Orient Pharm Exp Med (2013) 13:41–49 DOI 10.1007/s13596-013-0103-9
RESEARCH ARTICLE
Inhibition of arachidonic acid metabolism and pro-inflammatory cytokine production by Bruguiera gymnorrhiza leaf Rajib Barik & Ratul Sarkar & Prova Biswas & Ashok Pattnaik & Samir Kumar Samanta & KT Manisenthilkumar & Murari Pal & Sanmoy Karmakar & Tuhinadri Sen
Received: 18 July 2012 / Accepted: 7 January 2013 / Published online: 31 January 2013 # Institute of Oriental Medicine, Kyung Hee University 2013
Abstract Bruguiera gymnorrhiza is a mangrove plant of the family Rhizophoraceae. The roots and leaves of the plant have traditionally been used for treating burns and mild inflammatory lesions. In the present study an attempt was made to evaluate the anti-inflammatory activity of the Bruguiera gymnorrhiza leaves. The methanolic fraction of Bruguiera gymnorrhiza leaves (BRG) was evaluated using different in vitro and in vivo models to evaluate the antiinflammatory properties and also to obtain an insight on the probable mechanism of such activity. The leaf extract produced significant inhibition of both carrageenan induced rat paw oedema and acetic Acid induced peritoneal capillary permeability. The above observations were further supported by our findings from in vitro experimental models, where the plant extract produced significant inhibition of both cyclooxygenase (COX) and lipoxygenase (LOX) enzymes. Moreover, BRG inhibited the production of
R. Barik : R. Sarkar : P. Biswas : A. Pattnaik : S. K. Samanta : K. Manisenthilkumar : M. Pal : S. Karmakar : T. Sen Division of Pharmacology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032 West Bengal, India T. Sen School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032 West Bengal, India R. Barik : M. Pal Division of Biology, TCG Life Sciences Ltd, Salt Lake, Kokata 700091 West Bengal, India T. Sen (*) Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India e-mail:
[email protected]
proinflammatory cytokines (TNF- α, IL-6 and IL-1β) in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) in a dose dependent manner. It was also found to possess significant free radical (DPPH, superoxide and oxygen radical) scavenging activity. From the results obtained from the various in vitro and in vivo test models, it may be concluded that the methanolic extract of Bruguiera gymnorrhiza leaves display significant anti-inflammatory properties, probably mediated through blockade of both COX – LOX pathway, coupled with scavenging effect on free radicals. Keywords Bruguiera gymnorrhiza . Carrageenan . PBMC . Cytokine . Cyclooxygenase . Lipoxygenase
Introduction Bruguiera gymnorrhiza (Family: Rhizophoraceae) is a mangrove plant native to many countries of southern and eastern Africa, Asia and northern Australia (Hou 1970). The plant is traditionally used by local inhabitants to treat different ailments (Othman 1998). The leaves of this plant are popularly used by the inhabitants of Sundarban regions of West Bengal (India), for treating burns and mild inflammatory lesions. From survey of ethnomedical literature, it was also found that the fruit and bark of this plant have been used traditionally for the treatment of diarrhoea in China (Bamroongrugsa 1999) and the flowers are consumed as vegetable in Thailand (Bunyapraphatsara 2000). Three new dammarane triterpenes, bruguierins A-C, have been isolated from a petroleum ether extract of the flowers of Bruguiera gymnorrhiza and Bruguierin A has been reported to be a selective COX-2 inhibitor (Homhual et al. 2006). The
42
methanolic leaf extract of this plant has also been reported to possess significant antioxidant and antimicrobial activities (Haq et al. 2011). However, till date, no report is available regarding the anti-inflammatory activity of the leaves of this plant and related mechanism of action for validating the traditional uses. The conventional non-steroidal anti-inflammatory drugs (NSAIDs) possess several adverse effects, thereby limiting the widespread application of these agents. Development of newer anti-inflammatory compounds possessing fewer side effects still remains a challenge to the scientific community. It is now well established that both conventional NSAIDs and also selective COX-2 blockers are known to exert their anti-inflammatory properties through inhibition of prostaglandin biosynthesis. However, it may also be noted that metabolism of arachidonic acid also gives rise to other important lipid mediators like leukotrienes and lipoxins, which are known to play a vital role during the inflammatory process. Leukotrienes and prostaglandins are known to promote the inflammatory reaction whereas, the lipoxins are predominantly anti-inflammatory in nature. Recent advances in inflammation research provide an insight to the dual blockade of both cyclooxygenase and lipoxygenase enzymes where such blockade leads to synergistic anti-inflammatory effect with a better spectrum of activity. According to available reports, dual inhibitors like BW755C, SK&F86002, ER-34122 are known to exert superior anti-inflammatory properties (Higgs et al. 1984; Griswold et al. 1987; Horizoe et al. 1998) as compared to conventional NSAIDs. Interestingly, these dual inhibitors are better tolerated in the GI tract (probably due to reduced leukotriene biosynthesis) as compared to indomethacin or other conventional NSAIDs. Medicinal plants possessing anti-inflammatory activities may provide a viable alternative to synthetic molecules, particularly in terms of reduced toxicity and improved tolerance. Our laboratory has been actively engaged in studies related to screening of Indian medicinal plants, both mangrove and other local varieties, for novel antiinflammatory molecules. During the course of such investigations we were able to identify some local plants like Pluchea indica (Sen et al. 1993), Bryophylum pinatum (Pal and Nagchaudhuri 1991), Calotropis procera (Sen et al. 1998) and Acanthus ilicifolius (Manisenthilkumar et al. 2008) possessing simultaneous anti-inflammatory and antiulcer activities, attributed to probable blockade of COX and LOX enzyme systems. The present study has been designed to evaluate the antiinflammatory potential of the leaf extract of Bruguiera gymnorrhiza. An attempt has also been made to elucidate the possible mechanism of activity using various in vitro and in vivo models.
R. Barik et al.
Materials and methods Plant material and extraction procedure The leaves of Bruguiera gymnorrhiza were collected from Jharkhali (Sundarban) region of West Bengal, India. The plant material was taxonomically identified and authenticated by the Botanical Survey of India, Howrah, India (Authentication number CNH/I-I/(286/2008/Tech.II/328 dt.12/12/2008). The leaves were dried under shade, sliced and pulverized using a mechanical grinder. The powdered leaves (~2 kg) were defatted with petroleum ether (60–80 °C) by soaking them in the solvent followed by extraction. It was then dried and subsequently subjected to extraction with chloroform as above. Thereafter, the extraction was carried out with methanol. After exhaustive extraction, the methanolic extract was collected and concentrated under reduced pressure at 45–50 °C. A dark brown concentrated residue was obtained following evaporation of the solvent in a rotary evaporator. The yield of the methanolic extract (BRG) was found to be 12.5 % (w/w). The final product was then stored at 4 °C until further use. Drugs, chemicals and instruments TNF- α, IL-6, IL1β and LTB4 ELISA kits were purchased from Cayman Chemical (Ann Arbor, USA), Amplex red was purchased from Molecular Probes, USA. Unless otherwise mentioned, all other chemicals and reagents (analytical grade) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Animals Adult male Charles Foster rats (weighing 150–200 g) and male Swiss albino mice (20–25 g) were used for in vivo studies. The animals were housed at normal laboratory conditions (24±2 °C) with 12 h light and dark cycle for at least 10 days before using for pharmacological experiments. The animals were given standard pellet diet and water ad libitum. The care and use of laboratory animals were strictly in accordance to the guidelines prescribed by the Institutional Ethical Committee (constituted under the guidelines Committee for the Purpose of Control and Supervision of Experiments on Animals, India). Acute toxicity study Swiss albino mice (20–25 g) in different groups (n=10 in each group) were used for this experiment. Before the experiment, animals were fasted for 18 h with free access to water. Multiple doses (50–600 mg/kg) of BRG samples were dissolved in normal saline and administered orally to different groups of animal. Animal mortality was recorded
Anti-inflammatory mechanism of Bruguiera gymnorrhiza
after a period of 72 h. Besides the number of deaths, parameters such as alertness, sedation, ptosis, dyspnea, urination, convulsions, spontaneous motor activity, postural reflex, were observed for 72 h. Carrageenan induced rat paw oedema Experimental rats were divided into four groups (6 rats/group). The control vehicle (isotonic saline 0.9 %, w/v), BRG (150 mg/kg and 300 mg/kg) and standard drug (aspirin 100 mg/kg) were administered orally to different groups 30 min prior to intraplantar carrageenan injection (0.1 ml; 1 % w/v in normal saline) in the right hind paw of each rat. The paw volumes were measured using a Plethysmometer (Model No 7141; UGO Basile, Italy), before and 1, 3 and 5 h after carrageenan injection (Winter et al. 1962). The percentage inhibition of paw oedema was calculated by comparing the paw volumes with respect to the control group. Acetic acid-induced peritoneal inflammation The experiment was performed in male mice weighing 18– 22 g. Experimental animals were divided into four groups (6 mice/group). The control vehicle (isotonic saline 0.9 %, w/v), BRG (150 mg/kg and 300 mg/kg) and standard drug (aspirin 100 mg/kg) were administered (p.o.) to different groups. Thirty minutes later, 0.25 ml of 1.2 % acetic acid (in normal saline) was administered intraperitoneally to each animal. Three hours later, the animals were sacrificed and viscera were exposed by abdominal incision and the peritoneal exudates were collected. Thereafter, the protein content (Bradford 1976) and the total count of white blood cells (WBC) in the peritoneal exudates was determined.
43
disorders and were not exposed to NSAIDs for at least 4 days prior to blood collection. BRG samples and standard inhibitor (BWB70C) were incubated with blood for 1 h at 37 °C. Thereafter, A23187 (Calcium ionophore; 50 μM final) was added to the samples and incubated for 30 min at 37 °C. The samples were then centrifuged at 1,500 × g for 15 min and the plasma was separated and stored at −80 ° C until use.The plasma samples were analyzed for LTB4 levels using ELISA kit as per the procedure outlined by the manufacturer (Brideau et al. 1999). Effects on production of cytokines This assay determines the effect of test compounds on the production of cytokines in human Peripheral Blood Mononuclear Cells (PBMC). BRG samples and standard compounds were tested for their ability to inhibit the activity of TNF-α, IL-6 and IL-1β in human PBMC. PBMC were isolated from blood (from healthy volunteers) using BD Vacutainer CPT™ (Cell preparation tube, BD Bio Science, USA) and suspended in RPMI medium (Jansky et al. 2003). The test compounds were pre-incubated with PBMC (0.5 million/incubation well) for 30 min at 37 ° C and then stimulated with lipopolysaccharide (Escherichia coli: B4; 1 μg/ml) for 18 h at 37 °C under 5 % CO2. The cell culture supernatants were collected and stored at −80 ° C until analysis. Levels of each cytokine in cell culture medium were estimated using respective ELISA kits performed in a 96 well format as per the instructions provided by the manufacturer (Cayman Chemical, Ann Arbor, USA). Cytotoxicity assessment
Effect on COX-1 and COX- 2 using Amplex red-based fluorimetric assay The assay was performed following the method described by Robert Batchelor et al. (2003). Briefly, the test and the standard drugs were incubated in a black 96 well plate with the appropriate COX enzymes (COX1, 1.5U/well or COX2, 2.5U/well) in a reaction buffer (0.1 M Tris–HCl, 5 mM EDTA, 2 mM phenol and 2.5 mM hematin, pH 8.0) for a period of 15 min at 37 °C. Thereafter, 50 μM amplex red was added to the wells and mixed. The reaction was initiated by addition of arachidonic acid (100 μM/well), thereafter fluorescence (Ex 544 nm and Em 590 nm) was measured up to a period of 30 min. Effect on 5-Lipoxygenase (LOX) enzyme Fresh human blood from healthy male volunteers was collected in tubes containing sodium heparin (30 IU/ml of blood). The volunteers were free from any inflammatory
Cytotoxicity analysis was performed on human PBMC. After the cell culture supernatants were collected for the cytokine quantification, the peletted PBMCs were reconstituted and incubated with 500 μg/ml of (3-(4, 5Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) for 4 h at 37 °C. After centrifugation, the supernatant was removed and the purple precipitate was dissolved in DMSO and the absorbance of the samples were measured at 544nM (Mosmann 1983). 2,2-Diphenyl-1-picryl hydrazine hydrate (DPPH) scavenging activity Different concentrations of the test (BRG) and standard substances (vitamin C and quercetin) were prepared in methanol and incubated (in darkness) with DPPH (0.3 mM in methanol). After 30 min of incubation at room temperature, the absorbance was measured at 518 nm (Kurechi et al.
44
R. Barik et al.
1980). Radical scavenging effect of test samples was calculated as following:
“p” values less than 0.05 were considered to be statistically significant.
% Inhibition ¼ ðAbsorbance of Control Absorbance of testÞ
Results
100=Absorbance of Control ðEq:1Þ Superoxide scavenging activity Measurement of superoxide scavenging activity of the BRG was performed following the method described by Nishimiki and Rao (1972). One ml of Nitroblue tetrazolium (NBT) solution along with 1 ml of NADH solution were mixed with 0.1 ml of the samples (BRG or quercetin). Thereafter, 100 μM of Phenazine methosulphate (PMS) solution, prepared in phosphate buffer (pH 7.4), was added to each sample. The samples were then incubated at room temperature for 5 min before measuring absorbance at 560nM. Decrease of absorbance indicated superoxide scavenging activity (Eq. 1). Oxygen radical scavenging activity This assay was performed following the method described by Lau et al. (2002) with minor modification. In brief, heparinized blood was centrifuged at 1,000 × g for 10 min. After removing the supernatant, the pellet was washed successively for four times with 0.9 % (w/v) NaCl. Twenty percent erythrocyte suspension was prepared by adding PBS (pH 7.4). Thereafter, different concentrations of BRG and quercetin were added to the erythrocyte suspension followed by the addition of 100 mM 2, 2′-azo-bis (2-amidinopropane) dihydrochloride (AAPH). The samples were incubated at 37 °C for 3 h and then the absorbance was measured at 540nM. Preliminary phytochemical analysis Preliminary phytochemical analysis of BRG was carried out to identify the presence of different secondary metabolites such as terpenoids, steroids, flavonoids, alkaloids, tannins and glycosides following the methods described by Harborne (1973).
Acute toxicity study The BRG samples were well tolerated by the experimental animals and no mortality was observed even at the highest experimental dose (600 mg/kg). Moreover, no abnormality was detected in the experimental animals during the observation period. Carrageenan-induced rat paw oedema Methanolic fraction of Bruguiera gymnorrhiza leaves significantly inhibited carrageenan-induced paw oedema (Fig. 1). According to our findings, the percentage reductions in paw oedema were found to be 47.78±4.44 (150 mg/kg) and 58.8± 2.22 (300 mg/kg) after a period of 3 h. However, the percentage inhibition of paw oedema was found to increase after a period of 5 h (52.0±12.67, 150 mg/kg; 76.0±12.45, 300 mg/kg). The standard drug Aspirin produced an inhibition of 72.22±3.33 (3 h) and 80.04±12.0 (5 h) at a dose of 100 mg/kg. Acetic acid-induced peritoneal inflammation Pre-treatment with BRG (150 and 300 mg/kg; i.p.) and aspirin (100 mg/kg, i.p.) demonstrated significant inhibition (21.70 %, 69.67 %, 49.57 % respectively) of acetic acidinduced peritoneal protein exudation and also reduction in peritoneal leukocytes count (Table 1). Effect on in vitro COX assay BRG samples produced inhibition of both COX1 and COX2 enzymes (Fig. 2a and b). BRG samples showed more than 80 % inhibition of COX1 enzyme at concentration of 10 μg/ml and 100 μg/ml whereas COX2 activity was almost abolished at these two concentrations. In the whole blood assay, the test samples (BRG) produced significant but lesser inhibition of COX enzymes (Data not shown) compared to in vitro enzyme based assay. Effect on 5-lipoxygenase
Statistical analysis Results were expressed as mean ± S.E. (n=6) for in vivo and mean ± S.D. (n=3) for in vitro studies. Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test using prism software.
BRG samples, at a conc. of 100 μg/ml, produced 25 % inhibition of 5-lipoxygenase enzyme. However, no inhibition was observed at the lower doses (10 μg/ml and 1 μg/ml) (Fig. 3). The standard drug, BWB70C, produced inhibition of 5lipoxygenase enzyme activity in a dose dependent manner.
Anti-inflammatory mechanism of Bruguiera gymnorrhiza
45
Fig. 1 Effect of methanolic extract of Bruguiera gymnorrhiza leaves (BRG) on carrageenan induced rat paw oedema. Rats (n=6 per group) were administered with either BRG, aspirin or control vehicle 30 min prior to carrageenan administration, and the paw volumes were measured after 1,3 and 5 h. Percent inhibition of paw oedema was calculated with respect to the control group. **p
