drugs, their safety and, finally, high efficacy. So, it has repeatedly been shown (experimentally and clinically) that RA drugs are comparable in their efficacy to wellrecognized agents (e.g. insulin, rosiglitazone, glibenclamide, interferons, oseltamivir, diazepam), proving acceptable for treating serious diseases such as influenza, chronic heart failure, type 1 and 2 diabetes, prostatic hyperplasia etc. Conclusion: The new knowledge gained as a result of years-long investigation with the use of state-of-the-art means and methods established basis for utilizing Hahnemann’s technique of consecutive concentration decrease in terms of designing pathogenetic therapeutics. It seems most probable that the modifying effect of RA dilutions, as demonstrated in our studies, will find application not only in medical practice, but also in the field of technology.

472 REGULATION OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION BY DECOY RECEPTOR 3 Hsieh S. Most of cancer patients are immunocompromised and are susceptible to opportunistic infection, thus we are interested to understand whether DcR3 modulate host immune response to Candida albicans, which is one of the most common pathogens responsible for opportunistic infection in cancer patients. We found that DcR3 can suppress PMA-mediated activation of polymorphonuclear neutrophils (PMN) and inhibit the formation of neutrophil extracellular traps (NET), which can kill C. albicans and limit its spreading. We demonstrated that both recombinant DcR3.Fc has potent suppressive effect to inhibit NET formation when neutrophils were exposed to PMA, C. albicans, Gram-positive and Gram-negative bacteria. DcR3-mediated suppression is apparently via interacting with HSPG as DcR3_HBD.Fc, which comprises the HSPG-binding domain (HBD) of DcR3 and human IgG1 Fc portion, has the same effect as DcR3.Fc. Moreover, DcR3-mediated NET suppression is rescued by HDAC (histone deacetylase) inhibitor, suggesting DcR3-mediated inhibition of NET formation is via epigenetic regulation. Thus, DcR3 not only enhances tumor progression via modulating TAM differentiation, but also contributes to increased susceptibility to opportunistic infection in cancer patients.

473 REPEATED SERIAL CONCENTRATION REDUCTION: DISREGARDED TECHNOLOGY Epstein O., Tarasov S., Dugina J. Introduction: The procedure of repeated consecutive reduction of the initial substance (IS) concentration was first utilized in the XVIII century for the preparation of homeopathic remedies. Our numerous studies allowed us to arrive at a paradoxical conclusion that ultra-high (extreme) dilutions acquire new properties which we named ‘releaseactivity’ (RA). The distinctive feature of ‘release-active’ dilutions is their capability to modify the properties or effects of the initial substance or its biological targets. Methods: Review of results provided by experimental and clinical studies conducted since 1995 at numerous research sites of Russia, the USA, Europe and Asia to investigate the efficacy and safety of RA dilutions in the treatment of common diseases (infections of viral and bacterial etiology, diabetes mellitus, mental conditions, urogenital disorders etc.), as well as the mechanism of action of this class of drugs. Results: It was demonstrated that when administered concomitantly with the initial substance, RA dilutions of various drugs (aspirin, cy-

474 RGS2 PROTEIN DEGRADATION IS MEDIATED BY A NOVEL CULLIN 4B/F-BOX 44 E3 LIGASE COMPLEX Sjogren B., Swaney S., Neubig R. Background: Hypertension and heart failure are major health issues and there is a need to identify more effective treatments and novel drug targets. Regulator of G protein Signaling 2 (RGS2) is highly expressed in both heart and vascular smooth muscle and regulates Gaq signaling. RGS2-/- mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. Overexpression of RGS2 reduces cardiac hypertrophy and we have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling. Therefore, we hypothesize that enhancing RGS2 protein expression is a novel route in treating cardiovascular disease. RGS2 is rapidly degraded through the proteasome, but the enzymes involved are not known. Thus, the goal of the current study was to identify the molecular machinery responsible for RGS2 protein degradation. Methods: We developed a cell-based b-galactosidase complementation assay that efficiently quantifies levels of RGS2 protein in a 384well plate format. Using this assay we performed an siRNA screen against the human Dharmacon siGENOME SMART-POOL siRNA library to identify genes that regulate RGS2 protein levels. The hits in the screen were subjected to bioinformatics analysis with genes involved in protein degradation being the primary focus. Follow-up studies included co-immunoprecipitation and Western blot, with overexpression or siRNA knockdown of identified genes. Results: In our screen we identified components of a novel putative cullin-RING E3 ligase (CRL) responsible for RGS2 protein degradation. siRNA knock-down of the substrate-recognizing component Fbox 44 resulted in a significant increase in RGS2 protein levels and stability. Knock-down of cullin 4B (CUL4B), but not CUL4A or CUL1, had similar effects. Furthermore, over-expression of F-box 44 or CUL4B reduced RGS2 protein levels. Also, F-box 44 co-immunoprecipitated with both RGS2 and CUL4B, suggesting that these proteins exist in a complex together. Conclusion: RGS proteins are difficult drug targets due to their mode of action being through protein-protein interactions and enhancing function is particularly challenging. Inhibiting a more druggable protein within the degradation pathway would be a way to increase RGS2 protein levels and function. Our studies have identified several potential candidates that could be targets for inhibitor drug development.


145 475 ROLE OF ANDROGRAPHOLIDE IN ANALGESIC AND ANTIINFLAMMATORY ACTIVITY OF ANDROGRAPHIS PANICULATA: AN EXPERIMENTAL STUDY IN DIABETIC RODENTS Kumar V., Thakur A. Andrographis paniculata (Burm. F.) Wall. Ex Nees (Family: Anthaceae) is a traditionally known Ayurvedic medicinal plant and Andrographolide is quantitatively the major bioactive secondary metabolite of the plant identified to date. Although during more recent years many reports on anti-hyperglycemic and anti-inflammatory activity of this plant have appeared, as yet little attention has been paid to their therapeutic potentials for combating hyperalgesia and exaggerated inflammatory responses accompanying diabetic conditions. Therefore, an analytically characterized extract of Andrographis paniculata leaves (AP; Andrographolide >30% w/w by HPLC) and Andrographolide (99% pure by HPLC) were evaluated for their analgesic and antiinflammatory activity in Type-2 diabetic rodents. For such purposes, AP (100, 200 and 400 mg/kg/day) and Andrographolide (30, 60 and 120 mg/kg/day) were administered orally for ten consecutive days. Pentazocine and indomethacin were used as standard analgesic and anti-inflammatory drugs, respectively. As compared to non-diabetic animals, diabetic animals demonstrated significant abnormal pain-associated behaviors, measured as hyperalgesia to painful stimuli in tail flick test, hot plate test and formalin-evoked pain test, and exaggerated inflammatory responses in carragennan-induced paw edema and cotton pellet induced granuloma tests. AP and Andrographolide treatments in diabetic animals demonstrated significant analgesic and anti-inflammatory activity in dose dependent manner in all these tests, and their maximal efficacies were always comparable to those of the standard drugs used. However, unlike pentazocine or indomethacin, the body weight loss and hyperglycemia in diabetic rats were compensated, or partially reversed, in AP and andrographolide treated diabetic rats. Taken together, these observations not only reconfirm that Andrographolide is the major active constituent of Andrographis paniculata, but also strongly suggest that anti-inflammatory and analgesic efficacies of AP are entirely due to the presence of high contents of andrographolide present in it, and that andrographolide could as well be a structurally novel drug lead for prevention and cure of diabetes associated enhanced inflammatory responses.

476 SAFETY AND EFFECTS OF AQUEOUS ROOT BARK EXTRACT OF CITROPSIS ARTICULATA ‘OMUBORO’ ON SEXUAL FUNCTION IN MALE RATS Oloro J., Agaba A.G., Alele P.E., Amanya M., K Tanayen J., C Ezeonwumelu J.O. About 80% of the world’s population use herbal medicine for the treatment of various health conditions. Erectile dysfunction being one condition commonly treated using traditional herbs on large scale because the conventional medicines are very expensive for most people to afford. In this study we set out to determine the safety and effects of the aqueous root bark extract of Citropsis articulata on sexual function in male rats. Objectives: (i) To identify the secondary metabolites (phytochemicals) present in the aqueous root bark extract of Citropsis articulata; (ii) conduct acute toxicity test to determine the safety of the aqueous root bark extract from Citropsis articulata; (iii) determine the effect of the aqueous root bark extract of Citropsis articulata on improving the erectile function of male rats; and (iv) evaluate the effect of the aqueous root bark extract of Citropsis articulata on testosterone levels in male rats.

Methods: Extraction was done by warm maceration, phytochemical analysis conducted using chemicals of analytical grades; acute toxicity conducted following Lorke, 1983 method. Efficacy was evaluated using non-contact and contact models and testosterone analysis was performed using the AXSYM testosterone reagent by Abbott AXSYM system. Results: Phytochemical screening revealed the presence of saponins, proteins, free amino acids, arginine, terpenoids, phenolic compounds, tannins and fats andoils. The LD50 was estimated at 9486.833 mg/kg body weight. The extracts did not induce erection, had a significant effect on mounting (P-value = 0.013) and a significant effect on testosterone level (p-value = 0.02). Conclusion: Aqueous root bark extract of Citropsis articulata significantly increased mounting frequency and testosterone levels in male rats, was slightly toxic, contained a phytochemical, known to have effects on erection and also other phytochemicals which may explain the results we found.

477 SAFETY EVALUATIONS OF DEFATTED ETHANOLIC EXTARACT OF MORINGA OLEIFERA SEED IN ALBINO RATS Bello M. Moringa oleifera seed has wide acceptance and is consumed as herbal remedy for various diseased conditions. The study was designed to evaluate the sub-chronic toxicity of defatted ethanolic extract of Moringa oleifera seed (DESMOL) in albino rats. At 400, 800, 1600 mg/ kg of the extract, there was no significant changes in some biochemical parameters like the packed cell volume, (PCV), Red Blood cell (RBC), haemoglobin percentage (HB), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC) but significant changes in platelet levels, total and differential white blood cell (WBC).The extract, at 800 and 1600 mg/kg also caused significant changes in the levels of total protein, total and conjugated bilirubin, plasma alanine transferase (ALT). Aspartate transaminase (AST), Alkaline Phosphatase (ALP) and Gamma glutamyl transferase (GGT). The results of the histo-pathology showed that the extract at doses above 400 mg/kg caused kidney damage by inducing diffused and widespread cortical necrosis. The extract also caused 40% and 60% mortality at doses of 800 and 1600 mg/kg respectively.

478 SALICYLANILIDES - ACTIVE MOLECULES AGAINST MYCOBACTERIUM TUBERCULOSIS AND MDR-TB Vinsova J., Kratky M., Stolarikova J. Tuberculosis (TB) is an infection disease affecting more than eight million peopleworldwide, 1.3 million died from the disease in 2012(1). The rise of MDR-TB and XDR-TB cases put TB in a worrying status.The situation becomes even worse with the spread of HIV. A growing problem of the lethal combination of drug-resistant tuberculosis and HIV infection presents serious challenges, which stimulate searching of effective TB treatment and development of new drugs. We have prepared several series of salicylanilide derivatives substituted on salicylic part, having free or O- protected phenolic hydroxyl group and substituted aniline part (see general formula,Fig 1). To increase lipophilicity for better passing through the lipophilic mycobacterial cell wall, we designed and synthesizedesters with carboxylic acids, amino acids, benzene sulfonic acid, phosphorus-based acids and others, as well carbamatesand thiocarbamates(2).


146 Conclusion: These findings support the use of these products as treatments for diabetes mellitus and provide basis for more in-depth evaluation that will lead to their development for the prevention and treatment of diabetes mellitus. 480 SHIFT FROM THE ONE-DRUG-ONE-TARGET TO MULTI -DRUG TARGET PARADIGM: WHO WAS THERE FIRST? Ogbuehi I., Ebong P., Ogbuehi H.

All derivatives were evaluated for their in vitro antimycobacterial activity against Mycobacteriumtuberculosis 331/88 (H37Rv),M.avium 330/88 and two strains of M. kansasii: 235/80 and a clinical isolate, 6509/96. Those having MIC ≤1 lM were tested against five clinically isolated MDR-TB strainswith different resistance patternsand one XDR strain.The lowest reached MIC for drug-sensitive M. tuberculosis was 0.125 lM and for MDR-TB also 0.125 lM. The atypical bacteria were inhibited at the concentrations ≥0.25 lM.The most active structures will be presented. 1. Global tuberculosis report 2013. http://www.who.int/tb/publications/ global_report/en/ 2. http://portal.faf.cuni.cz/Groups/Development-new-drugs-antimicrobial-activity 3. Kratk y M., Vinsova J., Novotna E., Stolarıkova J. Salicylanilidepyrazinoates inhibit in vitro multidrug-resistant Mycobacterium tuberculosis strains, atypical mycobacteria and isocitratelyase. European J. Pharm. Sci. 2014, 53, 1–9. 4. Ferriz J.M., Vavrova K., Kunc F., Imramovsky A., Stolarıkova J., Vavrıkova E.,Vinsova J.Salicylanilidecarbamates: Antitubercular agents active against multidrug-resistant Mycobacterium tuberculosis strains. Bioorg. Med. Chem. 2010, 18, 1054–1061.

479 SCREENING OF SELECTED NIGERIAN MEDICINAL PLANT EXTRACTS FOR THEIR ANTIDIABETIC POTENTIAL IN VITRO AND IN VIVO Shittu H., Musallam L., Okhale S., Tijani A., Dzarma S., Tsalha S., Haddad P. Background: Diabetes mellitus is reaching epidemic status, especially in third world countries where a significant proportion of diabetics are asymptomatic and undiagnosed. Nigeria is one of 25 countries with the largest number of people with diabetes mellitus majority of whom use plant extracts because they can neither afford nor access appropriate therapies. Therefore, our overall aim was to develop phytomedicines from Nigerian medicinal plants traditionally used for the treatment of diabetes mellitus. Methods: (i) An ethnobotanical survey of medicinal plant recipes used by herbalists in the management of diabetes mellitus in the 6 geopolitical zones of Nigeria was done; (ii) Assessment of efficacy of the collected plants in vitro (glucose uptake in C2C12 muscle cells and inhibition of glucose production in H4IIE liver cells) and in vivo (OGTT after a 2 g/kg glucose load in Swiss albino mice) was done. Results: Seventy-five traditional healers were contacted resulting in the collection of 90 plant extracts and recipes (the dry plant materials). 18 recipes (maximum two plants) were extracted in water according to traditional usage. Of the 18 plant recipes, NC01 and NCO9E extracts significantly stimulated glucose uptake (43%) in C2C12 cells. Insulin-induced glucose uptake in the same cells was 33%. These two extracts also significantly prevented hyperglycaemia after glucose load in normal mice as efficiently as metformin. Only NC01 significantly inhibited glucose-6-phosphatase (G6Pase) activity in H4IIE cells.

The idea of synergy is employed liberally by herbalists as seen in their use of two or more different herbs to produce a medicine. Even when a single medicinal plant is prescribed, it is in the belief that it contains an orchestra of compounds, all acting in concert towards maintaining health and providing relief from disease. The therapeutic effect of the whole plant tends to be significantly more effective than the particular action of its known constituents. Little wonder, then that herbalists usually mix herbs to provide a multi -drug target and take further advantage of the synergistic curative potential of their plant medicines. Most pharmacological drug development research is geared towards identifying and validating a single compound known as the active principle. However, there may be lessons to be learned from traditional medicine as practiced by herbalists. And already, we have been copying their method, when dealing with life threatening diseases or in the face of rapidly evolving disease causing organisms e.g. malaria, HIV, AIDS, Tuberculosis, methicillin resistant Staphylococcus aureus, diabetes, hypertension etc. Pharmacologists now employ multi-drug regimens or combination therapy in the face of drug resistant organisms and to provide the best therapeutic option. The modern approach of combination therapy is a re-enactment of what was and still practiced in herbal medicine. A recent ethnobotanical study in south-east, Nigeria showed that more combination than single herbs is used in the treatment of malaria, traditionally. A pharmacokinetic study into one of the combination supports the principle of potentiation, whereby one is the principal herb while the other is an adjunct herb. The role of the adjunct herb is to potentiate the effect of the main herb. Another concoction, when scientifically evaluated was found to be derived from three plants, all had antimalarial properties of varying degrees but in addition contains either antipyretic, antioxidant or analgesic property. Can the inherent practice of polypharmacy in traditional medicine provide new clues to identifying potential effective combinations and multi-target interaction in conventional medicine?

481 SINGLE-DOSE INTRAVENOUS METFORMIN TREATMENT ATTENUATES RENAL INJURY IN AN EXPERIMENTAL MODEL OF ISCHEMIA-REPERFUSION IN RATS Medic B., Jovicic D., Savic Vujovic K., Srebro D., Vujovic A., Vuckovic S., Divac N., Stojanovic R., Prostran M. Background: Recent studies have shown that metformin, an oral antidiabetic agent, also posses anti-inflammatory and antioxidant effects. The aim of our study was to determine does pretreatment with metformin (in doses of 3 or 10 mg/kg) could attenuate acute kidney failure caused by renal ischemia-reperfusion (I/R) injury in anaesthetized rats. Materials and methods: Male adult Wistar rats (n=57, b.w. 250– 300 g) are anesthezied with intraperitoneal bolus injection of sodium thiopentone (120 mg/kg) and placed on their backs on thermostatically controlled heating mat to provide constant body temperature 37.5 1C. Trachea, carotid artery, jugular vein and urinary bladder are separated from surrounding tissue and inserted with appropriate tubes or cannulas. Rats were randomized into six experimental groups (N=6–10 per group) - I: Sham + saline, II: Sham + metformin 3 mg/kg, III: Sham + metformin 10 mg/kg, IV: I/R + metformin 3 mg/kg, 30 min before ischemia V: I/R + metformin 10 mg/kg, 30 min before ischemia VI : I/R + saline. Animals randomized into I/R injury groups were


147 subjected to bilateral clamping of renal pedicles for 45 min followed by reperfusion for 4 h. In addition, cardiovascular parameters are monitored continuosly, and anesthesia is maintained by supplementary injections of thiopentone sodium (10 mg/kg, i.v.), as required. Both mean arterial pressure and heart rate were monitored continuously. Selected parameters of glomerular and tubular function, as well as histological score, were obtained from the appropriate serum, urine or tissue samples at the end of reperfusion period. Results: Acute pretreatment with metformin (in doses of 3 and 10 mg/kg) significantly attenuated I/R-induced increase in specific parameters of glomerular function (serum urea and creatinine concentrations) and tubular function (fractional excretion of Na+). Also, metformin significantly reduced the total and tubular necrosis histological score. Conclusion: Our study shows that a single dose of metformin could afford significant protection of the injured rat kidney. Its use in the future should be considered to reduce in prevention of development of acute kidney failure in patients undergoing major surgical procedures or a kidney transplantation surgery.

482 STABILITY STUDY ON THE LEVAMLODIPINE TABLETS OF VARIOUS SALT FORMING MODE

biodiesel) has not been as investigated. The objective here was to compare JCSO with five commonly-used vegetable oils. Methods: JCSO was obtained by exhaustively defatting pulverised dried J. curcas seeds with petroleum ether (32% yield); while corn oil, groundnut oil, olive oil, palm oil and soy bean oil were purchased from groceries. Physicochemical parameters and vitamins levels were determined using IUPAC and UV-Visible spectrophotometric methods respectively. Data analysis was by ANOVA plus Dunnett’s posthoc, as well as correlation statistics. Results: Means for JCSO (followed by ranges for the other five oils) are presented. Refractive index and specific gravity were 1.467 (1.463–1.472) and 0.906 (0.910–0.913). The following data were for acid value: 2.666 (0.695–2.950) mg KOH/g, iodine value: 90.37 (82.10–142.10) g I2 /100 g, peroxide value: 2.85 (1.89–6.63) meq/kg and saponification value: 191.96 (174.08–285.36) mg KOH/g. Vitamins A, C and E contents in mg/L, were 15.909 (7.312–15.909), 0.120 (0.137–0.458) and 15.425 (9.367–17.937) respectively while the corresponding levels of vitamins B3, B9 and B12 were 2.584 (1.680– 2.971), 0.113 (0.017–0.304) and 8.164 (3.488–19.665). Overall, JCSO compared favourably with the other oils and for the parameters studied, correlating best with soy bean oil, and least with groundnut oil. Conclusions: Based on its similarity to these oils, JCSO which (unlike the seed extract), had been shown to be practically non-toxic in our previous study, may find application in the pharmaceutical, cosmetic and food industries.

Yang Y., Fan G. Objective: To establish a high performance liquid chromatography content determination method of levamlodipine in levamlodipine besylate tablets and levamlodipine maleic acid tablets. To carry out stability inspection of Shihuida and Xuanning with this method. Method: Chromatographic conditions of Levamlodipine Besylate is Elite Hpersil ODS2 chromatography column; mobile phase is methanol 0.03 M potassium dihydrogen phosphate solution(75:25); detection wavelength is at 238 nm; and the flow rate is 1 mL/min. Method: chromatographic conditions of Levamlodipine Maleic Acid is Shim-Pack CLC-ODS chromatography column; mobile phase is methanol 0.03 M potassium dihydrogen phosphate solution(72:28); detection wavelength is at 238 nm; and the flow rate is 1 mL/min. Stability study adopts accelerated test and strong light exposure test methods. Results: The line relation of Levamlodipine Besylate sampling concentration within 10~80 lg/ml with peak area is good (r = 0.9999), the average application of sample recovery of Levamlodipine in Levamlodipine Besylate is 99.24%, RSD = 0.38%. Results: the line relation of Levamlodipine Maleic Acid sampling concentration within 10– 80 lg/ml with peak area is good (r = 0.9999), the average application of sample recovery of Levamlodipine in Levamlodipine Maleic Acid is 99.22%, RSD = 0.35%. After 6-month accelerated test and strong light exposure test, there is no significant change for Levamlodipine content in Levamlodipine Besylate, although Levamlodipine content in Levamlodipine Maleic Acid presents no obvious change likewise after accelerated test, strong light exposure test has greater effect on its stability. Conclusion: The drug stability test shows that Levamlodipine Besylate is more stable than Levamlodipine Maleic Acid, which may be related to their salt forming modes.

484 SUPPRESSION OF NFKB SIGNALLING PATHWAY BY CATECHIN PREVENTS POST STROKE NEUROLOGICAL COMPLICATIONS IN RATS

483 STUDIES ON JATROPHA CURCAS LINN. (EUPHORBIACEAE) SEED OIL: II - SOME PHYSICOCHEMICAL PROPERTIES AND VITAMINS LEVELS COMPARED WITH THOSE OF COMMON VEGETABLE OILS

485 SURAMIN PROMOTES RECOVERY OF RENAL FUNCTION FOLLOWING I/R-INDUCED INJURY IN MICE AND RHABDOMYOLYSIS-INDUCED INJURY IN RATS

Bhandari R., Kuhad A., Kaur I., Chopra K. NFkB signaling pathway plays major role in the development of poststroke neurological complications. The aim of the present study was to explore the effect of catechin on neurological scoring, memory, mechanical hyperalgesia, allodynia, depression, oxidative-nitrosative stress inflammation and NFkB in collagenase-induced hemorrhagic stroke. Catechin possesses strong neuroprotective potential but limited penetration in brain. Therefore, we have also used catechin loaded in solid lipid nanoparticles, a technology to enhance brain delivery, and compared the neurological effects with plain catechin. Chronic treatment with plain catechin and catechin loaded in solid lipid nanoparticles for 4 weeks starting from 2nd day after collagenase injection significantly attenuated neurological, behavioral, biochemical and molecular changes associated with hemorrhagic stroke. Catechin loaded in solid lipid nanoparticles produced a pronounced effect as compared to plain catechin. The major finding of the study is that catechin alone ameliorated the post stroke neurological complications but only when it is delivered across the blood brain barrier. Moreover, catechin not only attenuated the neurological deficits associated with stroke but also reversed post stroke neurological complications through modulation of NFkB signaling pathway in the collagenased rats and thus catechin may find clinical application to treat neurological complications in the stroke patients.

Schnellmann R.

Kwanashie H., Ejiofor J., Inekwe V. Objective: Whereas Jatropha curcas leaves, seeds and their extracts have been relatively well investigated for medicinal and nutritional properties, J. curcas seed oil (JCSO) (prized primarily as a source of

Acute kidney injury (AKI) is a common and potentially life-threatening complication after ischemia/reperfusion (I/R) and exposure to nephrotoxicants. At this time there are no drug therapies to treat AKI. In this study, we examined the efficacy and mechanism(s) of suramin, a treatment for trypanosomiasis, in promoting recovery from I/R-


148 induced AKI in mice and rhabdomyolysis-induced AKI in rats. Following I/R in mice, serum creatinine was maximal 24 h later and decreased to control levels over 6 days. Increasing doses of suramin (0.1–10 mg/kg) administered 24 h after I/R, decreased serum creatinine levels to controls levels and diminished histopathologic tubular damage. Suramin also decreased renal cortical apoptosis and leukocyte infiltration, and increased proliferating tubular cells after I/R. After intramuscular administration of glycerol, to induce rhabdomyolysis-AKI in rats, serum creatinine was maximal betwen 24 and 72 h and then decreased over days. Suramin (1 mg/kg) administered 24 h after glycerol accelerated recovery of renal function by decreasing serum creatinine, NGAL, kidney injury molecule-1, apoptosis and histopathologic tubular damage. Mechanistically, suramin decreased early glycerol-induced proinflammatory (IL-1beta, NF-kB) and growth inhibitory (TGF-beta1) mediators, resulting in the prevention of late downstream inflammatory effects (ICAM-1 activation, leukocyte infiltration). Finally, suramin stimulated proximal tubule cell proliferation. These results in two different models of AKI and in two different species strongly support the potential of suramin as a treatment for AKI in humans. Suramin efficacy observed 24 h after the injurious insult is important for clinical use. Finally, the multiple positive actions of suramin, decreased apoptosis and leukocyte infiltration, and increase renal proximal tubule cell proliferation stimulates the recovery of renal function.

486 SURFACEN: INHIBITION BY MECONIUM, SERUM AND CHOLESTEROL AND REVERSION BY HYALURONIC ACID Blanco O., Lugones Y., Lopez-Rodriguez E., Cruz A., Echaide M., Perez-Gil J. Background: Recently Lopez-Rodriguez et al (2013) has optimized a protocol to assess lung surfactant inhibition by serum, cholesterol or meconium in the captive bubble surfactometer (CBS). These results were obtaining with native lung surfactant. There are some articles what showed the differences between native and clinical lung surfactant in relation with inhibition. Methods: In the present work, we have evaluated the inhibition of Surfacen, a clinical lung surfactant, by meconium and serum and its reversion by hyaluronic acid using a new methodology proposed in captive bubble surfactometer, also we studied the effect of medium (aqueous or organic) in the percentage the incorporation of cholesterol into membranes of Surfacen, as well as, its biophysical behavior. Results: Exposure of Surfacen to serum or meconium affects the biophysical behavior. Addition of hyaluronic acid to Surfacen fully reverses inhibition by serum or meconium. Exposure of membranes of Surfacen to cholesterol in organic medium is completely different to medium aqueous in relation to percentage de incorporation and therefore biophysical behavior. Conclusions: These results suggest that improved formulations of lung surfactants are possible and may be useful in reducing some types of surfactant inactivation in treating lung injuries.

487 SYNERGISM AND RULES OF A NEW COMBINATION DRUG YIQIJIEDU FORMULAE ON ISCHEMIC STROKE BASED ON AMINO ACIDS METABOLISM

(AAs) were chosen as the potential biomarkers and with partial least squares discriminant analysis (PLS-DA) analysis, the effect of a new combination drug, Yiqijiedu formulae (YQJD) composed of ginsenosides (A), berberine (B), and jasminoidin (C), on those 7 AAs were evaluated. Four amino acids, glutamic acid (Glu), homocysteine (Hcy), methionine (Met), and tryptophan (Trp), which changed significantly in YQJD-treated groups compared to vehicle groups (P < 0.05 or P < 0.01), were identified as the designated AAs to further explore the synergism of YQJD. The result of principal component analysis (PCA) showed the combination of these three could exhibit the strongest synergistic effect compared to other combination groups and ginsenosides might play a pivotal role, especially when combined with jasminoidin. We made a successful attempt to explore the synergetic mechanism of combination drug and provided a new mode to evaluate the integrated effects in the treatment of complex diseases. 488 SYNERGISTIC COMBINATIONS BETWEEN OESTRONE ANALOGUES AND GLYCOLYSIS INHIBITORS LONIDAMINE AND INDINAVIR ON MDA-MB-231 BREAST ADENOCARINOMA CELLS Van Tonder A., Cromarty A., Joubert A. Introduction: Cancer is often successfully treated in the clinical setting with combinations of effective therapies. The potential synergism of combinations of two in silico designed oestrone analogues, ESE-15ol and ESE-16, with commercially available glycolysis inhibitors were investigated and promising results were obtained for combinations with lonidamine or indinavir. This study assessed mechanistic aspects of these combinations; including influence on mitochondrial membrane potential (MMP), effect on intracellular reduced glutathione (GSH) concentration and activity of caspases 3, -8 and -9, using the MDAMB-231 breast adenocarcinoma cell line. Methods: MDA-MB-231 cells were used at a cell density of 5 9 103 cells/well. To assess the effect of synergistic combinations on MMP, cells were exposed to the combinations for 6–24 h and a fluorometric method using JC-1 was employed. The effect of the combinations on intracellular GSH was measured using a 6 h kinetic monochlorobimane method. Caspases 3, -8 and -9 activity was determined using for 16–24 h treatment using three fluorescent caspase-specific substrates. Results: Preliminary results indicate that treatment with combinations with lonidamine and indinavir after 16 h incubation caused increased hyperpolarisation of the mitochondrial membrane. The greatest increase in GSH concentration was also observed for the combinations with indinavir. A slight increase in caspase 3 activity was observed for all lonidamine combinations. The combination of ESE-16 and lonidamine caused a significant increase in caspase 8 activity. None of the combinations had a marked influence on caspase 9 activity. Conclusion: Results obtained suggest that the synergistic combinations between the oestrone analogues and lonidamine are most promising: these combinations increase hyperpolarisation of the mitochondrial membrane indicating inhibition of respiration and elicit an increase in caspase 3 activity indicating induction of apoptosis. However at 6 h no stress response, as measured by GSH concentration, was observed. Combinations with indinavir also increased hyperpolarisation of the mitochondrial membrane, but no effect on caspase activity or GSH concentration was detected. Even though these results show the potential of the combinations, especially those with lonidamine, further studies will be done to establish the mechanism of action.

Li S. The application of combination drugs is considered as a promising strategy to control complex disease such as ischemic stroke. The detection of metabolites has been used as a versatile tool to reveal the potential mechanism of diverse diseases. In this study, the levels of 12 endogenous AAs were determined quantitatively at the same time in middle cerebral artery occlusion (MCAO) rat brain by a rapid resolution liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry (RRLC-QQQ) method. Seven amino acids © 2014 The Authors Basic & Clinical Pharmacology & Toxicology © 2014 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 115 (Suppl. 1), 1–374

149 489 SYNTHESIS AND ANTIARRHYTHMIC ACTIVITY OF N-[2-(1ADAMANTYLAMINO)-2-OXOETHYL]-N-(AMINOALKYL) NITROBENZOIC CARBOXAMIDES Mokrov G., Kryzhanovskii S., Likhosherstov A., Stolyaruk V., Vititnova M., Tsorin I., Gudasheva T., Seredenin S. The problem of prevention and treatment of malignant cardiac arrhythmia and reduce the risk of sudden cardiac death is one of the most actual challenges facing modern cardiovascular pharmacology. It is believed that Class III antiarrhythmics in VaughanWilliams classification [1] are the most promising drugs for this purposes. However, the most known class III antiarrhythmics have a wide range of side effects, including arrhythmogenic [2].The original class III antiarrhythmic cardiocyclide (N-[2-(dicyclohexylamino)-2-oxoethyl]-N-[3-(diethylamino) propyl]-4-nitrobenzoic carboxamide hydrocloride) has been earlier synthesized and pharmacologically studied in the Institute of Pharmacology RAMS [3]. The studying of the drug safety showed that it has the phlebotoxic action. The novel cardiocyclide analogs - N-[2-(1-adamantylamino)-2-oxoethyl]-N-(aminoalkyl)nitrobenzoic carboxamides (I) - were synthesized in this work. The relationship between their chemical structure and antiarrhythmic activity has been studied [4].

It was hypothesized that new compounds required the special pharmacophoric elements that provide activity (Ph1-Ph3). The structure of dialkylamino group (Ph3) has the significant influence on the activity. The extension of the (CH2)n chain connecting the pharmacophore centers by one methylene leads to increasing of the activity. The aminoacetic acid derivatives are more active and more toxic than the aaminopropionic acid derivatives. The position of the nitro group in the benzene ring (Ph2) has the significant influence on the antiarrhythmic activity of the compounds. The studies revealed the most active compound to be N-[2-(adamantylamino)-2-oxoethyl]-N-[3-(diethylamino) propyl]-4-nitrobenzoic carboxamide hydrocloride which is comparable in its antiarrhythmic activity with prototype cardiocyclide and in contrast with latter, does not have the phlebotoxic action. References: 1. I. Savelieva, J.Camm, J. Interv. Card. Electrophysiol., 23 (1), 7-14 (2008). 2. C.E. Pollard, N.Abi Gerges, M.H.Brindgland-Taylor, et al., Br. J. Pharmacol., 159 (1), 12-21 (2010). 3. H. Poppe, R. Schindler, W. Sauer, D. Marks, R. Bartsch, N.V. Kaverina, A.M. Lichoscherstow, S.F. Sokolov, V.V. Lyskovtsev, S.B. Seredenin, S.A. Borisenko, Arch. Pharm. Pharm. Med. Chem., 332, 233-242 (1999). 4. S.B. Seredenin, A.M. Lichoscherstow, S. A. Kryzhanovskii, G.V. Mokrov, V.N. Stolyaruk, M.B. Vititnova, I.B. Tsorin, T.A. Gudasheva, A. V. Sorokina, A.D. Durnev, Russian Federation Patent № 2500666 (2013).

491 SYNTHESIS AND BIOLOGICAL EVALUATION OF 2-HIDROXI-1,4-NAPHTHOQUINONE-BASED DERIVATIVES AS NOVEL INHIBITORS OF SIGNAL TRANSDUCTOR AND ACTIVATOR OF TRANSCRIPTION (STAT) FAMILY Martın-Rodrıguez P., Guerra B., Hueso-Falcon I., Dıaz-Chico J., Lopez-Rodrıguez M., Gutierrez-Ravelo A., Amesty A., Fernandez-Perez L. Background: Aberrant activation of JAK/STAT signaling pathway has been reported in several types of cancer. Particularly, one STAT family member, STAT5, is constitutively active in many forms of hematologic cancers. Naphthoquinone (NFQ)-based derivatives have been shown to suppress STAT signaling pathway in non-cancer, inflammatory, and cancer cells. The Ihf-c6 is a new synthetic NFQ-based derivative that was discovered in our lab by high-through cell based screening of small molecule libraries). In this study, we hypothesized that Ihf-c6 might be explored for use as inhibitor of STAT-dependent cancers. Methods: Novel structures were obtained from 2-OH-1,4-NFQ by using multicomponent reactions. All cell lines were purchased from the American Type Culture Collection (ATCC). The mitochondrial metabolization of the tetrazolium salt 3-(4,5-methyltiazol-2yl-)-2,5diphenyl-tetrazolium bromide] (MTT) (Applichen, Germany) was used as indicator of cell viability. Cytostatic and cytotoxic effects were evaluated by using the Incucyte Live-Cell Imaging System (IncuCyte HD). Nuclear/cytosolic proteins were prepared using a Nuclear Extract Kit (Active Motif, CA, USA). STAT5- or STAT3-cytokine-stimulated DNA-binding activities were measured in nuclear extract by using a TransAM STAT kit (Active Motif Inc). Results: The NFQ derivative Ihf-c6 showed antitumoral effects in vitro. Ihf-c6 was highly effective in inhibiting human promyelocytic leukemia HL60 cells (IC50=0.4 lM), human eritroleukemia HEL cells (IC50=1 lM), and human chronic myelogenous leukemia K562 cells (IC50=1.6 lM), whereas non-blood tumor cells (e.g., MCF7 and SKBR3) were higher resistant to Ihf-c6-induced cytotoxicity. The constitutive Tyr-STAT5 phosphorylation (pY-STAT5) was abolished in K562 by Ihf-c6 (maximum inhibition at 3 lM 9 6 h). Ihf-c6 inhibited GH-induced pY-STAT5 (IC50=0.7 lM) and IL-6-induced pY-STAT3 (IC50=0.5 lM) in adenocarcinoma breast cells (T47D) in a time(maximal at 30 min) and dose-dependent manner. Finally, Ihf-c6 inhibited cytokine-induced DNA-binding of STAT5 and STAT3. Conclusion: Ihf-c6 is an effective inhibitor of constitutive (oncogenic) as well as cytokine-induced STAT5/3 phosphorylation. These findings suggest that NFQ-based derivatives might be potential therapeutic agents for leukemia and related hematologic malignancies.

492 SYNTHESIS, ANTINOCICEPTIVE AND SEDATIVE EFFECTS OF DICHLOROSUBSTITUTED PHENYL PROPANAMIDES IN MICE Malami S., Yaro A., Idris A. Background: Drug development has become a global activity; aimed at providing information on the optimal use of a new drug in the treatment or prevention of diseases. Efficacy and safety are the main criteria of interest in drug development both from natural and synthetic origin. Derivatives of 2,3- and 2,5- dichloro phenyl propanamides were synthesized by uncatalysed nucleophilic reaction using their respective aniline derivatives and acrylamide as reactants. The aim of this study was to evaluate antinociceptive and central depressant effects of these compounds in mice. Methods: The compounds were synthesized in a single step reaction according to Michael’s reaction. The Pharmacological screening was conducted using some animal models: acetic acid-induced writhes for peripheral pain activity; diazepam-induced sleep and hole-board for exploratory behavior to study their possible sedative effects. All the experiments were conducted using three graded doses (50, 25 and


150 12.5 mg/kg) of each compound and drugs were administered through intraperitoneal route (i.p.). Results: The compounds produced a significant reduction in the number of abdominal constrictions at the highest and intermediate doses (P < 0.05); suggesting antinociceptive activity. The 2,3- substituted derivative showed a significant decrease in both the onset and duration of sleep at the highest dose (P < 0.05). Where as, the 2,5 derivative showed only significant activity at the onset of sleep but did not produce significant activity in the duration of sleep. Also, the number of head dips for the exploratory experiment for both compounds (50 mg/ kg only) have significantly (P < 0.05) decreased. Conclusion: Therefore, these derivatives could be said to have pharmacophoric moiety capable of reducing pain and could be exploited as antinociceptives with minimal sedative effect.

493 TAMOXIFEN AMELIORATES SYMPTOMS OF MUSCULAR DYSTROPHY IN MDX MICE Ruegg U., Reutenauer-Patte J., Ismael H., Decosterd L., Dahmane E., Dorchies O. Background: s part of our research focusing on the identification of substances for the symptomatic therapy of Duchenne muscular dystrophy (DMD), we have investigated tamoxifen (TAM), a drug used since more than 20 years to treat estrogen-dependent breast cancer, on dystrophic mice. Methods &Results: TAM caused remarkable improvements of muscle force and of the structures of diaphragm and heart in the mdx5Cv mouse model of DMD. Oral tamoxifen treatment from 3 weeks of age for 15 months at a dose of 10 mg/kg/day stabilized myofiber membranes, normalized whole body force, and increased force production and resistance to repeated contractions of the triceps muscle above those of normal mice. TAM improved the structure of leg muscles and diminished cardiac fibrosis by ~50%. TAM also reduced fibrosis in the diaphragm, while increasing its thickness, myofiber count and myofiber diameter, thereby augmenting by 72% the amount of contractile tissue available for respiratory function. TAM conferred a markedly slower phenotype to the muscles. TAM and its metabolites were present in nanomolar concentrations in plasma and muscles, suggesting signaling through high affinity targets. Interestingly, the estrogen receptors ERa and ERb were several times more abundant in dystrophic compared to normal muscles, and TAM normalized the relative abundance of ERb isoforms. In addition, TAM was very potent: Even with doses as low as 0.3 mg/kg/day, the full effects on muscle were observed. Conclusions: Because of these encouraging findings, the well-known profile of activity of TAM, its high specificity and potency, and ready availability, we suggest carrying out a clinical trial with TAM in DMD boys.

494 TD-7442 IS AN ORALLY ACTIVE, POTENT AND PERIPHERALLY-SELECTIVE OPIOID RECEPTOR ANTAGONIST Armstrong S., Campbell C., Richardson C., Vickery R., Tsuruda P., Long D., Beattie D., Hegde S.

of TD-7442, a novel, orally-bioavailable, peripherally-selective, opioid receptor antagonist with potential for the treatment of OIC. Methods: In vitro opioid receptor affinity was assessed using radioligand binding assays with membranes prepared from Chinese hamster ovary cells stably-transfected with human m- and d-, or guinea pig kopioid receptor cDNA. In addition, opioid receptor antagonist potency was evaluated using electrically stimulated guinea-pig ileum and hamster vas deferens preparations mounted in tissue baths. The in vivo peripheral GI potency of test agents to inhibit loperamide-induced delays in gastric emptying of a charcoal meal, and attenuation of castor oil-induced diarrhea in rats was determined. To assess in vivo CNS potency, inhibition of morphine-induced antinociceptive activity was evaluated using the hot-plate and tail flick tests in rats. Results: TD-7442 had high affinity for opioid receptors (l pKi = 9.7, d pKi = 9.3, k pKi = 8.9) and exhibited potent antagonist activity (l pA2 = 10.7, d pKb = 9.4, k pKb = 9.5) in the in vitro tissue preparations. In vivo, TD-7442 exhibited potent oral activity reversing loperamide-induced delay in gastric emptying (ED50 = 0.09 mg/kg) and attenuating castor oil-induced diarrhea (ED50 = 0.03 mg/kg) in the rat. With respect to CNS activity, TD-7442 failed to inhibit morphineinduced antinociception up to the highest dose tested (30 mg/kg). Conclusions: The in vitro and in vivo data indicate that TD-7442 is a potent opioid receptor antagonist with peripheral GI activity following oral administration. The lack of CNS activity, at much higher doses, supports the utility of this compound as a potential treatment for OIC patients.

495 THE ANTI-CANCER AGENT GUTTIFERONE-A PERMEABILIZES MITOCHONDRIAL MEMBRANE: ENSUING ENERGETIC AND OXIDATIVE STRESS IMPLICATIONS Pardo Andreu G., Nu~nez Figueredo Y., Cuesta Rubio O., Delgado Hernandez R., Curti C., Alberici L. Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1–25 lM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca2+ efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: (i) GA interaction with mitochondrial membrane promoting its permeabilization; (ii) mitochondrial membrane potential dissipation; (iii) NAD (P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; (iv) ROS accumulation inside mitochondria and cells; (v) additional mitochondrial membrane permeabilization due to ROS; and (vi) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds.

Background: Opioid analgesics play an important role in chronic pain management. Despite their effectiveness, these drugs commonly induce opioid-induced constipation (OIC) in patients (Walsh 1990). OIC results from opioid agonist action on enteric neurons and smooth muscle throughout the gastrointestinal (GI) tract leading to a reduction in coordinated contractions associated with transit and secretion. First-generation opioid antagonists such as naloxone and naltrexone attenuate OIC in patients but readily penetrate the CNS and interfere with pain control (Pappagallo 2001). Here we describe the in vitro and in vivo properties © 2014 The Authors Basic & Clinical Pharmacology & Toxicology © 2014 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 115 (Suppl. 1), 1–374

151 496 THE ANTI-HYPERTENSION EFFECTS OF SANCAO JIANGYA DECOCTION AND ITS INFLUENCES ON THE NO AND ET-1 LEVEL IN VIVO AND IN VITRO Wang X., Lu Y., Cheng F., Wang Q., Wang D., Zhao W. Background: In clinic, hypertension is treated very successful with the combination of traditional Chinese medicine (TCM) and Western medicine in China. Even more some TCM decoctions show stable effects of anti-hypertension by themselves and offer a better protection of the important organs such as heart, brain and kidney. Sancao jiangya decoction (SCD) is one of them. Methods : To develop it into a Chinese Herbal Drug we had extracted the components of SCD by the enrichment and purification technology of macroporous resin and four components were gotten that is SCDDW、SCD-10ET、SCD-50ET and SCD-95ET. Experiments for Pharmacology were carrying out on SHR model and HUVEC. Nifedipine were selected as the positive control. Negative control of animals and blank well were settled during experiment. The blood pressure and behavioral indicators of SHR were observed in a 4 weeks test cycle. The level of ET-1 and NO were detected both in plasma and supernatant of cell culture. Results: In this study SCD-95ET shows the best effects of anti-hypertension in SHR which was similar to positive control of Nifedipine (P > 0.05) and has significant difference with the normal control group and model group. The maximum drop of blood pressure is 35 mmHg which was appeared in 2 h after intragastric administration and this can be remained at least 2 more hours. Moreover, a stable 20 mmHg drop of blood pressure was maintained by one administration per day in longterm treatment. In SHR SCD-95ET can also extended rotation time, reduce the the number of standing, increase appetite, diuretic, relieve the state of stasis, increased pain thresholds. Except this, it can increase the NO level and decrease the ET-1 level both in SHR serum and in the supernatant of Human umbilical vein endothelial cells. Conclusions These results indicate that SCD can be a candidate of the therapeutic TCM drugs to Hypertension.

497 THE ANTIDEPRESSANT PROPERTIES OF AZURE B AND ETHYL-THIONINIUM CHLORIDE, A SYNTHETIC METHYLENE BLUE ANALOGUE Delport A., Petzer J., Petzer A., Harvey B. Background: The shortcomings of current antidepressant agents prompt the design of novel multimodal antidepressants and the identification of new antidepressant targets. Such antidepressants should possess a faster onset of action as well as an improved safety profile. Methylene blue (MB) possesses diverse pharmacological actions and is attracting increasing attention for the treatment of a variety of disorders including Alzheimer’s disease, bipolar disorder, anxiety and depression. MB acts on both monoamine oxidase (MAO) and the nitric oxide (NO) pathway, and possesses antidepressant activity in rodents. The present study investigated the antidepressant properties of a synthetic MB analogue (ethyl-thioninium chloride; ETC) as well as azure B, the major metabolite of MB, in rats using the acute forced swim test (FST). Methods: ETC was synthesized from diethyl-p-phenylenediamine with 6% yield. ETC was firstly evaluated as a potential inhibitor of recombinant human MAO-A and MAO-B. Azure B and ETC were evaluated for antidepressant-like activity over a dosage range of 4-30 mg/kg using a behavioural sampling technique, and the results compared to those obtained with saline, imipramine (15 mg/kg) and MB (15 mg/ kg) under the same treatment conditions. Locomotor activity was assessed using an automated animal activity monitor to exclude locomotor-associated false positive responses. Behaviour was analysed using a one-way ANOVA followed by Tukey’s post-test. A value of P < 0.05 was deemed statistically significant.

Results: The results document that ETC inhibits MAO-A and MAOB with IC50 values of 0.510 lM and 0.592 lM, respectively. Furthermore, ETC inhibits MAO-A and MAO-B reversibly. In the acute FST, azure B and ETC were more effective than imipramine and MB in reversing immobility without inducing locomotor effects. Importantly, azure B and ETC significantly increased swimming behaviour without affecting climbing behaviour, indicative of enhanced serotonergic neurotransmission. Conclusion: These results suggest that azure B may be a major contributor to the antidepressant effect of MB, and acts via increasing serotonergic transmission. Even though ETC is a less potent MAO-A inhibitor than MB, it still possesses antidepressant-like activity in the FST with a similar serotonergic profile. Further studies investigating possible dual actions on MAO and the NO pathway are indicated.

498 THE ANTIDIABETIC ACTIVITY OF A POLYHERBAL TEA MIXTURE AND ITS CONSTITUENTS IN VITRO Paddy V., Van Tonder J., Steenkamp V. Background: Type 2 diabetes mellitus (T2DM) is a global epidemic currently affecting an estimated 382 million individuals. T2DM is associated with debilitating pathologies and co-morbidities, mainly due to hyperglycemia-induced damage mediated by reactive oxygen species (ROS). Current treatments of T2DM are associated with unwanted side-effects and unpleasant regimen requirements. Diabetea is a polyherbal tea, commercially available to individuals suffering from T2DM. The antidiabetic efficacy of this tea has not yet been studied in terms of its antioxidant and hypoglycaemic activities. Methods: The aim of this study was to evaluate ROS activity in Ea.hy926 cells and insulin stimulated glucose uptake into C2C12 myotubes for the hot water (HW) and dichloromethane (DCM) extracts of diabetea and its seven herbal constituents: Achillea millefolium L. (Yarrow), Barosma betulina Bartl. &Weidl. (Buchu), Salvia officinale. L. (Sage), Taraxacum officinale L. (Dandelion), Thymus vulgare. L. (Thyme), Trigonella foenum-graecum L. (Fenugreek) and Urtica urens L. (Nettle). Results: None of the DCM extracts had any antioxidant activity. The HW extracts of T. vulgare, S. officinale and U. urens possessed significant antioxidant activity at all test concentrations. The HW extracts of the diabetea and T. foenum-graecum aggravated the production of ROS in Ea.hy926 cells. All HW and DCM extracts caused an increase of glucose uptake into C2C12 myotubes except for the HW extract of A. millefolium. The HW extract of S. officinale and DCM extract of T. vulgare caused a significant (P < 0.05) increase in glucose uptake compared to the activity of insulin. Conclusions: Both the HW and DCM extracts of diabetea caused a significant over production of ROS. The HW extract solutions were found to to be more active than the DCM extracts. Of all extracts tested the HW extract of S. officinale possessed the most promising antidiatebic activity and requires further investigation.

499 THE ANTIMALARIAL PROPERTIES OF NOVEL QUINAZOLINES AND THEIR METAL COMPLEXES Mahlangu T., Van Zyl R., Juneja A. Background: Malaria remains one of the most important life-threatening parasitic diseases in humans with 600,000 deaths attributed to a Plasmodium. Falciparum infection. The current antimalarial drugs have a limited range of cellular targets with a lack of structural diversity. In addition, the emergence and spread of multiple-drug resistant malaria parasites poses an imminent threat to the current therapies. Hence there is an urgent need for new antimalarial drugs to complement or replace artemisinin.Quinazoline derivativeshave been shown to be effective


152 against protozoa, with their bioactivity profile being enhanced when the ligands are chelated with metals. As such the antimalarial activity of these new electron rich donor centered quinazolines and transition metal complexes were investigated. Methods: The four 2, 3-dihydroquinazolin-4(1H)-one ligands (Q1-Q4) and their transition metal (cobalt II, nickel II and zinc II) complexes (Q5-Q16) were synthesized by a three step reaction procedure and characterized by FTIR and 1HNMR.The sixteen compounds were evaluated for antimalarial activity on a chloroquine-sensitive 3D7 strain of P. falciparum using the [3H]-hypoxanthine incorporation assay, with their haemolytic properties. Their ability to inhibit b-haematin formation was determined at an acidic pH. The anti-oxidant activity of the uncomplexed and metal complexes of select quinazolines were evaluated for their iron chelating and reductive properties. Results: The affinity of the quinazoline ligands for iron (II) was 40% that of EDTA, with the ligands possessing a greater specificity for cobalt (II), nickel (II) and zinc (II). Whilst only three quinazoline metal complexes, Zn(II)-Q3 (Q13), Co(II)-Q1 (Q5) and Zn(II)-Q1(Q7) (range: 114.33–183.50%) showed a Trolox equivalent iron reduction capacity greater than that of Trolox at 100%.The quinazolines displayed potential as antimalarial agents with no effect on the host red blood cell membrane integrity. Only two derivatives, Ni (II)-Q2 (Q9) and Co (II)-Q1 (Q5) and inhibited b-haematin formation (IC50:24.903.44 mM 49.804.45 mM, respectively) in a similar manner to chloroquine (IC50:21.382.65 mM) and quinine (IC50:49.524.53 mM). Conclusions: This study has opened a new avenue in the search of potential antimalarials where structural modifications of these quinazolines complexes may lead to more potent antimalarial agents.

500 THE ANTINOCICEPTIVE EFFECTS OF VERBASCUM EXUBERANS HUB.-MOR Eyiis E., Kaygisiz B., Yilmaz H., Ayhanci A., Kilic F. Background: The extracts, decoctions and infusions of Verbascum L. genus, commonly known as “mullein”, have been used in traditional medicines in almost all of the world for centuries. Verbascum species have immunomodulatory and antiinflammatory activities. The effects of this species are mostly attributed to their antioxidant capabilities. Moreover, it is reported that Verbascum species have antinociceptive, antitumor, anticancer, antiulcerogenic, antihepatotoxic, antihyperlipidemic, antitussive and antigerminative activities. Verbascum exuberans Hub.-Mor, which is endemic in Manisa province of Turkey, has never been studied before. Therefore we investigated the central and peripheral antinociceptive activities of Verbascum exuberans in mice. Methods: Air dried and powdered aeriel parts of Verbascum exuberans was extracted and filtered with methanol. Combined extract was evaporated under vacuum to give crude extract. The methanol extract was dissolved in distilled water and partitioned with equal volume of petroleum ether, and the water fraction was lyophilized. 21 male Swiss albino mice were divided into 3 groups: control (saline), 250 mg/kg extract and 500 mg/kg extract groups. The aqueous extract was injected intraperitoneally and tail clip, tail flick, and hot plate tests were applied to measure the central antinociceptive activity. Furthermore, the acetic acid-induced writhing test was applied for the investigation of peripheral antinociceptive activity. Maximal potent effect % (MPE %) was calculated for central antinociceptive tests. Results: The number of writhings at 250 and 500 mg/kg doses were significantly decreased compared to control (P < 0.001). The decline was higher at 500 mg/kg dose of the extract (P < 0.05). Both doses of the extract showed no significant effect on central antinociceptive tests compared to control (P > 0.05) whereas there was a significant peripheral effect observed. Conclusion: We suggest that Verbascum exuberans exerts a peripheral but not a central antinociceptive activity. In the later stages of this study, the role of nitrergic, opioidergic, and serotonergic pathways in the antinociceptive effect of the extract will be investigated. Additionally, carrageenan induced paw edema and serum levels of TNF-a and interleukine 1-b will be studied in rats.

501 THE ANTISTEOTOTIC PROPERTIES OF FOUR AFRICAN HERBAL REMEDIES IN AN IN VITRO OLEIC ACID HEPATOSTEATOSIS MODEL Cordier W., Steenkamp V. Background: Hepatosteatosis, or fatty liver, is a common form of hepatotoxicity caused by high-fat diets, drug-induced liver injury or alcoholism. Due to impairment of liver function individuals suffer from decreased quality of life and have the risk of liver failure. Herbal remedies have been reported to increase fatty acid metabolism which in turn may reduce hepatotoxicity induced by steatotic changes. The aim of the study was thus to assess the effect of extracts of four African herbal remedies on hepatosteatosis using an in vitro model. Methods: Hot water and methanol extracts (10% w/v) were prepared using standard brewing and ultrasonic maceration techniques. Inherent cytotoxicity of extracts and oleic acid were determined in the HepG2 hepatocarcinoma cell line after 72 h exposure using the sulphorhodamine B assay. Prevention of oleic acid-induced hepatotoxicity was assessed by pre-treating cells with extracts for 1 h, after which exposure to 0.5% oleic acid for 72 h was commenced. Fatty acid concentration was determined using the nile red assay. Results: Oleic acid induced a significant (P < 0.05) increase in fatty acid content (7-fold). Preliminary results indicate that supplementation with both hot water and methanol extracts of Leonotis leonurus leaves, Moringa oleifera leaves, Terminalia sericea leaf-root mixture and Ziziphus mucronata roots decrease oleic acid-induced fat accumulation dose-dependently. Methanol extracts resulted in a greater decrease than hot water extracts. The largest decrease was observed with the methanol extract of T. sericea, where a 2-fold fat accumulation was seen at 32 lg/ml. Conclusion: Herbal remedies are potential antisteototic sources. Of the four plants assessed, the methanol extract of T. sericea leaves and roots presented with the highest activity. T. sericea contains a variety of polyphenolic compounds, which could be responsible for the activity seen. Further experimentation is required to determine to what extent damage is prevented.

502 THE CHEMISTRY, BIOLOGICAL ACTIVITIES AND MECHANISM OF ACTION OF (-) CLAUSENAMIDE Zhang J. From clausena lansium (Lour) skeels isolated 7 novel compounds, amides, one of them is clausenamide which contains 4 chiral centers having 16 enantiomers. After identify of the configuration of 16 enantiomers, according to the biogenetic, the synthesis of 16 enantiomers, optical active clausenamide and asymmetric synthesis of (+) and (-) clausenamide were successfully completed. During this study, many stereochemical and synthetic difficulties were solved and Baldwin principle was repaired. Now, the production scale is good enough to satisfy the clinical therapeutic need. For pharmacology study, using many models and indications showed that (-) clausenamide is effective enantiomer, while (+) clausenamide is ineffective and more toxicity than (-) clausenamide. The phamacological effects of (-)clausenamide are mainly increasing cognition in ten models of memory impairment and inhibiting Ab toxicity, blocking neurofibrary tangle formation via inhibiting high phosphorylation of tau protein. The anti-dementia mechanism is characterized by increasing synaptic plasticity both in efficacy and structure that provide support to the new theory ‘synaptic loss is the main cause of dementia’ (-)clausenamide is then considered as a promising drug for treatment of AD and other neurodegenerative disorders.


153 503 THE CONTRACEPTIVE AND FAMILY PLANNING POTENTIAL OF RICOM-1013-J Okwuasaba F., Ekwere, McNeil, Sokomba, Usar, Ekwenci, Ekpenyong, Iranloye, Ogaji, Ochekpe, Isichei, Ramyil, Musa, Okafor, Salawu, Tijani, Azija The use of plants as fertility regulating agents is very well documented in various cultures. A survey of the seed RICOM-1013-J revealed that it is highly acceptable as a means of family planning amongst Nigerian women. The LD50 which was greater than 2 g/kg revealed a wide margin of safety with no evidence of organ damage (liver, kidney, GIT, heart, ovary, fallopian tube, and uterus). Determination of blood urea, sodium (Na+), Potassium (K+), Chloride (Cl-), Bicarbonate (HCO3+) as a measure of renal function and ALP, GPT, GOT, and GGT as a measure of liver function, show that both renal and liver function profiles were normal compared with the control (P < 0.05). Haematological indices were not significantly different from the control (P < 0.05). In the pre-trial clinical observational studies, RICOM-1013-J administered as a single dose of 2.3–2.5 mg once/12 months, protected against pregnancy in 50 women volunteers for a period of 9–12 months. Clinical observations reveal very minimal side effects. In addition, lipid profiles were not significantly affected. Further studies in women volunteers show that the prevalence in cervical intraepithelial neoplasia (CIN) between control and volunteers on RICOM-1013-J was not statistically different. Regular menstrual cycle was not disrupted over the study period. There was no evidence of weight gain, nausea and vomiting, increase in blood pressure or glucose intolerance. The anthropometric evaluation in children of mothers who had taken RICOM-1013J was within the 5th and 95th percentile range for both control and the study group for both sexes. In other to determine its mechanism of action, hormonal profile of women volunteers were evaluated. Results indicated a state of hyperprolactinaemia and significant depression of LH and FSH. The chemical constituents of RICOM-1013-J through GC-Mass spectrometer are non-steroidal in structure. The data are indicative that the probable mechanism of action may in part be due to direct effect on the reproductive organs/or on the hypophyseal-pituitary-gonadal axis.

504 THE CYTOTOXIC EFFECTS OF AZAINDOLES AND IMIDAZOPYRIDINES ON COLON CANCER CELL LINES Ragie S., Harmse L., de Koning C., Dam J. It is estimated that colorectal cancer accounts for over 9% of total cancer incidence and once it has metastasized it becomes difficult to treat. The chemotherapeutic agents used to treat advanced colorectal cancer cause severe adverse reactions, resistance and therapeutic failure is common. Therefore, the search for new agents are important. In this context novel azaindoles and imidazopyridines were tested for activity against colorectal cancer cell lines. Caco-2 and HT-29 cell lines were maintained aseptically in culture according to standard operating procedures. The compounds were synthesized using multi-component reactions to assemble a variety of imidazopyridines and azaindoles. The effect of novel compounds on cell viability was determined by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) cytotoxicity assay. IC50 values were determined for the test compounds that caused a decrease in cell viability of more than 80% after exposure to 200 lM for 48 h. The potential of the novel compounds to induce apoptosis was evaluated by phase contrast microscopy and fluorescence microscopy making use of ethidium bromide and acridine orange as fluorochromes. Four test compounds JD88, JD119, JD122 and JD137 were active against both HT-29 and Caco-2 cells. Two compounds, TL75 and JD81 were selectivity for either Caco-2 or HT-29 cells.. HT-29 cells were inhibited by JD 81, JD88, JD119, JD122 and JD137 with IC50 values of 9.080.24, 11.411.52, 8.751.76, 28.220.85 and 4.20.7 lM respectively. Caco-2 cells were inhibited by TL75 and JD

88, JD119, JD122, and JD137 with IC50 values of 7.240.77, 6.625.76, 38.60.97, 31.19 and 15.110.16 lM respectively. Preliminary apoptotic studies of HT-29 cells showed that JD88 and JD122 are able to induce apoptosis. A condensed nucleus and blebbing at the cell surface was observed by phase contrast microscopy. This was confirmed by fluorescence microscopy. In contrast to JD88 and JD22, JD137 showed features associated with necrotic cell death. Six compounds, one azaindole and five imidazopyridines were identified as potential lead compounds for colon cancer. Initial studies suggest that these compounds are able to induce apoptotic cell death. Further studies will be performed to confirm the mode of cell death and will include studies on animal models.

505 THE DESIGN OF BETA 2 ADRENOCEPTOR AGONISTS WITH HIGH EFFICACY AND FUNCTIONAL SELECTIVITY THROUGH DOCKING STUDIES Soriano-Ursúa M., Andrade-Jorge E., Lopez-Cabrera Y., Cuevas-Hernandez R., Trujillo-Ferrara J., Correa-Basurto J. Background: The beta-2 adrenoceptor is the most studied G-protein coupled receptor, being a target for the treatment of several diseases. The prevalent thinking some years ago was that a single signaling pathway of beta-2 adrenoceptors modifies the levels of a specific second messenger that is responsible for the effects of a ligand. Recently, it has been described that the effects triggered by beta-2 adrenoceptor activation are mediated by multiple signaling pathways. By taking this into account, some well-known beta-2 adrenoceptor ligands have been tested to determine their selectivity for the various activated pathways responsible for generating a cellular response. Several studies are currently being carried out with the aim of obtaining insights into the functional selectivity of GPCR ligands. Methods: We acquired data from the Protein Data Bank for 3D models of human beta-2 adrenoceptors in the active state. Then we carried out docking simulations by using a cluster of well-known beta-2 adrenoceptor ligands as well as a cluster with proposed structures. The latter ligands were designed for high efficacy and functional selectivity based on a modification of previously tested compounds with functional selectivity in cell-based systems. Results: Docking simulations suggested specific binding modes and key residues linked to the reported activity of well-known ligands on AMPc, beta-arrestin or calcium release pathways. Based on this information, we generated a map of a binding pocket and marked the residues that seem to be related to the activity of a specific signaling pathway. Finally, we suggest specific moieties that could enable ligands to contact these key residues. Conclusions: Our theoretical studies have identified pivotal features for the functional selectivity of ligands on beta-2 adrenoceptors, including key residues in these receptors and key moieties in the ligands. Moreover, the new data have allowed for the design of a new compound with high intrinsic activity (105% comparatively to isoprenaline), although its functional selectivity has not been tested.

506 THE EFFECT OF SYNTHETICALLY-DERIVED AROMATIC COMPOUNDS ON THE ROLE OF INDUCING CELL DEATH IN BREAST CANCER CELLS Davison C., Frost C., De Koning C., Davids H. Breast cancer is the most frequently diagnosed cancer in women worldwide. While breast cancer has long been recognised as a major public health burden in high-income countries, the majority of cases occur in low- and middle-income countries. A treatment regime, both effective and safe, is required and this can only be achieved once more effective chemotherapeutic agents are found. These ‘drugs’ need to induce cell death (apoptosis) in the cancer cells, yet not affecting the


154 normal healthy cells. Apoptosis occurs normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues. Apoptotic cell death allows a cell to “commit suicide” in genetically- controlled or programmed mechanism(s). Anti-proliferative and cytotoxic effects of the 5 synthetically-derived quinolone compounds were assessed using the MTT assay for a 24 h period on breast cancer cells (MDA-MB-231 and MCF-7) as well as isolated white blood cells. The ability of the 5 compounds to induce apoptosis was determined by Annexin V/Propidium iodide staining, cell cycle analysis and mitochondrial cell membrane potential. When the analysis of these assays is combined, one can determine the mechanism by which a compound may induce apoptosis. The compounds toxicity was screened and determined to be at 100 lM where above 60% cytotoxic affects were observed in both the breast cancer cell lines and white blood cells. Four of the compounds displayed a significant (P < 0.05) time-dependent increase in apoptosis induction across the various exposure (6, 12 and 24 h) periods, with approximately 4-fold increase observed in the MCF-7 cells relative to the untreated cells, with necrosis induction seen to be minimal. In the MDA-MB-231 a 4- fold significant (P < 0.05) effect was observed for necrosis, with apoptosis induction playing a minor role relative to the untreated cells over the different exposure times. Preliminary data suggests that the cell cycle is arrested in the G1 phase for 4 of the compounds, and that the mitochondrial membrane potential is also affected for both breast cancer cell lines. Four of the 5 compounds tested show potential as candidates for breast cancer treatment.

507 THE EFFECTS OF NOVEL 7-AZAINDOLES AND 6-SUBSTITUTED IMIDAZOLES ON MCF-7 BREAST CANCER CELLS Kurebwa T., de Koning C., Harmse L., Dam J. Background: Breast cancer is characterized by the unregulated growth of cells and globally is a leading cause of mortality in women. Current treatment of metastatic breast cancer is prone to treatment failure and severe adverse effects. Biological agents like trastuzumab are effective treatments with low toxicity profiles but their use is limited by their prohibitive cost. Therefore the need for new, effective, safer and cost-effective therapeutic agents are of great importance. In this context this study has investigated novel compounds based on either a 7-azaindole or a 6-substituted imidazole scaffold for activity against breast cancer cells. Methods: The MCF-7 breast cancer cell line was maintained aseptically in culture according to standard procedures.Cells were exposed to 200 lM of test compound for 48 h in 96 well plates and the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was performed to measure cell viability. Compounds that caused more than 80% cell death were further investigated to determine their half maximal inhibitory concentration (IC50). Active compounds were further assessed by phase contrast and fluorescence microscopy to determine if the cells display features of apoptosis or necrosis by using a combination of acridine orange and ethidium bromide dyes as fluorochromes. Results: The study screened 30 compounds and identified three azaindole and ten imidazole compounds as effective (80% cell death). The IC50 value of the most active azaindole, III-76, was 24.42.1 lM. In the imidazole group, three compounds; JD-88, JD-122 and JD-137, were the most active with IC50 values of 0.620.05, 19.83.1 and 32.61.5 lM respectively. Phase contrast microscopy images of cells treated with JD-88 and JD-122 showed characteristics consistent with apoptotic cell death while cells treated with JD-137 showed necrotic characteristics. Fluorescence microscopy images suggest apoptotic induction in cells treated with JD-88 and JD-122 while those exposed to JD-137 showed early loss of membrane integrity which indicates necrotic cell death. Conclusion: Two compounds JD-88 and JD-122 show great promise as potential lead compounds for breast cancer therapy. However, further testing to determine the mode of cell death as well as animal stud-

ies are required to confirm the efficacy and toxicity of these compounds.

508 THE EFFECTS OF ZNF185 ON MOUSE SPERM FERTILITY WITH RNA INTERFERENCE Feng W. Background: Zinc finger protein 185 (ZNF185) is a newly found gene that is highly expressed in mouse testis, but biological function and molecular mechanism of ZNF185 are not clear. In this study, we investigated the effects of ZNF185 on mouse sperm fertility with RNA interferences. Methods: Three siRNAs targeting ZNF185 mRNA (si-ZNF185-1, siZNF185-2, si-ZNF185-3) were synthesized and transfected into sertoli cells with the mediation of Lipofectamine2000, and those sertoli cells without transfection, supplemented with only Lipofectamine2000 and transfected with siRNA-scramble were taken as blank control group, reagent control group and negative control group, respectively. The expression of ZNF185 was detected by real-time PCR and western blotting. The most effective fragment of siRNA targeting ZNF185 was constructed into lentivirus vector. The recombinant lentivirus vector and control vector infected sertoli cells. The interferences efficiency was detected by real-time PCR and western blotting. The recombinant lentivirus vector and control vector were injected into mouse testes. The expression of ZNF185 of testis was determined by real-time PCR and western blotting. HE staining of reproductive organs tissue sections was performed. The sperm fertility was evaluated by different indices, including litter size in mated female mice, sperm motility, sperm count and in vitro fertilization rate. Results: The expression of ZNF185 was significantly lower in sertoli cells transfected with ZNF185 siRNA-2. The recombinant lentivirus vector could markedly reduce the expression of ZNF185 in sertoli cells and attenuate the expression of ZNF185 in mice testis. There was no pathological change in the testes tissues in treated group compared with control group. The injection of the recombinant lentivirus vector reduced the in vitro fertilization rate and the conception rate in vivo significantly (P < 0.05), although the sperm count and the sperm motility decreased insignificantly (P > 0.05). Conclusions: RNA interference targeted ZNF185 could reduce the sperm fertilization ability of mouse, and ZNF185 might act as a gene target for reproduction and contraception.

509 THE ENDOPLASMIC RETICULUM-ORIENTED DRUG DEVELOPMENT FOR FAMILIAL AMYLOID POLYNEUROPATHY Kai H., Chosa K., Sato T., Yokoyama T., Mizuguchi M., Suico M., Shuto T. Familial amyloid polyneuropathy (FAP) is one of the hereditary amyloidoses caused by a point mutation in the human plasma protein, transthyretin (TTR). Amyloid fibrils derived from TTR variants accumulate in peripheral nerves and visceral organs. TTR variants are easily dissociated from tetramer to monomer because of having low energetic stability of their tetrameric structure in comparison with wild-type (WT) TTR. Previously, we demonstrated that endoplasmic reticulum (ER) quality control system and ER associated degradation (ERAD) of TTR are pathological determinants of FAP (EMBO J. 2007, J. Biol. Chem. 2009). The monomeric mutation introduced in the vast majority of TTR mutants that are normally secreted resulted in the ER retention and efficient degradation of these monomeric mutants by ERAD. Therefore, inhibition of TTR tetramerization in ER lumen and suppression of TTR variants secretion could be a promising therapeutic strategy for FAP. Here, we employed structure-based virtual screening, and we identified some candidate compounds with a poten-


155 tial as destabilizer of intracellular TTR tetramer. The selected compounds were assayed using HEK293 cells stably expressing the most common variant, V30M TTR. Our data indicated that some of these compounds may inhibit extracellular secretion of V30M TTR, suggesting a possible use for preventing TTR amyloid pathogenesis. 510 THE GPR30 AGONIST G-1 INDUCES MITOTIC ARREST AND APOPTOSIS IN HUMAN VASCULAR SMOOTH MUSCLE CELLS Gui Y., Shi Z., Gao J., Li D., Walsh M., Zheng X. The g-protein coupled estrogen receptor (GPER) has been implicated in the regulation of cardiovascular functions. However, the results regarding the role of GPER in cell apoptosis are inconsistent. The GPER selective agonist G-1 is a useful tool for exploring the biological roles of GPER in a variety of experimental settings. The present study investigated whether activation GPER by G-1 induces apoptosis of vascular smooth muscle cells (hVSMCs). Treatment with G-1 (1– 10 mM) increased mitotic index and apoptosis in hVSMCs. G-1induced apoptosis was concurrent with an increase in cleaved-caspase 3. Furthermore, G-1 induced microtubule disruption, mitotic spindle damage, and tubulin depolymerization. The MAPK kinase inhibitor PD 98059 or down-regulation of Erk1/2 with siRNA transfection did not prevent the effect of G-1 on apoptosis. We conclude that G-1 induces mitotic arrest and apoptosis in hVSMCs, apparently involving microtubule disruption and independent of the MAPK pathway. Our results suggest that G-1 may be an effective therapeutic agent to treat proliferative vascular diseases, such as atherosclerosis and restenosis.

511 THE HIV INFECTION-PROMOTING AMYLOID PEPTIDE SEVI: IDENTIFICATION OF AMYLOID FORMING REGIONS AND THEIR CELL TOXICITY Elias A., Carver J., Musgrave I. Several debilitating and incurable human diseases including Alzheimer’s disease, Parkinson’s disease, systemic amyloidosis and type 2 diabetes are due to extracellular deposition of highly structured protein aggregates termed amyloid fibrils. Amyloid fibrils arise when a specific protein or protein fragment loses their correct folding status, aggregate and form insoluble deposits. The precipitating factors can be due to stresses like elevated temperature, low pH, infection, reactive oxygen species and inherited mutations. Semen contains a fibril forming peptide that significantly increase the ability of HIV to infect the cells by helping the virus to attach to the cells. The peptide is a fragment of prostatic acidic phosphatase and its fibrils are termed Semenderived enhancer of virus infection (SEVI). The aims of this study were (i) to identify potential amyloidogenic regions in SEVI and determine whether these putative amyloidogenic regions are able to form amyloid fibrils; and (ii) to determine whether SEVI and its fragments were toxic to neuronal PC 12 cells, a standard model for the toxicity of amyloid b (Ab, the putative causative agent in Alzheimer’s disease), and human epithelial CACO-2 cells as a model of epithelial cells. Our results determined the fibril froming regions of SEVI. The results of this study also indicate SEVI and its fibril forming fragments are toxic to neuronal cells but not to confluent epithelial cells. These findings imply that although SEVI assists the attachment of HIV-1 to immune cells, it does not facilitate HIV entry by compromising the epithelial cell layer that presents a barrier to HIV. Potentially, the presence of SEVI fragments in semen could also promote HIV infection along with SEVI itself.

512 THE IN VITRO ANTIMALARIAL ACTIVITY OF NOVEL 2-[4(7-CHLOROQUINOLIN-4-YL)PIPERAZIN-1-YL] ACETAMIDE DERIVATIVES Van Zyl R., Jansen van Vuuren N., Chen C., Inam A. Background: The reported cases of resistance to artemisinin by the malaria parasite, Plasmodium falciparum has truly awakened the scientific community to the realization that we may soon lose this drug from our arsenal. Although the number of cases of malaria has decreased over the past few years, there are still an unacceptably high number of cases especially involving pregnant women and children. In an attempt to counter this problem, a novel series of 2-[4-(7-chloroquinolin-4-yl)piperazin-1-yl] based compounds were synthesised in an attempt to enhance their potential antimalarial activity and reduce the toxicity profile. Methods: Twenty-seven derivatives of 2-[4-(7-chloroquinolin-4-yl) piperazin-1-yl] were synthesised and characterised. The tritiated hypoxanthine incorporation assay was used to measure the sensitivity of the 3D7 parasite strain to the derivatives compared to chloroquine and quinine. Whilst susceptibility of the host red blood cell membrane to lysis was quantified by the amount of released haemoglobin. The toxicity of the compounds to human neuroblastoma (SH-SY5Y) cells was determined using the tetrazolium viability assay. To elucidate a possible mechanism of action, the b-haematin formation inhibition assay was performed. The concentration required to inhibit 50% of the effect (IC50) was determined from a log sigmoid dose response curve. The Mann-Whitney statistical test was performed, with a p value HP-b-CD >b-CD >c-CD. Based on spectral data obtained from 2D ROESY Rotational nuclear Overhauser Effect Spectroscopy, a reasonable geometry for the complex was proposed implicating the insertion of 3,5-dihydroxyhept-6-enoic acidic and flurophenyl ring of rosuvastatin into the wide end of the torus cavity of M-b-CD, whereas in case of b-CD, HP-b-CD and c-CD different mode of inclusion were revealed. Molecular modelling studies was conducted to confirm the exact geometry of inclusion. Indeed the best docked complex in terms of binding free energy supports the model proposed from NMR experiments. Moreover, M- b-CD &HP-b-CD inclusion complexes performed better than RST in reducing total cholesterol and triglyceride levels against triton-induced dyslipidemia. In conclusion, the association of cyclodextrins with rosuvastatin leads to great enhancement in dissolution rate and improvement of therapeutic efficacy of the drug.

521 THERAPEUTIC ROLE OF ARISTOLOCHIA INDICA AND ITS ACTIVE PRINCIPLE BY INVIVO AND INVITRO STUDY Mittal D., Joshi D. Aim: The present study was carried out to observe the hepatoprotective effect and antioxidant activity of the aqueous extract of the roots

523 THIENOQUINOLINS EXERT DIURESIS BY STRONGLY INHIBITING INNER MEDULLARY COLLECTING DUCT UT-A UREA TRANSPORTERS Yang B., Ren H., Zhou H. Urea transporters (UT) play an important role in the urine concentration mechanism by increasing urea reabsorption across the inner medullary collecting duct (IMCD), suggesting that inhibition of these proteins could have therapeutic use as a novel class of diuretic. Recently we found a thienoquinolin UT inhibitor, PU-14, which exhibited diuretic activity. In this study, a more potent UT inhibitor, PU-48, was identified that has better pharmacological characteristics. Inhibition rate of PU-48 on UT-A1-mediated urea transport was 69.5% with an IC50 of 0.32 mM. PU-48 (10 lM) significantly inhibited basal urea transport by 31.1% in perfused rat terminal IMCDs. PU-48 (1, 5, and 10 lM) completely inhibited the vasopressin-stimulated component of urea transport in rat terminal IMCDs. Subcutaneous administration of PU-48 at 12.5 and 50 mg/kg caused significant diuresis with low urinary osmolality and urea concentration. There was no significant difference in blood Na+, K+, Cl-, and urea after the 6-day PU-48 treatment. Total osmolality was significantly reduced in inner medul-


159 lary tissue of PU-48-treated rats, which was due to a reduction in inner medullary urea concentration. Ten mM PU-48 caused no significant inhibition of the potassium channel hERG, sodium channel NaV1.5, or L-type calcium channel CaV1.2, as determined by electrophysiolic experiments. PU-48 was not cytotoxic to MDCK cells and had no acute toxicity in mice. Our data provide proof of concept for the potential utility of IMCD UT-A inhibitors as a novel class of diuretics.

Baseline mean intercourse satisfaction domain values of International Index of Erectile Function 12 reached 17 at 10-week treatment in groups 1 and 2, respectively (P < 0.05). There was some non-significant withdrawal effects with tramadol after the tested period. In conclusion, Tramadol seems to provide significantly better results in delaying ejaculatory latency time and enhancing intercourse satisfaction in both normal and patients with premature ejaculation. Further studies should be performed to evaluate its safety and efficacy in long-term use.

524 TOXICOLOGICAL, NUTRITIONAL, AND IMMUNOLOGICAL EFFECTS OF A LECTIN-PROTEASE INHIBITOR FRACTION OF TEPARY BEAN SEEDS (PHASEOLUS ACUTIFOLIUS) ORALLY ADMINISTERED IN RATS

526 TRYPANOCIDAL EFFECTS OF AQUEOUS ROOT EXTRACT OF SECURIDACA LONGEPEDUNCULATA IN MICE INFECTED WITH TRYPANOSOMA BRUCEI

Garcia-Gasca T., Moreno-Celis U., Noriega-Giron H., Lopez-Martinez J., Ferriz-Martinez R., Andrade-Portillo V., Aranda-Vargas P., Cervantes-Jimenez R., Guerrero-Carrillo M., Rodriguez-Mendez A., Mendiola-Olaya E., Blanco-Labra A. Studies of our work group have shown that lectins and protease inhibitor (PI) of Tepary beans have individual effects on colon cancer cells. When tested in cancer cells, PI decreased cell invasion ability, inhibited extracellular matrix degradation and increased cell adhesion, while a lectin rich fraction LRF presented differential cytotoxic effect by apoptosis induction. Orally administered LRF to rats showed low toxicity and antinutritional effects and also the ability to reduce tumor incidence in colon. As both type of proteins exhibit anticancer properties, and in order to study the adverse effects of introducing both proteins together, the aim of this study was to determine the in vivo effect of a lectin-protease inhibitor fraction LIP-60 orally administered to rats. LIP-60 was tested for its subchronic toxicity at a daily schedule using a dose of 100 mg/body weight kg for 28 days. In order to know the effects on the hematopoietic system, blood samples were obtained on 0, 1, 3, 9, 14, 19 and 24 days and animals were sacrificed at day 30. Spleen, thymus and femur bone marrow were obtained for histopathological analysis and blood samples were used in order to determine markers for renal, hepatic and pancreatic damage and also the nutritional status. The animals showed piloerection, yellow hair pigmentation, diarrhea, abdominal distention, irritability, partial food intake decrease and stationary body weight gain with a 10% decrement. Blood markers for toxicity or nutritional status did not show significant alterations. Complete blood count showed decrease in lymphocytes while granulocytes increased, suggesting allergic process. Histopathological analyzes showed reduced white pulp of the spleen, related with an increase in the release of mature leukocytes suggesting an allergy-like response. Taking together, these results suggest that LIP-60 presents systemic effects with low toxicity and is suitable to be tested against colon cancer as a natural drug with lower adverse effects.

Haruna Y., Kwanashie H., Atawodi S., Anuka J., Hussain I. Background: Human African trypanosomiasis (HAT) or sleeping sickness is a major threat to public health in Africa. It is responsible for the death of half a million persons annually, and is the main reason why 9 million square kilometres (about half of the cultivable land in Africa) cannot be used for intensive livestock farming. At present, the only available trypanocides are as old as 50 years and have very severe side effects. Since ethno-botanical compounds have recently been scientifically proven to be effective trypanocides; this study investigated the trypanocidal activity of Securidaca longepedunculata in mice. Methods: Aqueous root extract of S. longepedunculata was obtained by cold maceration. Thirty Swiss albino mice were divided into 6 groups of 5 each; with five of the groups receiving 1X107 Trypanosoma brucei, i.p. in 0.2 ml blood in PBS. Parasitaemia was determined 3 days post-inoculation and daily, subsequently. Treatment commenced at peak parasitaemia as follows: group A served as a positive control (no infection, no treatment), group B was the negative control (infected, not treated), group C received 3.5 mg/kg of diminazine aceturate once, while other groups received 0.97, 1.93 and 3.86 mg/kg of the extract respectively for 7 days; all intraperitoneally. Data were analysed using ANOVA, and P < 0.05 was taken to be statistically significant. Results: All mice in the negative control group died on day13, complete clearance of all parasites in the diminazine treated group occurred on day1 post-treatment, and a gradual reduction in parasitaemia was observed in all extract-treated groups dose dependently. Subsequently, by day7 post treatment (12 days post-inoculation), parasitaemia (no./ field) was significantly (P < 0.05) reduced by extract to 5.800.21 (3.86 mg/kg), 7.500.35 (1.93 mg/kg) and 8.440.37 (0.97 mg/kg) against 33.060.79 for control (0 mg/kg). Relapse parasitaemia was observed in all extract-treated groups but the extract prolonged the life span of mice to at least 20 days post-inoculation. In contrast, mice in diminazine aceturate-treated group showed no relapse parasitaemia, and all survived the study period. Conclusions: S. longepedunculata possesses trypanocidal activity against T. brucei brucei and is a potential candidate trypanocidal agent for drug development.

525 TRAMADOL AND PREMATURE EJACULATION Assy A., Shaker M. Tramadol is a potent centrally-acting analgesic. It mainly act at opioid receptors. We first reported in July 2007 the use of tramadol to retard ejaculation in healthy men. The present study was performed to evaluate the efficacy and safety of tramadol in delaying ejaculation in normal men and in patients with premature ejaculation. About 150 potent men with normal and premature ejaculation were randomly assigned to receive 50 mg tramadol (group 1, n =75) or placebo (group 2, n = 75) approximately 3 h before planned sexual activity, for 10 weeks. The efficacy was assessed using responses to International Index of Erectile Function, intravaginal ejaculatory latency time evaluation, and several general assessment questions. The mean intravaginal ejaculatory latency time after tramadol and placebo increased from 22 and 27 seconds to approximately 37 and 39 seconds, respectively (P < 0.001).

527 UGANDA’S MEDICINAL PLANTS MAY HOLD A SOLUTION FOR RIFAMPICIN¬-RESISTANT TUBERCULOSIS Kirimuhuzya C., Bunalema L., Kakudidi E., Tabuti J., Waako P., Orodho J., Magadula J., Otieno N., Okemo P. Management of drug-sensitive tuberculosis is by a lengthy omplex regimen of five drugs. Worse still, treatment of XDR-TB is still a gamble while that for MDR-TB involves use of the more expensive and less safe, second-line drugs which are beyond the reach of most communities. As a result, a large percentage of patients have been using herbal remedies for which the efficacy and toxicity are uknown. There aim of this study was to scientifically validate the ethnobotanical claims of anti


160 TB activity, isolate the active ingredients in these plants, and to determine safety profiles of the plants used and the derived compounds. Methods: Surveys were conducted and then followed by collection of samples of the plant species, drying, pulverizing and extractions done using solvents of various polarities. The extracts were screened against various strains of Mycobacterium tuberculosis including the rifampicinresistant TMC-331. Diffussion methods were used for sensitivity tests while MICs were mainly determined using the microtitre plate method. Acute toxicity tests were also conducted on mice. The plants found to be active aganist rifampicin-resistant TB were subjected the isolation process using various chromatographic techniques and the process is continuing and is expected to be concluded with formulation of standardised products which can subsequently be taken through clinical trials. Results: Over 50 plant species were documented as being used by Traditional Medicine Practitioners (TMPs) in the treatment of symptoms of tuberculosis and screening revealed that the crude extracts of some of the selected plants were active against rifampicin-resistant tuberculosis. They included Lantana camara, Cryptolepis sanguinolenta, Erythrina abyssinica, Warburgia ugandensis and Mangifera indica with MIC values of 0.015, 1.56, 1.17, 1.56 and 0.59 mg/ml respectively, for rifampicin-resistant TB.The active extracts that were tested for toxicity in mice and were found to be in the safe range of dosage.The isolation process on Erythrina abyssinica and Warburgia ugandensis has so far shown that the ethyl actate fraction has the highest activity against rifampicin-resistant MTB. Conclusion: Uganda’s plants have potential to produce lead molecules that could be used to tackle the escalating multidrug-resistant M.tuberculosis and probably prevent the threat of evolution of TDR.

528 UNEXPECTED REACTION OF OXIDATION HYDRAZONES CLEAVAGE UNDER BASIC TREATMENT: EVALUATION OF SCOPE AND LIMITATIONS Okorochenkov S., B€urglova K., Hlavac J. We surprisingly discovered that hydrazone contained 4-(4-Formyl-3methoxyphenoxy)butyric acid (BAL-linker) on solid support under base treatment with potassium trimethylsilanolate in organic solvents is converted to unexpected product with very high purity. Further studies demonstrated this product is BAL-linker with aldehyde group oxidized to carboxylic function. Also we showed that ‘hydrazine’ part of starting hydrazone remains in solution, but the hydrazon group is cleaved. Moreover, it was founded that this reaction doesn’t proceed in solution as well as on solid phase in inert atmosphere, so, oxygen absorbed on solid support is required as an oxidizer. Experiments with different substrates demonstrated that reaction of oxidative cleavage of hydrazones gives the better yields if starting hydrazone contains electron-donating or weak electron-withdrawal groups in ‘hydrazine’ part and electron-donating groups in “aldehyde” part. Studies of BAL-hydrazones stability on solid support showed that hydrazone bond remains unaffected under treatment with weak acids and bases, reduction agents (e.g. sodium borohydride), alkali in presence of water. Hence, reaction of oxidative cleavage of hydrazones could be used for development of traceless linkers as well as for conversion of aldehydes to carboxylic acids in very mild conditions. 529 WILL THE REAL DRUGS PLEASE STAND UP? Southan C., Sharman J. Background: A comparison of database subsets of approved drugs in 2009 recorded only 807 exact structures in-common (PMID:20298516). Factors contributing to low overlap included semantic naming inconsistencies, ambiguity in structure representation and the fact that neither regulatory bodies nor pharmaceutical companies directly verify public electronic chemical database records. This work is a current comparison of drug sources inside PubChem.

Methods: We selected submitters that nominally included small-molecule drug collections and International Non-proprietary names (INNs) and/or US approved names (USANS). Unions, intersects and differences were derived by using the Entrez query history interface to perform Boolean operations on retrieved sets. Additional filters were explored, including salt-stripping by selecting a covalent unit count of 1. Results: DrugBank 3.0 declares 1541 small-molecule drugs and the term ‘approved’ returned 1424 substances (SIDs) in PubChem. These collapse to 1392 compounds (CIDs), and removal of mixtures reduces to 1325. The Therapeutic Target Database (TTD) declares 1540 approved drugs on their website. The CID overlap with the DrugBank 1325 was 1108, and the equivalent figure for ChEMBL_17 was 1141. The threeway consensus (from the DrugBank starting point) was 1003. The term INN retrieves 7916 CIDS, reducing to 7180 single-components. USAN brings back 5494 of which only 3204 are single-component (i.e. more salt forms are designated as USANs). Of the 1108 3-way set, 927 have an INN or USAN. The ‘same connectivity’ query indicates, on average, each of the 927 have nearly 20 canonically-related CIDs. Issues associated with these metrics will be outlined and, depending on new source releases, the numbers will be updated. Conclusions: A surprising degree of non-overlap persists in drug structures. Our results are not a criticism of the valuable sources but further analysis is needed of the multiplicity of structural representations and fuzzy naming of essentially the same canonical drugs inside PubChem. This important issue in cheminformatics extends beyond the INNs to all pharmacologically active structures. It also rationalises our IUPHAR/BPS Guide to PHARMACOLOGY strategic choice of focusing on consensus sets for curation. This work indicates definitive drug lists will remain elusive until there is more collective engagement for provenance, standardisation and cross-mapping.

530 b-MANGOSTIN SUPPRESSESS LIPOPOLYSACCHARIDEINDUCED EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN RAT HAPI MICROGLIAL CELLS Thampithak A., Suksamrarn S., Sanvarinda Y. Excessive production of nitric oxide (NO) and proinflammatory mediators from activated microglia play an important role in human degenerative disorders including Parkinson’s disease and Alzheimer’s disease. Inhibition of microglial activation may have potential anti-inflammatory effects. Garcinia mangostana (mangosteen) has traditionally been used in Thailand for treatment of skin infection, diarrhea and wounds for several years. However, the molecular mechanisms underlying its anti-inflammatory action remain unclear. In this study, the effect of bmangostin, a xanthone compound purified from mangosteen, on lipopolysaccharide (LPS)-induced inflammation was investigated using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and Griess assay on rat HAPI microglial cells. Results are expressed as means SEM. Statistical analysis was performed using one-way ANOVA followed by a Tukey comparison test. In the present results, b-mangostin at the concentration of 0.1, 0.5, 1, 5 and 10 lM significantly suppressed the LPS (0.1 lg/ml)-stimulated activation of microglia by reducing the production of NO in a concentration-dependent manner. Moreover, the protein and mRNA expression of inducible nitric oxide synthase (iNOS) were also markedly inhibited by bmangostin. These results demonstrate that b-mangostin exerts antiinflammatory effect probably by suppression of iNOS expression and suggest that this compound may offer substantial therapeutic potential for treatment of neurodegenerative diseases that are accompanied by microglial activation.


161 1206 EXPLOITING SECONDARY PHARMACOLOGY IN DRUG DISCOVERY: IN SILICO GUIDED WET LABORATORY SCREENING OF GENERIC DRUGS FOR ANTIMALARIAL ACTIVITY

1208 RESEARCH ON MECHANISMS OF LAPPACONITINE ANTIHYPERALGESIA Zhang S., Zhang J., Jia Z., Wu J.

Bello S., Aminu C., AbdulMajeed Y. Background: Generic drug are potentially cheap and available alternatives to current antimalarial agents. If identified and optimized they may fill in gaps in drug discovery for orphan diseases. In silico screening may greatly reduce the cost of such wet laboratory screening in this regard. Method: One hundred (100) commercially available drugs not used for malaria were selected from Drugbank and the potential binding affinities against ten selected known or putative plasmodium molecular targets (from PDB)were analyzed using molegro virtual docker software. Ten (10) drugs with the best affinities were selected and their suppressive, curative and prophylactic antimalarial activities were confirmed using plasmodium berghei mice model. Result: Fluconazole,indomethacin,loratidine,lisinopril,meloxicam,promethazine,nifedipine,clarithromycin,piroxicam and flucloxacillin produced the least binding energies against the ten plasmodium targets. Nifedipine (73.8%,55.8% and 48.8%) clarithromycin (77.6%,46.1% and 41.8%), flucloxacillin (77.6%,46.1% and 44.1%) and lisinopril (71.5%,53.4% and 41.8%) produced significant, suppressive, curative and prophylactic activity respectively while meloxicam (70.8% and 53.4%) and piroxicam(69.2% and 41.2%) produced only significant suppressive and curative activity respectively. All the tested drugs produced significant suppressive activity(>40%). Additionally chloroquine, meloxicam, nifedipine and lisinopril produced a statistically significant (P < 0.01) mean survival time of 27.17, 16.5, 15.83, and 15.67 days respectively while promethazine produced statiscally significant (P < 0.05) mean survival time of 12.3 days. Conclusion: Nifedipine,clarithromycin,flucloxacillin, lisinopril, meloxicam and piroxicam are potential chemoprophylatic and chemotherapeutic agents against malaria.

Lappaconitine (LA) is the alkaloid extracted from the aconitum roots of a ranunculaceae plant. For a long time, the Chinese and Japanese used aconite to help to comfort the pain. As the non-narcotic analgesics, LA was first discovered in China and has been accepted as the three-step analgesic medication for the cancer patients. Objective: Our research observed the effects of LA on chronic inflammatory pain and hyperalgesia rats induced by CFA (complete Freund’s adjuvant). The mechanical pain threshold, the expression of GFAP (glial fibrillary acidic protein), OX-42(CD 11 b/c), c-fos, NOS in spinal cord and P2X3 receptor in dorsal root ganglion (DRG) were explored for the anti-hyperalgesia mechanism of LA. Methods: The model rats were hypodermic injected with of 100 ll into the plantar surface of the right hind paw were. LA was given to the model rats once a day for 6 days since the CFA injected. The mechanical pain threshold were measured before molding day and on the 1st day, the 3rd day, the 6th day, the 9th day, the 12th day and the 13rd day. The expression of GFAP, OX-42, c-fos, NOS in L4-6 of spinal cord and P2X3 receptor in L4-6 of DRG were explored on the 14th day. Results: The mechanical pain threshold of modal rats decreased obviously after the CFA injected. LA can increase the mechanical pain threshold of modal rats obviously in 3rd and 6th day of administration 16 mg/kg. LA can inhibited the rise of the expression of GFAP, OX42, c-fos, NOS in L4-6 of spinal cord and P2X3 receptor in L4-6 of DRG (compare with modal group P < 0.05). Conclusion: LA can inhibited chronic inflammatory pain of rats. The inhibition of expression of colloid cells, c-fos, NOS in spinal cord and P2X3 receptor in DRG is one of the mechanisms of LA inhibiting chronic inflammatory pain and anti-hyperalgesia. Key words: LA, hyperalgesia, P2X3 receptor, GFAP, OX-42, c-fos, NOS.
