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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

IN VITRO ANTIOXIDANT ACTIVITY OF FLAVONOID FRACTIONS OF CYNODON DACTYLON AND

TERMINALIA CATAPPA LEAVES Saroja. M* and Annapoorani. S

Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore-43, Tamil Nadu, India

Article Received on: 11/05/12 Revised on: 29/06/12 Approved for publication: 10/08/12

ABSTRACT In vitro antioxidant activity of flavonoid fractions of Cynodon dactylon (CdFf) and Terminalia catappa (TcFf) were assessed by studying its ability to scavenge DPPH, nitric oxide and hydrogen peroxide. Scavenging of DPPH, nitric oxide and hydrogen peroxide by CdFf and TcFf were found to be dose dependent and the IC50 of CdFf and TcFf were found to be lower than that of the reference compound butylated hydroxyl toluene (BHT) These results showed the more effective antioxidative potential of CdFf and TcFf than that of BHT. This study indicates that CdFf and TcFf are a potential source of natural antioxidant. Thus, the CdFf and TcFf can be recommended as a potent antioxidant to the patients suffering from various oxidative degenerative diseases such as diabetes, arthritis, cardiovascular diseases and cancer. Key words: Cynodon dactylon,Terminalia catappa, flavonoid fraction, nitric oxide, DPPH, hydrogen peroxide. INTRODUCTION Natural antioxidants present in the plants scavenge harmful free radicals from our body. A free radical can be defined as any atom or molecule possessing one or more unpaired electrons1. In the human body the free radicals are continuously produced due to the oxygen utilization by the cells of the body 2.This generates a series of reactive oxygen species (ROS) like super oxide anion (O2

-) and hydroxyl (HO·) radicals and non free radical species such as H2O2, singlet oxygen (O2) and nitric oxide (NO). Recent studies have investigated the potential of plant products as antioxidants against various diseases induced by free radicals such as atherosclerosis, inflammatory injury, cancer, and cardiovascular disease.3Furthermore, natural products of plant origin have been proposed as a potential source of natural antioxidants with strong activity, which is mainly due to the presence of phenolic compounds like flavonoids, phenols, flavonols and proanthocyanidins4,5. Antioxidants are needed for diminishing the undesired effect and many plant-derived antioxidants have been reported6. In recent years, flavonoids have attracted the interest of researchers as a potential source of useful antioxidants.7-9 Cynodon dactylon (Family; Poaceae), commonly known as ‘doob’ (Hindi),’aroogum pillo’ (Tamil), is called creeper in Iindia. Recent studies indicated the diuretic10, anti-arthritis11, antioxidant and antidiabetic,12 DNA protective and immunomodulatory properties of shoots13 , CNS depressant14, preventive against aluminium induced neurotoxicity15, carbofuran induced oxidative stress16, aphrodisiac and male fertility activity17 were also reported in the species. Terminalia catappa (Combretaceae) is also known as Indian Almond, Malabar Almond, and Tropical Almond. The leaf juice of Terminalia catappa is used to relieve rheumatic joint pain, leprosy and skin diseases and bark is used as astringent, diuretic and cardiotonic18. The extract of T. catappa leaves inhibit Lewis lung carcinoma cells that contribute to lung cancer19. Ethanolic extract of T. catappa leaves has been shown to protect against acute liver injury produced by some hepatotoxicants20. Morioka et al 21 found that Terminalia catappa inhibit the occurrence of preneoplastic lesions in rat colon carcinogenesis. Hence the present study has been made to investigate the in vitro radical scavenging efficacy of

flavonoid fractions of Cynodon dactylon and Terminalia catappa. MATERIALS AND METHODS Collection of plant material Fresh leaves of Cynodon dactylon and Terminalia catappa were collected from the outskirts of Coimbatore district, Tamilnadu. The collected leaves were washed thoroughly in tap water, shade dried and finely powdered. Preparation of Flavonoid fractions of Cynodon dactylon and Terminalia catappa In the preliminary screening, the direct ethyl acetate extract of Cynodon dactylon and Terminalia catappa with powdered magnesium + conc.HCl. developed an orange to magenta color indicated the presence of flavonoid showed a characteristic color reaction in shinoda test.Knowing the presence of flavonoid in ethyl acetate extract, the extraction was undertaken with 20 g of powdered plant material and 200ml. of light petroleum ether (b.p. 40

0 – 60

0 C) in a Soxhlet

apparatus for 18 hours to remove the chlorophyll, non flavonoid components and lipid de waxing. 22 The treated material was dried and extracted with ethyl acetate using Soxhlet apparatus.23 This fraction is referred as CdFf and TcFf. Assessment of in vitro radical scavenging efficacy of CdFf and TcFf DPPH radical scavenging assay This was assayed as described by Elizabeth and Rao24. The reaction mixture contained Methanol-50 ml. DPPH (Diphenyl-2-picryl hydrazyl radical)- 1mM 3 ml of 1mM DPPH in methanol was added to 100μl of plant extract with concentrations ranging from 10μg to 100μg. DPPH solution with methanol was used as a positive control and methanol alone acted as a blank. When DPPH reacts with antioxidant in the sample, it was reduced and the color changed from deep violet to light yellow. This was measured at 518 nm. The percentage scavenging activity was calculated by the following formula. Scavenging activity (%) = A518 (control)-A518 (sample) ---------------------------------X 100 A518 (control) Nitric oxide radical scavenging activity Nitric oxide radical scavenging activity was measured by Green et al25. Three ml of reaction mixture containing sodium

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nitroprusside in PBS and extract was incubated at 25°C for 150 minutes. Controls were kept without test compound in an identical manner. After incubation, 0.5 ml of Griess reagent was added. The absorbance of the chromophore formed was read at 546nm.The percentage inhibition of nitric oxide generation was measured by comparing the absorbance values of control and those of test compounds. Hydrogen peroxide scavenging assay Ruch et al.26 proposed an assay for the determination of antioxidant activity of compounds by their ability to scavenge the oxidant hydrogen peroxide. The reaction mixture contained Phosphate buffer (pH-7.4) hydrogen peroxide in phosphate buffer (40mM). A solution of hydrogen peroxide (40mM) was prepared in phosphate buffer. Plant extracts at the concentration of 10mg/10μl was added to a hydrogen peroxide solution (0.6ml,40mM).The total volume was made up to 3ml.The absorbance of the reaction mixture was recorded at 230nm.The blank solution contained phosphate buffer without hydrogen peroxide. The percentage of hydrogen peroxide scavenged by the plant extract was calculated as follows: Percentage of scavenged H2O2 = A0 - A1 --------- X100 A0 A0- Absorbance of control A1- Absorbance in the presence of plant extract RESULT AND DISCUSSION In vitro antioxidative role of CdFf and TcFf was evaluated by assessing their effect on the scavenging of DPPH radical, NO and H2O2 against the standard antioxidant Butylated hydroxyl toluene (BHT). Scavenging of DPPH radical The model of scavenging the DPPH radical is a widely used method to evaluate the free radical scavenging ability of various samples27.Figure 1 shows the effect of CdFf, TcFf and BHT on the scavenging of DPPH radical. CdFf, TcFf and BHT showed a dose dependent free radical scavenging activity and reflected their antioxidative potential. The concentration of BHT required for IC50 (75 µg) was found to be more than that of CdFf (44.9 µg) and TcFf (50 µg).This indicated that the antioxidative role CdFf and TcFf was more effective than that of BHT.The antioxidant activity of CdFf and TcFf could be attributed to the presence of aromatic hydroxyl group which possess the ability to donate hydrogen radicals to DPPH for it becomes a stable diamagnetic molecule. Effect on scavenging of nitric oxide (NO) Figure 2 shows the dose dependent effect of CdFf, TcFf and BHT on the scavenging of NO. From the present study, it is very clear that the flavonoid constituents of CdFf and TcFf have the property to counteract the effect of NO formation and in turn prevent the ill effects of excessive NO generation in vivo. The IC50 of CdFf, TcFf and BHT were found to be 43 µg, 37 µg and 71 µg respectively. Effect of CdFf and TcFf on scavenging of NO was found to be more pronounced than that of BHT. Further, the scavenging activity may also help to arrest the chain reactions initiated by excessive generation of NO that are detrimental to human health. NO has been associated with a variety of physiologic processes in the human body since it was identified as a novel signal molecule. In addition to reactive oxygen species, nitric oxide is also implicated in inflammation, cancer and other pathological conditions28.

Nitric oxide plays an important role in various inflammatory processes but the overproduction of nitric oxide contributes to various diseases. The toxicity of NO increases greatly when it reacts with superoxide radical, forming the highly reactive peroxynitrite anion29. The flavonoid constituents compete with oxygen to react with NO thereby inhibiting the formation of nitrite. Scavenging of H2O2 Figure 3 shows the effect of CdFf, TcFf and BHT on the scavenging of H2O2 in a concentration dependent manner and IC 50 was found to be 40 µg, 43 µg and 71 µg respectively. Scavenging of H2O2 by CdFf and TcFf was found to be more effective than that of BHT. Scavenging of H2O2 by CdFf and TcFf might be due to the donation of electrons to H2O2, thus neutralizing it to water. Hydrogen peroxide, although not a radical species, plays a role in oxidative stress. Scavenging of H2O2 by flavonoid fractions may be attributed to their phenolics, which can donate electrons to H2O2, thus neutralizing it to water. Hydrogen peroxide is highly important reactive oxygen species because of its ability to penetrate biological membrane. Although hydrogen peroxide itself is not very reactive, it can sometimes cause cytotoxicity by giving rise to hydroxyl radicals in the cell 30.Free radical scavenging activity of CdFf and TcFf in the above three in vitro methods demonstrated the antioxidative that is, the free radical scavenging activity of CdFf and TcFf at low concentrations than the reference compound BHT. Scavenging of the stable free radical by CdFf and TcFf indicated their most effective antioxidative potential. The above results and discussion suggested that flavonoid fractions of Cd and Tc might counteract the excess free radicals produced in tissues by its antioxidative potential. CONCLUSION On the basis of the results obtained in the present study, it concludes radical scavenging efficacy of flavonoid fractions of Cynodom dactylon and Terminalia catappa thus gives scientific basics for its traditional uses as potent antioxidant to the individuals under oxidative stress such as inflammation, aging, mutagenicity and carcinogenicity. Further investigation on the isolation and identification of antioxidant component(s) in the plant may lead to chemical entities with potential for clinical use. REFERENCES 1. Madhavi DL, Deshpande SS, Sulunkhe DK . Food antioxidants: technological, toxicological and health perspectives,1996, New York, Marcel dekker. 2. Vilasrao JK., Yadunath MJ, Harshad PS and Tej AJ .Free radical scavenging activity of aqueous Solution of Black Salt. Int J Pharma Pharmaceutical Sci,2010,2(2): 95-96. 3. Hou WC, Lin RD, Cheng KT, Hung YT, Cho CH, Chen CH, Hwang SY, Lee MH. Free radical-scavenging activity of Taiwanese native plants. Phytomed,2003, 10: 170-175. 4. Pietta PG. Flavonoids as antioxidants. J Nat Prod,2000, 63: 1035- 1042. 5. Surendra Kr Sharma and Naveen Goyal. In Vitro antioxidant activity of root extracts of Heliotropium eichwaldi Stued. ex DC. RJPBCS,2012,(3) 1: 444-449. 6. Larson R (1988). The antioxidants of higher plants. Phytochemistry 27(4): 969-978. 7. Peng ZF, Strack D, Baumert A, Subramanium R, Goh NK, Chia TF, Tan SN, Chia LS.Antioxidant flavonoids from leaves of Polygonum hydropiper L. Phytochemistry,2003, 62: 219-228. 8. Bakar MFA, Mohamed M, Rahmat A, Fry J. Phytochemicals and antioxidant activity of different parts of bambangan (Mangifera pajang) and tarap (Artocarpus odoratissimus). Food Chem,2009, 113: 479-483 9. Lin KW, Liu CH, Tu HY, Ko HH, Wei BL. Antioxidant prenylflavonoids from Artocarpus communis and Artocarpus elasticus.Food Chem,2009, 115: 558-562.

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Figure 1: Percent DPPH scavenging activity of CdFf and TcFf

Figure 2: Percent nitric oxide scavenging activity of CdFf and TcFf

Figure 3: Percent hydrogen peroxide scavenging activity of CdFf and

TcFf

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