Molecular and Cellular Biochemistry 273: 193–208, 2005.
cgSpringer
2005
Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection Raman Chawla,1 Rajesh Arora,1 Raj Kumar,1 Ashok Sharma,1 Jagdish Prasad,1 Surendar Singh,1 Ravinder Sagar,1 Pankaj Chaudhary,1 Sandeep Shukla,1 Gurpreet Kaur,1 Rakesh Kumar Sharma,1 Satish Chander Puri,2 Kanaya Lal Dhar,2 Geeta Handa,2 Vinay Kumar Gupta2 and Ghulam Nabi Qazi2 1
Division of Radiopharmaceuticals and Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, New Delhi, India; 2 Natural Products Chemistry Division, Regional Research Laboratory (CSIR), Jammu, India Received 12 October 2004; accepted 18 January 2005
Abstract Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2 -bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389 ± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 µg/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 µg/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly ( p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress. (Mol Cell Biochem 273: 193–208, 2005) Key words: 2,2 -bipyridyl, free radical, lipid peroxidation, polyphenols, radioprotection, oxidative stress, Podophyllum hexandrum
Address for offprints: R.K. Sharma, Division of Radiopharmaceuticals and Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, Brig. SK Mazumdar Road, New Delhi 110054, India (E-mail:
[email protected])
194
Introduction Low linear energy transfer (LET) radiation causes damage to biological systems by generating reactive oxygen/nitrogen species (ROS/RNS) like superoxide radicals (O•2 ), hydrogen peroxide (H2 O2 ) singlet oxygen (O), hydroxyl radicals (OH• ), nitric oxide (NO• ) and peroxynitrite [1]. ROS/RNS interact with various macromolecules like DNA, proteins and lipids leading to lipid peroxidation (LPO), which results in leaky membrane, DNA lesions, removal of sulfhydryl groups from cellular proteins eventually leading to protein fragmentation and denaturation [2–4]. This can result in loss of cellular homeostasis and even cell death [5]. Oxidative stress has also been implicated in the generation of several pathological conditions like arthritis [6], Alzheimer’s and Parkinson’s disease, ageing, glycated oxidation in case of diabetes mellitus [7], low-density lipoprotein (LDL) oxidation in atherosclerosis [8], red blood cell hemolysis in glucose-6-phosphate dehydrogenase deficiency [9] and possibly play a causative role in Acute Mountain Sickness [11]. The medicinal use of Podophyllum hexandrum Royale syn. P. emodi Wall (Himalayan Mayapple; family: Berberideceae), a high-altitude plant species native to the alpine and sub alpine areas of Himalayas, dates back to ancient times [11]. The plant has been described as ‘Aindri’ – a divine drug in the traditional Indian system of medicine – the Ayurveda [11], and has also been used in traditional Chinese System of Medicine [12] for treatment of a number of ailments. In the modern allopathic system of medicine, the plant has been successfully used for the treatment of various metabolic disorders [13], monocytoid leukemia, Hodgkin’s and non-Hodgkin’s lymphomas, bacterial and viral infections [14, 15], venereal warts [16], rheumatoid artharalgia associated with limb numbness and pycnogenic infections of skin tissue [12], AIDS-associated Kaposis sarcoma and different cancers of brain, lung and bladder [17]. The roots and rhizomes of P. hexandrum are known to synthesize a plethora of secondary metabolites and bioactive components like podophyllotoxin, epi-podophyllotoxin, podophyllotoxone, and other aryl tetrahydronaphthalene lignans, flavonoids like quercetin, quercetin-3-glycosides, 4 -demethylpodophyllotoxin glycoside, podophyllotoxin glycoside, 4 -demethylpodophyllotoxone, deoxypodophyllotoxin, dehydropodophyllotoxin, kaempferol and astragalin or kaempferol-3-glucoside [11, 12] with a diverse array of biological activities. Etoposide and teniposide, semi synthetic derivatives of glycoside from podophyllotoxin, form an integral part of the therapeutic regimen used for chemotherapy [18], and have also triggered further studies in the design and synthesis of other potentially useful anticancer compounds [19–21]. The radioprotective effects shown by this plant have been attributed to its ability to reduce the generation of reactive oxygen and nitrogen
species, stabilize the membrane potential and augment the levels of glutathione [22], scavenge free radicals and protect mitochondria [23] against oxidative damage. The radioprotective effect of P. hexandrum was first reported by our group [23, 25, 26]. Earlier studies in our group have shown that the aqueous extract of P. hexandrum provides over 80% radioprotection to Hep G2 cells [24], and also to Strain ‘A’ mice subjected to whole-body lethal (10 Gy) gamma radiation [23]. The crude (whole) extract of P. hexandrum rhizomes has also been reported to modulate antioxidant enzyme levels, protect against ionizing radiationinduced DNA damage, protect gastrointestinal, reproductive, and central nervous system, against radiation-induced damage [23]. The role of antioxidant compounds in mitigating oxidative damage to biological systems has been reported by several workers [27–30]. However, the precise mode of action of these compounds, e.g., whether they reduce free radical generation by chelating transition metal ions or by scavenging free radicals and/or modulating intrinsic antioxidants is required to be delineated to explain their role in rendering radioprotection. Therefore, in the present investigation, we fractionated the plant material of high-altitude P. hexandrum (HAPH) using solvents of differing polarity. The bioactivities of five different fractions of P. hexandrum were evaluated under in vitro conditions by measuring reduction of radiationinduced LPO, chelation of metal (ferrous) ions, antioxidative properties and their ability to reduce DNA strand breaks.
Materials and methods Materials All chemicals and reagents used for the study were of high purity. Ferric chloride, sodium sulphite, ferrous sulphate, 2-2 -bipyridyl, potassium di-hydrogenorthophosphate, di-potassium hydrogen orthophosphate, potassium ferricyanide, trichloroacetic acid, di-sodium ethylene diamine tetra acetic acid, ascorbate, thiobarbituric acid (TBA), electrophoresis grade plasmid (pBR322) DNA, bromophenol blue, xylene cyanol FF, 15% Ficoll, glycerol, ethidium bromide, and molecular biology grade di-sodium tris-acetateethylene diamine tetra acetic acid (TAE), were purchased from Sigma Chemicals (St. Louis, MO, USA). Metal-free micro-centrifuge tubes, pipette tips and gamma rays sterilized 35-mm petridishes were obtained from Tarsons (Kolkata, India). Dimethylsulfoxide and hydrochloric acid were obtained from BDH Chemical Co. (Toronto, Ontario, Canada). The rest of the chemicals utilized for this study were of analytical reagent (AR) grade and were obtained from reputed local suppliers in India.
195 Plant material (high-altitude Podophyllum hexandrum (HAPH)) Collection, authentication and processing The rhizome of P. hexandrum Royale (syn. Podophyllum emodi Wall) was collected from the high-altitude regions (>3000 m) of Leh and Ladakh, Jammu and Kashmir, India. The plant material was identified by Dr. Om Prakash Chaurasia, Ethnobotanist, Field Research Laboratory (FRL), Leh (J&K), and further authenticated on the basis of botanical characteristics by Dr. Rajesh Arora, INMAS, Delhi. Voucher specimens have been deposited in the repositories, both at INMAS, Delhi and RRL, Jammu [voucher no.: INM/FRLPH/Leh-2004]. Care was taken to free the plant material of foreign matter like soil, dust, insects and other extrinsic contamination. Organoleptic examination was conducted and the rhizomes were found to be brittle and snapped upon applying pressure. They possessed a phenolic odor with bitter taste. The powdered plant material was also subjected to analysis for determination of microbial counts, total fungal counts, etc.
Phytochemical screening The qualitative and phytochemical analysis of the powdered sample was carried out for the presence of various secondary metabolites such as polyphenols, lignans and glycosides (Table 2) using the method of Harborne [31]. Fractionation of HAPH The powdered plant material was transferred to a Soxhlet apparatus and consecutively extracted with solvents of increasing polarity viz., hexane, chloroform, alcohol, alcohol–water and water for a minimum of three times using proportionate amount of solvent over the course of 24–72 h and the respective filtrates were combined. The pooled filtrates were filtered through Whatman Paper No. 3, and concentrated by solvent evaporation under reduced pressure in a rotary evaporator (Buchi, Switzerland) and dried. The yield was determined on w/w basis separately for each fraction. The dried fractions were pulverized through a micropulverizer and passed through a number 40 sieve. The yield of extracts from nhexane, chloroform, alcohol (ethanol), 50% alcohol, and water on w/w basis was 0.56, 2.14, 18.28, 14.29, 8.00%, respectively (Table 1). The extracts were designated as HE, CE, AE, HA, and WE, respectively.
Standardization and quality control of HAPH Standardization and quality control of herbal material of P. hexandrum was carried out as per FAO/UNDP/WHO norms (Table 1). The rhizome (3.5 kg) was air dried in shade after collection and powdered mechanically till a moderately fine powder was obtained, and then stored at room temperature till the material was subjected to extraction.
Table 1. Standardization and quality control of herbal material of Podophyllum hexandrum 1. Loss on drying at 105 ◦ C
5.94%
2. Total ash content
4.0679%
3. Acid insoluble ash
0.2938%
4. Microbial counts; Escherichia coli (CFU/g) Total fungal content (CFU/g)
HA (2.32 mg%) > WE (1.42 mg%) > CE (1.25 mg%) > HE (0.46 mg%). In the liquid chromatographic separation, it was found that a gradient of methanol (A) and water (B) in the following manner: 1–60 min (A:B; 65:35), and finally 60–70 min (A:B; 65:35) v/v at a flow rate of 0.6 ml/min was the most optimal mobile phase. The LC/MS pattern of reconstructed ion chromatogram of three samples of P. hexandrum, collected at different times, matched except for a very slight variation in the relative intensity of the peaks. HPLC profiles of different fractionated extracts is represented in Fig. 1. A number of aryltetrahydronapthalene and related lignans were identified in the different fractions by analyzing the fragmentation patterns, as revealed in corresponding tandem mass spectra. The major fragment ions observed in the respective tandem mass spectra are summarized in Table 2. The molecular weights of two selected components (podophyllotoxin and podophyllotoxinβ-D-glucopyranoside) obtained online from the full scan ion spray mass spectrum at their corresponding retention time, are represented in Figs. 2(a) and 2(b).
Metal chelation activity of fractions of HAPH In this test method, the metal ion chelating activity of different fractions of HAPH was measured by 2,2 -bipyridyl method. The metal chelation activity was found to increase concomitantly with increase in concentration (µg/ml) of the different fractions in all the samples tested (Fig. 3). Maximum metal chelation ability was evaluated as percentage inhibition of iron–2,2 -bipyridyl complex (chromogen) formation. The maximum percent inhibition observed in case of HE, CE, AE, HA, WE and quercetin (used as standard) was 26.6% (40 µg/ml), 41.59% (10 µg/ml), 31.25% (2.5 µg/ml), 21.71% (5 µg/ml), 20.48% (2.5 µg/ml) and 34.9% (50 µg/ml), respectively. All the values were found to be significant ( p < 0.05) vis-a-vis control (0% inhibition).
Statistics Each experiment was performed in triplicate and repeated three times. All results are expressed as mean ± S.D. or as percentage. Statistical analysis of data was performed by using Student’s t-test and a p value < 0.05 was considered significant.
Antioxidant activity of fractions of HAPH The antioxidant activities of natural compounds are known to have a direct correlation with their power to act as reducing agents. The reducing power of the extract was, therefore,
199
Fig. 1. LC profiles of (a) n-hexane, (b) chloroform, (c) alcohol and (d) hydro-alcoholic fractions from high-altitude Podophyllum hexandrum.
compared with butylated hydroxyl toluene (BHT), a standard antioxidant compound, which served as control. The concentration to attain unit absorbance at 700 nm was 0.230541 ± 0.091607 for BHT and 0.331 ± 0.07092, 0.0750 ± 0.0021, 0.0389 ± 0.00717, 0.044 ± 0.0063, 0.351 ± 0.0359 in case of HE, CE, AE, HA and WE, respectively. The antioxidant activity of AE and HA was found to be much higher than the other fractions of HAPH as well as BHT (Fig. 4).
Modulation of radiation-induced DNA damage assay A comparative study was designed to evaluate the radioprotective effect of different fractions, viz., HE, CE, AE, HA and WE against 250-Gy gamma ray–induced DNA damage in pBR322 plasmid DNA. DNA-strand damage was semiquantitatively measured by converting double stranded supercoiled DNA (fast migrating) into nicked open circular form (slow migrating) [37] (Figs. 5 and 6). Quercetin (a known radioprotector) was used as a standard compound for comparison. The untreated control (pBR322 DNA) comprised of more than 95% supercoiled form, while upon exposure to 250-Gy gamma radiation, nearly 75% relaxed form (open circular DNA) was observed. The different doses of fractions (in the range of 10–50 µg/ml) were evaluated for their protective efficacy against radiation (data not shown), in terms
of percentage of supercoiled form of DNA retained. The doses that showed appreciable results were chosen for the purpose of comparison (HE: 10 µg/ml, CE: 30 µg/ml, AE: 40 µg/ml, HA: 10 µg/ml, WE: 10 µg/ml). A comparative analysis of pBR322 DNA pretreated (−1 h) with different fractions and then irradiated (250 Gy) revealed that the bioconstituents present in CE and HE helped in, retention of 68.09 and 68.02% SC form respectively, which was higher than that of quercetin (30 µg/ml) treated group, which retained 63.64% of SC form. In comparison to non-polar fractions, the polar fractions viz., AE and WE showed 60.59 and 51.13% retention of SC form, which was significantly protective ( p < 0.05) as compared to radiation control (25% SC form). Among the different fractions, the densitometric analysis of gels showed that the protection (%SC form) against radiation-induced damage follows the order: CE (68.09%) > HE (68.02%) > quercetin (63.64%) > AE (60.59%) > WE (51.13%) > HA (36.6%) Modification of radiation (250 Gy)-induced lipid peroxidation The radiation-induced LPO activity in the liver homogenate was found to decrease in a dose-dependant manner and
200
(a)
(b) Fig. 2. MS/MS of (a) podophyllotoxin and (b) podophyllotoxin-β-D-glucopyranoside.
maximum inhibition i.e., 91.686, 90.135 and 92.249, 93.914, 91.487% was observed at 2000 µg/ml in case of HE, CE, AE, HA and WE, respectively (Figs. 7a–7e). All the values were found to be significant ( p < 0.01) with respect to control (0% inhibition). The ratio of decrease in LPO activity (nm of MDA formed per hour per gram of tissue) in the range of 50–2000 µg/ml was 8.4, 2.29, 1.33, 6.96, 7.57 for HE, CE, AE, HA and WE, respectively. Lower ratio is indicative of effectiveness of the fraction even at lower concentration. Modification of iron/ascorbate-induced lipid peroxidation The iron/ascorbate-induced LPO activity in the liver homogenate was found to decrease in a dose-dependant manner and maximum inhibition, i.e. 99.35, 97.22, 99.76, 88.10, 87.45% was observed at 2000 µg/ml for HE, CE, AE, HA and WE, respectively (Figs. 7a–7e). All the values were found to be significant ( p < 0.01) with respect to control (0%
inhibition). The ratio of decrease in LPO activity (nanomoles of MDA formed per hour per gram of tissue) at 50–2000 µg/ml for HE, CE, AE, HA and WE was 149.8, 12.5, 312.6, 6.9 and 6.54, respectively. Lower ratio is an indication of effectiveness of the fraction even at lower concentration. Modification of iron/ascorbate- and radiation (250 Gy)-induced lipid peroxidation The combined stress of iron/ascorbate with radiation (250 Gy) mimics the biological condition maximally. The initiators, radiation-induced (free radicals) and the amplifiers (iron) worked in a coordinated manner to generate a lethal stress, hence maximum LPO activity was observed in control (0% inhibition). The different concentrations of the fractions decreased the activity in a dose-dependant manner and maximum inhibition i.e., 95.66, 93.92, 92.08, 93.05, 91.4% was observed at 2000 µg/ml for HE, CE, AE, HA
201
Fig. 3. Effect of different concentrations of various fractions of HAPH on chelation of metal ion. The percentage inhibition of M2+ –2,2 -bipyridyl chromogen complex containing all reagents, but without the drug was considered as 0% inhibition. ∗ Maximum percentage inhibition of chromogen complex formation was observed with respect to control (0% inhibition) ( p < 0.05). Quercetin was used as a positive control.
Fig. 4. Evaluation of reducing power of different fractions of HAPH. The absorbance at 700 nm was recorded in triplicate and each experiment was repeated thrice. The values are expressed as mean ± S.D. BHT, a standard synthetic antioxidant was used as control. ∗ Fractions of Podophyllum hexandrum with respect to control ( p < 0.05).
and WE, respectively (Figs. 7a–7e). All the values were found to be significant ( p < 0.01) with respect to control (0% inhibition). The ratio (activity at 50: 2000 µg/ml) of decrease in LPO activity (nanomoles of MDA formed per
hour per gram of tissue) for HE, CE, AE, HA and WE was 18.31, 6.15, 13.70, 12.20, 9.14, respectively. Lower ratio is an indication of effectiveness of fraction even at lower concentration.
202
Fig. 5. Densitometric analysis: 1, control (pBR322 plasmid DNA); 2, 250-Gy-treated pBR322 plasmid DNA; 3, pBR322 plasmid DNA + quercetin (30 µg/µl); 4, pBR322 plasmid DNA + quercetin (30 µg/µl) + 250 Gy; 5, pBR322 plasmid DNA + HE (10 µg/µl); 6, pBR322 plasmid DNA + HE (10 µg/µl) + 250 Gy; 7, pBR322 plasmid DNA + CE (30 µg/µl); 8, pBR322 plasmid DNA + CE (30 µg/µl) + 250 Gy; 9, pBR322 plasmid DNA + AE (40 µg/µl); 10, pBR322 plasmid DNA + AE (40 µg/µl) + 250 Gy; 11, pBR322 plasmid DNA + HA (40 µg/µl); 12, pBR322 plasmid DNA + HA (40 µg/µl) + 250 Gy; 13, pBR322 plasmid DNA + WE (30 µg/µl); 14, pBR322 plasmid DNA + WE (30 µg/µl) + 250 Gy.
Fig. 6. Effect of different fractions of HAPH against radiation (250 Gy)-induced DNA damage. %SC form (supercoiled form) of pBR322 DNA retained represents percentage protection, while %OC form (open circular form) represents percentage damage. Each experiment was performed in triplicate and was repeated three times. ∗ p < 0.05. Drug + radiation (250 Gy) vs. radiation (250 Gy).
Discussion Antioxidant compounds are known to influence peroxidation, DNA damage process mainly due to their free radical scavenging, divalent ion chelation properties [38, 39]. Homeostatis is maintained within the cell by the combined action of nutritionally occurring antioxidants, antioxidant enzymes like superoxide dismutase, catalase, glutathione-Stransferase (which catalyze the free radicals into unreactive products), and certain high molecular weight proteins i.e., ceruloplasmin, transferrin, albumin, which bind with transition metals and clear them from the extracellular milieu, due to their inherent capacity to react with hydrogen peroxide to form ferryl, perferryl species, which can initiate LPO [40]. In the present study, the iron chelation ability of different fractionated extracts of P. hexandrum was tested using
2,2 -bipyridyl assay [33]. The activity of CE fraction was found to be substantially higher at even lower most concentration (1 µg/ml: 34.86%) and was almost equal to the metal chelation ability of quercetin (34.9%) and CE (35.4%) at 50 µg/ml respectively. The inhibition of formation of chromogen complex by the different fractions followed the order: CE (41.59%) > quercetin > AE (31.25%) > HE (26.6%) > HA (21.17%) > WE (20.48%). The aforementioned results depict that CE fraction has highest metal chelation activity as compared to other fractions, which is likely due to the antagonistic and/or synergistic effect of the chloroformsoluble constituents, lignans including podophyllotoxin and its derivatives (Table 2). Exposure to ionizing radiation leads to generation of free radicals, which increases LPO and also enhances the degradation of hemoglobin, ultimately leading to increase in free cytosolic pool of iron [6], which acts as a secondary initiator. As secondary initiators, iron ions
203
(a)
(b) Fig. 7. Effect of different concentrations of (a) HE, (b) CE, (c) AE, (d) HA, and (e) WE against iron/ascorbate, radiation (250 Gy) and both [iron/ascorbate + radiation (250 Gy)] as a combined stress-mediated lipid peroxidation evaluated in liver homogenate of strain ‘A’ mice. Each experiment was performed in triplicate and was repeated three times and the lipid peroxidation activity expressed as nanomoles of MDA (malonialdehyde) formed × 106 . Lipid peroxidation in control represents 0% inhibition (maximum activity). (a) ∗ p < 0.05; HE + radiation (250 Gy) vs. radiation (250 Gy). ∗∗ p < 0.05; HE + iron/ascorbate vs. iron/ascorbate. ∗∗∗ p < 0.05; HE + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate. (b) ∗ p < 0.05; CE + radiation (250 Gy) vs. radiation (250 Gy). ∗∗ p < 0.05; CE + iron/ascorbate vs. iron /ascorbate. ∗∗∗ p < 0.05; CE + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate. (c) ∗ p < 0.05; AE + radiation (250 Gy) vs. radiation (250 Gy). ∗∗ p < 0.05; AE + iron/ascorbate vs. iron/ascorbate. ∗∗∗ p < 0.05; AE + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate. (d) ∗ p < 0.05; HA + radiation (250 Gy) vs. radiation (250 Gy). ∗∗ p < 0.05; HA + iron/ascorbate vs. iron/ascorbate. ∗∗∗ p < 0.05; HA + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate. (e) ∗ p < 0.05; WE + radiation (250 Gy) vs. radiation (250 Gy). ∗∗ p < 0.05; WE + iron/ascorbate vs. iron/ascorbate. ∗∗∗ p < 0.05; WE + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate. (Continued on next page)
204 catalyze OH• radical formation thereby accelerating LPO [41, 42]. The extent of initial damage caused by free radicals is further amplified by Fenton reaction generated hydroxyl radicals in the presence of superoxide and hydrogen peroxide [43]. Thus, the redox state and concentration of iron ions in the cellular milieu plays a crucial role in amplification of damage [44] as they interact with membranes to generate alkoxyl and peroxyl radicals, thereby inflicting further damage to the cellular system [45]. Several workers have reported that chelating agents like diethylenetriamine
pentaacetic acid exhibit antioxidant potential since they are able to occupy all the aqua-coordination sites of transition metals required for OH• generation [46]. The polyphenolic components present in all fractions of P. hexandrum contributed via their dual ability to donate hydrogen atoms (chain-breaking antioxidants) and chelate transition metal ions (secondary antioxidants). The results clearly indicated that the fractionated extracts are able to modulate the concentration of free iron in biological system and are thereby able to tackle the radiation-induced oxidative stress.
(c)
(d) Fig. 7. (Continued)
205
(e) Fig. 7. (Continued)
Antioxidant activity of different fractions was assayed by using the method of Oyaziu [34]. The antioxidant activities of natural compounds have a direct correlation with their reducing ability (electron donation capacity). The antioxidant activity of different fractionated fractions extract, as compared to BHT on the basis of their absorption unit values, was in the following order: AE > HA > CE > BHT > HE > WE (Fig. 4). AE showed maximum antioxidant potential, which could be attributed partially to its polyphenolic content, which was maximally present in this fraction. On the other hand, the fractions, viz., HE and WE, exhibited lesser antioxidant potential (even lesser than that of BHT), which correlated with the lower polyphenolic content of the fraction. CE showed intermediate antioxidant potential, overlapping with that of BHT, which could be attributed to its polyphenolic content (>1%) and also due to the presence of lignans such as 4 -demethylpodophyllotoxin, epi- podophyllotoxin, podophyllotoxin, its glycoside and other compounds. This gives an indication that polyphenols and lignans play a vital role in enhancing the overall electron donation capacity of extract acting in synergism and antagonism along with other constituents. The reducing power was found to increase with increasing fraction concentration. These results correlate well with other published reports [30] through with different extracts indicating that some of the compounds in the extracts were electron donors and could react with free radicals to terminate radical chain reactions and, therefore, were able to boost the natural antioxidant defence mechanism [47, 48].
Gamma radiation induces damage in biological systems mainly via free radical generation, which interact with biomolecules including DNA and lipid present in cell membranes [49, 50]. Plasmid relaxation assay was used for semiquantitative assessment of the ionizing radiation-induced oxidative damage to DNA. It is a more sensitive method to assess the DNA damage as compared to chromatographic analysis of 8-OHdG (8-oxo-7,8-dihydro-2 -deoxyguanosine) estimation [51]. Plasmid (pBR322) DNA in supercoiled form was nicked to generate open circular form, which was the result of singlestranded cleavage of supercoiled DNA. Wang and co-workers have suggested that single-strand damage in DNA is primarily due to the generation of OH• radical in free solution, while the intrastrand cross-links might be formed following metal ion binding to phosphate groups [52]. Radiation (250 Gy) caused extensive oxidative damage resulting in DNA fragmentation and degradation (Figs. 5 and 6). Exposure of pBR322 to 250 Gy gamma-radiation caused complete conversion of supercoiled pBR322 DNA (fast mitigating), into open circular form (slow mitigating). In the present study, evaluation of the effect of herbal fractions against 250-Gy gamma rays–induced DNA damage in pBR322 plasmid DNA revealed their protective efficacy against gamma radiation. Quercetin (a wellknown standard flavonoid), tested along with different fractions, at a dose of 30 µg/µl, was found to render protection against DNA damage. Non-polar fraction, viz., CE showed maximum protection (68.09% SC form), which was even higher than that of quercetin (63.64% SC form) and polar fractions, viz., WE (51.13%). This can be attributed to the
206 synergistic effect of polyphenolic compounds and lignans present in the different fractions. Quercetin and kaempferol, present in P. hexandrum are known to attenuate DNA damage [22, 24]. Anti-lipid peroxidation activity of HAPH was evaluated on the basis of TBARS estimation [36]. The effect of different fractions on induction of LPO in liver homogenate by non-enzymatic (iron/ascorbate) method, radiation (250 Gy) and both iron and radiation in combination further unraveled the mechanism by which the fractions act at tissue level. The percentage inhibition of iron/ascorbate-induced LPO offered by AE, HE and CE was similar i.e., 99.76, 99.35 and 97.22%, respectively, while that of HA and WE was comparatively lower i.e, 88.10 and 87.45%, respectively. AE showed maximal inhibition (99.76%), which is in conformity with its higher metal chelation activity (31.25%), maximal polyphenolic content (9.26 mg%) and antioxidant potential. However, on comparing the range of inhibition, polar fractions were found to be effective at all the concentrations, tested in the present study, while non-polar fractions and intermediately polar fractions were mainly effective at higher concentration. AE showed maximum protection (92.25%) against radiation-induced LPO. It can be concluded that AE provided protection both via metal chelation and through electron donation. It is known that during irradiation, as a cascading effect due to increase in high-energy electrons, followed by interaction with water, hydroxyl, superoxide radicals and hydrogen peroxide are formed [53, 54]. These reactive molecules attack lipid molecules and again form new free radicals and the chain continues. Iron ions further amplify the cascade of reaction causing leaky membranes, which leads to ion exchanges, initiating the cell death pathway. In the state of combined stress (iron/ascorbate and radiation), HE inhibited the LPO maximally, but exhibited metal chelation activity to a moderate extent and had higher inhibitory ratio (149.83). It was, therefore, effective mainly at higher concentration. On the contrary, CE also inhibited the LPO (iron/ascorbate- and radiation-induced) in a similar way to HE, but showed lower inhibitory ratio, maximum metal chelation activity (41.59%), high antioxidant activity and ability to provide DNA protection (68.09% SC form retention). Similarly, the inhibitory effect of different fractions on iron/ascorbate-induced LPO can be related to the presence of polyphenolic compounds like quercetin, and kaempferol in the fractionated extracts, which exhibit antioxidant properties [55, 56] along with lignans (known for their potent free radical scavenging activity) and numerous glycoside derivatives. Polyphenols are composed of one (or more) aromatic rings bearing more than one hydroxyl groups and are capable of scavenging free radicals by forming resonance stabilized phenoxy and quinone radicals [57]. Quercetin, which is known to have O-dihydroxy and 4,5-hydroxy ketone as substituent, exerts a strong scav-
enging activity effect, which is reported to be even better than that of trolox [58]. Polyphenolic compounds have been reported to exhibit the ability to donate their hydrogen atom in the initial stage of LPO to compete with polyunsaturated fatty acids, thereby breaking the propagation chain. The plausible mechanism by which these fractions inhibited LPO is indicative of free radical scavenging and to a certain extent metal ion chelation capacity. Similar results have been reported by other workers [55, 56]. These results clearly indicated that fairly polar fraction (AE) has highest potential to mitigate the oxidative stress induced by radiation. Further studies are needed at in vitro/in vivo level to unravel the site-specific molecular mechanisms involved and other inherent activities that are relevant to combat radiation stress.
Acknowledgements We are thankful to the Directors of INMAS and RRL, Jammu for providing research facilities and to Col. (Dr.) B. Raut, Director, FRL, Leh and Dr. Om Prakash Chaurasia, Scientist, FRL, Leh for kindly providing the rhizomes of Podophyllum hexandrum. The work was supported by research funds obtained from the DRDO’s CHARAK programme.
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