INTRODUCTION:

The etiology and pathogenesis of human diseases like diabetes mellitus, atherosclerosis, hypertension, ischemic diseases, Alzheimer’s diseases, Parkinsonism, cancer and inflammatory conditions are primarily due to the imbalance between pro-oxidants and antioxidants homeostasis ¹. Pro-oxidant reactive oxygen species (ROS), for example are normal products of aerobic metabolism ². However, excess free radicals can result from tissue damage and hypoxia, overexposure to environmental factors (smoking, ultraviolet radiation and pollutants) a lack of antioxidants or destruction of free radical scavengers ³.

Oxygen derived free radicals such as superoxide anions, hydroxyl radicals and hydrogen peroxide are cytotoxic and give rise to tissue injuries. In addition, oxidative stress causes inadvertent enzyme activation and oxidative damage to cellular system.

Cells are equipped with different kinds of mechanisms to fight against ROS and to maintain the redox homeostasis of cell. For example, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) play important roles in scavenging the free radicals and preventing cell injury⁴. Molecules such as ascorbic acid and α-tocopherol inhibit peroxidation in cell. When the mechanism of antioxidant protection becomes unbalanced in human body, antioxidant supplement may be used to help reduce oxidative damage.

Medicinal plants are an important source of antioxidants⁵. Natural antioxidants increase the antioxidant capacity of the plasma and reduce the risk of certain diseases such as cancer, heart diseases and stroke⁶. The secondary metabolites like phenolics and flavonoids from plants have been reported to be potent free radical scavengers. They are found in all parts of plants such as leaves, fruits, seeds, roots and bark⁷. There are many synthetic antioxidants in use. It is reported, however, they have several side effects⁸, such as risk of liver damage and carcinogenesis in laboratory animals^9-11. There is therefore a need for more effective, less toxic and cost effective antioxidants. Medicinal plants appear to have these desired comparative advantages, hence the growing interest in natural antioxidants from plants.

Several medicinal plants (Rasayana) have been extensively used in the Indian traditional (Ayurveda) system of medicine for the treatment of number of diseases ¹². Some of these plants have shown potent antioxidant activity^{13, 14}. However, majority of plants have not yet been screened for such activity. So, in order to contribute further to the knowledge of Indian traditional plants, our present study is focussed on three plants namely Flemingia chappar, Flemingia macrophylla and Flemingia strobilifera to determine their antioxidant and free radical scavenging properties. Flemingia is a genus of flowering plants in the legume family, Fabaceae. About 15 species occur in India, out of which three are available in Uttarakhand. Flemingia chappar Ham.ex Benth is commonly known as Salpan. Flemingia strobilifera R.Br. commonly known as Kusrunt and Flemingia macrophylla (willd.) Kuntze is commonly known as Barasalpan¹⁵.The literature survey showed scanty information available on these plants and thus prompted us to analyze these traditionally used plants. In the present study, we carried out a systematic record of the relative free radical scavenging activity of ethanol extract of F.chappar, F. macrophylla and F.strobilifera. Further an attempt has also been made to find the relationship between flavonoid and phenolic content with antioxidant activity of these plants.

MATERIALS AND METHODS:

Plant material and extract preparation:

Fresh roots of three Flemingia species were, collected from Regional Research Institute, tarikhet, Ranikhet, India and were preliminary identified by Dr. G.C. Joshi, Research Incharge, Regional Research Institute, tarikhet, Ranikhet, India and which were later on confirmed from National Botanical Research Institute (NBRI) Lucknow, India (vide access no. NBRI/CIF/227/2011 and NBRI/CIF/347/2012). Voucher specimen of these three Flemingia species have been kept in Laboratory of School of Pharmaceutical Sciences, IFTM University for future reference. The roots were air dried, pulverized to a coarse powder in a mechanical grinder, passed through a 40 mesh sieve, and extracted in a soxhlet extractor with ethanol. The extracts were decanted, filtered with Whatman No. 1 filter paper and concentrated at reduced pressure below 40 ºC through rota-vapor (Rotavapor RII, Buchi Labortechnik AG, Switzerland) to obtain dry extracts. The extracts were subjected for preliminary phytochemical screening to show the presence of steroids, flavonoids, tannins, carbohydrates and fatty acids according to standard methods^16-17.

Total Phenolic content (Folin-ciocalteau assay):

Total phenolic contents of EFS, EFM and EFC were determined using Folin-ciocalteau assay¹⁸. Briefly, 100mg of fractions were individually dissolved in 10 ml of methanol. Then, 0.1ml of these solutions were mixed with 2.5 ml of 10 fold diluted Folin-ciocalteau reagent, and 2.0 ml of 7.5% sodium carbonate (Na₂CO₃). After incubation at 40 ºC for 30 min, the absorbance of the reaction mixtures was measured at 760 nm by using a spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). Gallic acid was used as a standard and TPC of the extracts were expressed in milligram gallic acid equivalents (mg GAE/g extract).

Total flavonoid content:

Total flavonoid content was determined by the aluminium calorimetric method¹⁹, using quercetin as standard. Briefly, the test samples were individually dissolved in DMSO. Then, the sample solution (150 μl) was mixed with 150 μl of 2% AlCl₃. After 10 min of incubation at ambient temperature, the absorbance of the supernatant was measured at 435 nm by using a spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). Three replicates were made for each test sample. The total flavonoid content was expressed as quercetin equivalents in microgram per gram extract (μg QRT/g extract).

In vitro antioxidant assays:

ABTS assay:

The antioxidant potential was measured by 2, 2´-azinobis – (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay that measures the relative ability of antioxidant substances to scavenge the ABTS⁺ cation radical generated in the aqueous phase. 3.5 ml reaction mixture contained 0.17 mM ABTS, 25 – 250 μg/ml EFS/ EFM/ EFC/ ascorbic acid and phosphate buffer (pH 7.4). The method used was based on Miller and Evans 1996²⁰ modified by Lister and Wilson 2001²¹. The absorbance at 734 nm was measured using UV–Vis spectrophotometer. The antioxidant capacities of samples were measured against the standard.

Determination of DPPH radical scavenging activity:

The free radical scavenging activity was evaluated by the DPPH assay²². In its radical form, DPPH absorbs at 517nm, but upon reduction by an antioxidant or a radical species, the absorption decreases. Briefly, 1ml of 0.25mM solution of DPPH in methanol was added to 1ml of EFC/ EFM/ EFS solution in methanol (25 – 250 μg/ml). After 20min, the absorbance was measured at 517nm. Ascorbic acid was used as a positive control. The percentage DPPH decolorisation of the sample was calculated by the equation,

% of DPPH scavenging = [(A_control – A_extarct)/ A_control] × 100

Where A is the absorbance

Nitric oxide radical scavenging (NO) assay:

The nitric oxide radical inhibition activity was measured²³ using Griess reagent. Briefly, sodium nitroprusside (10mM) in phosphate buffered saline was mixed with different concentrations of EFC/ EFM/EFS and ascorbic acid dissolved in methanol and incubated at room temperature for 150 min followed by addition of 0.5 ml of Griess reagent (1% sulfanilamide, 2 % H₃PO₄ and 0.1% N-(1 naphthyl)ethylenediamine dihydrochloride). The absorbance of the chromophore formed was read at 546 nm.

Hydroxyl radical scavenging (OH) assay:

Hydroxyl radical scavenging activity was determined based on the ability to compete with deoxyribose for hydroxyl radicals²⁴. Hydroxyl radicals produced by the reduction of H₂O₂ by iron, in presence of ascorbic acid degrade deoxyribose to form products, which on heating with 2-thiobarbituric acid (TBA) form a pink colored chromogen. Briefly, the reaction mixture, of a final volume of 1.0ml, containing 0.4 ml of 20mM sodium phosphate buffer (pH 7.4), 0.1 ml of 25 – 250 μg/ml of EFC/ EFM/EFS, 0.1 ml of 60 mM deoxyribose, 0.1 ml of 10 mM H₂O₂, 0.1 ml of 1 mM ferric chloride, 0.1 ml of 1 mM EDTA and 0.1 ml of 2 mM ascorbic acid, was incubated at 37º C for 1h. The reaction was terminated by the addition of 1 ml of 17 mM TBA and 1 ml of 17 mM trichloroacitic acid (TCA). The mixture was boiled for 15 min, cooled in ice, and the absorbance measured at 532 nm. Ascorbic acid was used as a positive control. Distilled water in place of test fractions or ascorbic acid was used as control and the sample solution without deoxyribose as sample blank.

Ferric reducing antioxidant power (FRAP) assay:

The reductive potential was determined based on the chemical reaction of Fe³⁺ to Fe²⁺ ²⁵. To 100 – 500 μg/ml EFS/ EFM/EFC and ascorbic acid standard in 1 ml of methanol, 2.5 ml each of phosphate buffer (0.2 M, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (1% w/v) was added and the mixture incubated at 50 ºC for 20 min, followed by addition of 2.5 ml of TCA (10% w/v). The mixture was centrifuged for 10 min at 1000g, the upper layer (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1% w/v), and the absorbance was measured at 700 nm.

Statistical analysis:

The data are expressed as Mean ± Standard Deviation (S.D.). All data were analysed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test with equal sample size. The difference was considered significant when p value < 0.05.

RESULTS:

Phytochemical screening:

The results of the preliminary phytochemical screening of the EFS, EFM and EFC showed the positive results for steroids, flavonoids, tannins, carbohydrates, proteins etc.

Total phenolic content and total flavonoid content:

The content of phenolics compounds in the different fractions were determined through a linear gallic acid standard curve (y = 0.009x + 0.044, r² = 0.997). The total phenolic content of the fractions varied form 9.072± 1.021 to 14.745± 0.315 mg GAE /g extract. EFS found to have more phenolic content than EFM and EFC. The total phenolics in EFS was 14.745 mg /GAE/g in EFM was 12.725 mg GAE/g and in EFC was 9.072 mg /GAE/g . Total phenolics content of the extracts are arranged in the following ascending order: EFC < EFM<EFS (p<0.01) (Table 1). In this study, the total flavonoid content (TFC) of the extracts were evaluated by aluminum colorimetric assay. Quercetin (QRT) was used as a standard and the total flavonoid content of ethanol extracts of Flemingia species were expressed in microgram of quercetin equivalents per gram of extract (μg QRT/g extract) (y=0.0125x + 0.023, r² = 0.9773). The data presented in Table 1 indicates that the highest flavonoid content of 16.715μg QRT/g extract was observed in the EFS and comparatively lower content were observed in EFM (13.853μg QRT/g extract) and in EFC (10.657 μg QRT/g extract )(p<0.001). TFC of the extracts is arranged in the following sequence: EFS>EFM>EFC (p<0.01).

Table 1: Total Phenolic content and Flavonoid content of EFS, EFM and EFC (n=3)

Sl. No.	Samples (extracts)	Total Phenolic content (mg GAE/g extract)	Total flavonoid content ( μg QRT/g ext)
1	EFS	14.725a***	16.715a***
2	EFM	12.725	13.853
3	EFC	9.072	10.657

Values are expressed as mean ± standard deviation (S.D.) from triplicate determination; a: EFS compared to EFC and EFM; *** p>0.001. Analysis of variance (ANOVA) followed by dunnet’s t- test was used for statistical comparison.

In vitro antioxidant assay:

Analysis of the free radical scavenging activities of the extracts revealed a concentration-dependent antiradical activity resulting from reduction of ABTS⁺ (fig. 1), DPPH (fig. 2), NO (fig. 3) and OH^- (fig. 4) radicals to non-radical form. The scavenging activity of ascorbic acid, a known antioxidant used as positive control, was however higher and scavenging potential was in the order: ascorbic acid > EFS > EFM>EFC Fig.1- 5 presents the reduction potential of EFS, EFM and EFC.

IC₅₀= 5.076 μg/ml IC₅₀= 28.435 μg/ml

IC₅₀=66.734 μg/ml IC₅₀=86.654 μg/ml

Fig. 1: Inhibition of ABTS radical by STD (Ascorbic acid), EFS, EFM and EFC; Data are represented as mean ±S.D. of two independent experiments each

IC₅₀=4.854 μg/ml IC₅₀= 33.491 μg/ml

IC₅₀=74.892 μg/ml IC₅₀= 121.72 μg/ml

Fig. 2: Inhibition of DPPH radical by STD (Ascorbic acid), EFS, EFM and EFC; Data are represented as mean ±S.D. of two independent experiments each

IC₅₀= 3.30 μg/ml IC₅₀= 20.364 μg/ml

IC₅₀= 36.78 μg/ml IC₅₀= 72.072 μg/ml

Fig.3: Inhibition of nitric oxide radical by STD (Ascorbic acid), EFS, EFM and EFC; Data are represented as mean ±S.D. of two independent experiments each

IC₅₀=5.340 μg/ml IC₅₀= 8.366 μg/ml

IC₅₀= 54.143 μg/ml IC₅₀=68.528 μg/ml

Fig.4: Inhibition of hydroxyl radical by STD (Ascorbic acid), EFS, EFM and EFC; Data are represented as mean ±S.D. of two independent experiments each

Fig.5: The reduction potential of STD (ascorbic acid), EFS, EFM and EFC (mean±S.D; n=6);

* Significantly different from EFS,

EFC and EFM (p<0.05) ; *** Significantly different from EFS, EFC and EFM (p<0.001); ^ɵ : EFS significantly different from

EFC and EFM (p<0.05). Data were analysed by applying analysis of variance (ANOVA) followed by Dunnet’s t-test.

DISCUSSION:

These results suggest that the higher levels of antioxidant activity were due to the presence of phenolic components. The same relationship was also observed between phenolics and antioxidant activity in roseship fractions²⁶. Phenols are very important plant constituents because of their scavenging ability due to their hydroxyl groups²⁷. The phenolic compounds may contribute directly to antioxidative action²⁸. It is known that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans when ingested up to 1 g daily from a diet rich in fruits and vegetables²⁹. Phenolic compounds from plants are known to be good natural antioxidants. However, the activity of synthetic antioxidants was often observed to be higher than that of natural antioxidants³⁰. Phenolic compounds, at certain concentrations, markedly slowed down the rate of conjugated diene formation. The interests of phenolics are increasing in the food industry because they retard oxidative degradation of lipids and there by improve the quality and nutritional value of food³¹. The 2, 2´ -azinobis (3-ethylbenzothiazoline 6-sulfonate) (ABTS) formed from the reaction ABTS-e → ABTS+ reacts quickly with ethanol/hydrogen donors to form colorless ABTS. The reaction is pH – independent. A decrease of the ABTS+ concentration is linearly dependent on the antioxidant concentration. All fractions at tested doses (25 – 250 μg/ml) revealed good scavenging activity for ABTS+ in a dose dependent manner, but the activity was higher in case of EFS (IC50 = 28.435 μg/ml)than EFM (IC50 = 66.734 μg/ml) and EFC(86.654 μg/ml) (Fig. 1). The DPPH radical is considered to be a model for a lipophilic radical. A chain in lipophilic radicals was initiated by the lipid autoxidation. DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule ³². The reduction capability of DPPH was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. Positive DPPH test suggests that the samples were free radical scavengers. The scavenging effect of EFS and ascorbic acid on DPPH radical was compared. On the DPPH radical, EFS had significant scavenging effects with increasing concentration in the range of 25–250 μg/ml and when compared with that of ascorbic acid, the scavenging effect of EFM and EFC were lower. The IC50 values were found to be 4.845, 33.491, 74.892 and 121.72 μg/ml for Standard, EFS, EFM and EFC respectively (Fig.2). A higher DPPH radical-scavenging activity is associated with a lower IC50 value. Nitric oxide plays an important role in various types of inflammatory processes in the body. In the present study the extracts of the roots of Flemingia species were checked for its inhibitory effect on nitric oxide production. Nitric oxide radical generated from sodium nitroprusside at physiological pH was found to be inhibited by the extracts. The EFS at varied concentrations showed remarkable inhibitory effect of nitric oxide radical- scavenging activity compared to EFM and EFC (Fig. 3). Results showed the percentage of inhibition in a dose dependent manner for all the extracts tested. The concentration of EFS needed for 50% inhibition (IC₅₀) was found to be 20.364 μg/ml, whereas 36.78 μg/ml and 72.072 μg/ml were needed for EFM and EFC respectively. The hydroxyl radical is an extremely reactive free radical formed in biological systems and has been implicated as a highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells ^33-34. This radical has the capacity to join nucleotides in DNA and cause strand breakage, which contributes to carcinogenesis, mutagenesis and cytotoxicity. Hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity ^35-36. The highly reactive hydroxyl radicals can cause oxidative damage to DNA, lipids and proteins ¹⁷. The effect of the extracts from Flemingia species on the inhibition of free radical-mediated deoxyribose damage were assessed by means of the Iron (II)-dependent DNA damage assay. The Fentone reaction generates hydroxyl radicals (OH) which degrade DNA deoxyribose, using Fe²⁺ salts as an important catalytic component. Oxygen radicals may attack DNA either at the sugar or the base, giving rise to a large number of products. Ascorbic acid was highly effective in inhibiting the oxidative DNA damage. As shown in Fig. 4, the fractions displayed potential inhibitory effect of hydroxyl radical-scavenging activity. All results showed hydroxyl radical scavenging activity in dose dependent manner. IC₅₀ values were found to be 8.366, 54.143 and 68.528 μg/ml for EFS, EFM and EFC respectively (Fig. 4). The ability of the above mentioned extracts to quench hydroxyl radicals seems to be directly related to the prevention of propagation of the process of lipid peroxidation and seems to be good scavenger of active oxygen species, thus reducing the rate of the chain reaction. Ascorbic acid was used as reference standard. It is explained that high molecular weight and the proximity of many aromatic rings and hydroxyl groups are more important for the free radical-scavenging activity by phenolics than their specific functional groups ³⁶. Generally, the reducing properties are associated with the presence of compounds which exert their action by breaking the free radical chain by donating a hydrogen atom ²⁸. The results of the ferric reducing assay indicated that EFS had stronger reducing power than EFM and EFC (Fig. 5). This could be due to the presence of more reactive concentration of bioactive constituents in EFS than EFM and EFC.

In the present study, there exists a positive correlation between the total phenolics content and the antioxidant activity which is in accordance with the earlier findings ³⁷. We found higher in vitro antioxidant activity in EFS with higher polyphenols. The higher radical scavenging efficacy of EFS may be due to retention of antioxidant phytochemicals in this extract. Moreover, these results suggest that Flemingia species may offer effective protection from free radicals and support that Flemingia species are promising sources of natural antioxidant. However, further work is required on the isolation and identification of the antioxidant components present in it since it is also a precondition for a more extensive understanding of the mechanisms involved in the antioxidant capacity.

ACKNOWLEDGEMENT:

The author is grateful to Mr. G.C. Joshi for the identification of the plant material used in this study and Dr. R.M. Dubey for providing necessary help and facilities at College of Pharmacy, IFTM, Moradabad

REFERENCES:

1. Halliwell B and Whiteman M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br. J. Pharmacol.142 (2); 2004: 231-255.

2. Lillian B et al. 2007. Total phenols, ascorbic acid, β-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food Chem. 103: 413-419.

3. Heath DS et al. A review of free radicals and antioxidants for critical care nurses. Intensive Critical Care Nursing. 21; 2005: 24-28.

4. Bergendi L et al. Chemistry, Physiology and pathology of free radicals. Life Science. 65; 1999: 1865 –1874.

5. Rice-Evans C. Flavonoids and isoflavones: absorption, metabolism and bioactivity. Free Radical Biology and Medicine. 36; 2004: 827 – 828.

6. Prior RL and Cao G. Antioxidant phytochemicals in fruits and vegetables: Diet and health implications. Horticultural Science. 35; 2000: 588 – 592.

7. Mathew S and Abraham TE. In-vitro antioxidant activity and scavenging effects of cinnamomum verum leaf extract assayed by different methodologies. Food and Chemical Toxicology, 44; 2006: 198 – 206.

8. Ito N et al. Carcinogenicity of butylated hydroxyanisole. Journal of Natural Cancer Institute.70; 1983: 343–347.

9. Gao JJ, Igalashi, Nukina KM. Radical scavenging activity of phenylpropanoid glycosides in Caryoptaris incana. Bioscience Biotechnology and Biochemistry. 63; 1999: 983 – 988.

10. Williams GM, Iatropoulos MJ, Whysner J. Safety assessment of butylated hydroxytoluene as antioxidant food additives Food and Chemical Toxicology. 37; 1999: 1027 – 1038.

11. Osawa T and Namiki M. A novel type of antioxidant isolated from leaf wax of eucalyptus leaves. Agricultural and Biological Chemistry. 45; 1981: 735–739.

12. Chopra RN, Nayer SL, Chopra IC. Glossary of Indian medicinal plants. 3^rd ed. Council of Scientific and Industrial Research, New-Delhi, India; 1992.

13. Kaur C and Kapoor HC. Antioxidant activity and phenolic content of some Asian vegetables. International Journal of Food Science and Technology 37; 2002: 153-161.

14. Aqil F, Ahmad I, Mehmood Z. Antioxidant and Free Radical Scavenging Properties of Twelve Traditionally Used Indian Medicinal Plants. Turk. J. Biol. 30; 2006: 177-183.

15. Anonymous.The Wealth of India, Raw Materials, Publication and Information Directorate, CSSIR, New Delhi, 1993. vol.4th pp.45-48.

16. Sofowora EA. Medicinal Plants and Traditional Medicine in Africa, Wiley, Chichester, 1982; p. 256

17. Evans WC. Trease and Evans Pharmacognosy. 15th Edition. W. B. Saunders, Edinburgh. 2002; p.150.

18. Meda A et al. Determination of the total phenolic, flavonoid and praline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 91; 2005: 571–577.

19. Quettier-Deleu C et al. Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum sculentum Moench) hulls and flour. J. Ethnopharmacol. 72; 2000: 35–42.

20. Miller NJ and Rice-Evans C. Spectrophotometric determination of antioxidant activity. Redox Rep. 2; 1996: 161 – 171.

21. Lister E and Wilson P. Measurement of Total Phenolics and ABTS Assay for Antioxidant Activity (Personal Communication). Crop Research Institute, Lincoln, New Zealand.

22. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature. 26; 1958: 1190 – 1200.

23. Garratt CJ. Effect of iodination on the biological activity of insulin. Nature 28; 1964: 1324 –1325.

24. Halliwell B, Gutteridge JMC and Arugma OI. The deoxyribose method: a simple test tube assay for the determination of rate constants for reactions of hydroxyl radicals. Anal. Biochem. 165; 1987: 215 – 219.

25. Oyaizu M. Studies on products of browning reaction prepared from glucose amine. Jap. J. Nutr. 44, 1986: 307–314.

26. Gao X et al. Changes in antioxidant effects and their relationship to phytonutrients in fruits of sea buckthorn (Hippophae rhamnoides L.) during maturation. J. Agric. Food Chem. 48; 2000: 1485–1490.

27. Hatano T, Edamatsu R and Mori A. Effects of interaction of tannins with co-existing substances. Chem. Pharm. Bull. 37; 1989: 2016–2021.

28. Duh PD, Tu YY and Yen GC. Antioxidative activity of water extracts of Hamg jyur (Chrysanthemum morifolium). Lebnesmittel–Wissenschaft Technol. 32; 1999: 269–277.

29. Tanaka M, Kuei CW and Nagashima, Y. Application of antioxidative maillard reaction products from histidine and glucose to sardine products. Bull. Japanese. Society. 47; 1998: 1409–1414.

30. Ningappa MB, Dinesha R and Srinivas L. Antioxidant and free radical scavenging activities of polyphenol-enriched curry leaf extract (Murraya koenigii L.). Food Chem. 106; 2007: 720-728.

31. Aneta W, Jan O and Renata C. Antioxidant activity and phenolic compounds in 32 selected herbs.Food Chem. 105; 2007: 940–949.

32. Soares JR et al. Antioxidant activities of some extracts of Thymus zygi. Free Rad. Res. 26; 1997: 469–478.

33. Hochestein P and Atallah AS. The nature of oxidant and antioxidant systems in the inhibition of mutation and cancer. Mutant Res. 202; 1988: 363–375.

34. Shajiselvini CD and Kottai Muthu A. Arch. Applied Sci. Res., 2; 2010: 54-60.

35. Babu BH, Shylesh BS and Padikkala J. Antioxidant and hepatoprotective effect of Alanthus icicifocus. Fitoterapia. 72; 2001: 272–277.

36. Pal R, Girhepunje KN Shrivastav, M.M. Hussain, N. Thirumoorthy, Annals Biol.Res., 2011, 2, 127-131.

37. Vani T et al. Antioxidant properties of the ayurvedic formulation triphala and its constituents. Int. J. Pharmacog. 35; 1997: 313– 317.

Received on 21.02.2013 Modified on 03.03.2013

Research J. Pharm. and Tech. 6(5): May 2013; Page 516-523