|Iranian Journal of Pharmaceutical Research
((2008), 7 (3): 211-215
Received: May 2007
Accepted: November 2007
Copyright ? 2008 by School of Pharmacy
Evaluation of Antioxidant Properties
Elaeocarpus ganitrus Roxb. Leaves
T. Sathish Kumara, S. Shanmugama, T. Palvannanb and V. M. Bharathi Kumara*
aDepartment of Biotechnology, Kumaraguru College of Technology, Coimbatore 641 006, India. bDepartment of Biochemistry, Periyar University, Salem 636 011, India.
Ethanolic extract of leaves of Elaeocarpus ganitrus was analyzed for their total antioxidant capacity, reducing power, metal chelating, ABTS+ (2, 2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical scavenging and hydroxyl radical scavenging activities. The extract at 500 μg/ml showed maximum Iron chelating activity (76.70%) followed by the scavenging of the ABTS+ radical (55.77%) at the same concentration. However, the extract showed only moderate hydroxyl radical scavenging activity (13.43%). Total antioxidant capacity was found to be 24.18 mg ascorbic acid equivalents at 500 ?g/ml extract concentration. There was a positive correlation between the total phenolic content and antioxidant capacity, R2 = 0.8547, whereas the correlation between the total flavonoids and antioxidant capacity was determined to be R2=0.8413. The results suggest that phenolics and flavonoids in the leaves provide substantial antioxidant activity.
Oxygen is essential for the survival of all living creatures on this earth. During the process of oxygen utilization in normal physiological and metabolic processes approximately 5% of oxygen gets univalently reduced to oxygen derived free radicals (1, 2) like superoxide, hydrogen peroxide, hydroxyl and nitric oxide radicals. All these radicals known as reactive oxygen species (ROS) exert oxidative stress towards the cells of human body rendering each cell to face about 10000 oxidative hits per second (3). When generation of ROS overtakes the antioxidant defense of the cells, the free radicals start attacking the cell proteins, lipids and carbohydrates (4-6) and this leads to a number of physiological disorders. Many plants often contain substantial amounts of antioxidants including vitamin C and E, carotenoids, flavonoids and tannins etc., and thus can be utilized to scavenge the excess free radicals from human body (7).
Elaeocarpus ganitrus (syn: Elaeocarpus sphaericus; Elaeocarpaceae) is a large evergreen broad-leaved tree which grows in the area from the Gangetic Plain to the foothills of the Himalayas. Elaeocarpus ganitrus is commonly known as Rudraksha tree in India. Rudraksha is used in Ayurveda for mental diseases, epilepsy, asthma, hypertension, arthritis and liver diseases (8). There is no information about the antioxidant activity of plant mentioned above. The purpose of the present study is to evaluate the antioxidant properties of ethanolic extract of leaves of Elaeocarpus ganitrus.
All chemicals used were of analytical grade. 2,2-azinobis-(3-ethylbenzothiozoline-6-sulphonate) (ABTS) was obtained from Sigma Chemicals, USA. Rutin, 2-thiobarbituric acid, mannitol, 2-deoxyribose, ferrozine, ferrous chloride, vitamin C, Folin-Ciocalteu reagent, 2,4- dinitrophenyl hydrazine, ferric chloride, potassium ferricyanide, hydrogen peroxide, aluminum chloride, sodium carbonate, sodium nitrite, ammonium persulphate, sodium hydroxide and ethanol were obtained from HiMedia Chemicals, Mumbai, India.
Plant material and its extraction
The leaves of Elaeocarpus ganitrus Roxb. were collected from Nilgiris, India. The species was identified and confirmed at Botanical Survey of India (BSI), Southern Circle, Coimbatore, India and the Voucher specimen (No. MPG 04) was retained in our laboratory for future reference. About 5 g of air dried leaves were dissolved in 50 ml of ethanol and kept in an orbital shaker for overnight. The obtained extracts were filtered with Whatman no.1 filter paper and the filtrate was collected. The ethanol was then removed under reduced pressure at 50?C to obtain the concentrated extract.
Antioxidant activities assays
Determination of total antioxidant capacity
The method described by Prieto et al. (9) was used to determine the total antioxidant capacity of the plant extract. The tubes containing 0.2 ml of E. ganitrus (100-500 ?g/ml), 1.8 ml of distilled water and 2 ml of phosphomolybdenum reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) were incubated at 95?C for 90 minutes. After the mixture had cooled to room temperature, the absorbance of each solution was measured at 695 nm using an UV/Vis spectrophotometer (Beckman DU-530). The antioxidant capacity was expressed as ascorbic acid equivalent (AAE) by using the standard ascorbic acid graph.
Reducing power assay
A method developed by Oyaizu (10) was adopted for the determination of reducing power. 2.5 ml of different concentrations of E. ganitrus (100-400 ?g/ml) were mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50?C for 20 minutes, then rapidly cooled, mixed with 2.5 ml of 10% trichloroacetic acid and centrifuged at 3000 rpm for 10 minutes. 2.5 ml of supernatant was taken. 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride were added to it. Mixed well and allowed to stand for 10 minutes. The absorbance was measured at 700 nm.
Ferrous ion chelating ability
The method proposed by Decker and Welch (11) was espoused to determine the metal chelation ability of the plant extract. 2 ml of E. ganitrus (100-500 ?g/ml) was mixed with 0.1 ml of 2 mM FeCl2 and 0.2 ml of 5 mM ferrozine solutions and allowed to react for 10 minutes at room temperature. The absorbance at 562 nm of the resulting solutions were measured and recorded. The FeCl2 and ferrozine acted as control solution. The percentage inhibition of the ferrous ion was calculated by comparing the results of the test with those of the control using the formula (12)
[1−(absorbance of test/absorbance of control)]?100................................................... (1)
ABTS+ radical scavenging assay
ABTS+ radical scavenging activity was determined according to Re et al (13). ABTS+ radical was freshly prepared by adding 5 ml of 4.9 mM ammonium persulfate solution to 5 ml of 14 mM ABTS solution and kept for 16 h in dark. This solution was diluted with ethanol (99.5%) to yield an absorbance of 0.70?0.02 at 734 nm and the same was used for the assay. To 950 ?l of ABTS radical solution, added 50 ?l of E. ganitrus (100-500 ?g/ml) and the reaction mixture was vortexed for 10 sec. After 6 minutes the absorbance was recorded at 734 nm and compared with the control ABTS solution. Percentage inhibition was calculated from the formula (1).
Hydroxyl radical scavenging activity
A modified deoxyribose method determining thiobarbituric acid reactive substances (TBARS) proposed by Res_at Apak et al. (14), 2006, used to determine the hydroxyl radical scavenging activity. To a test tube added 3 ml of phosphate buffer (pH 7.0), 1 ml of 10 mM 2-deoxy-D-ribose, 0.5 ml of 20 mM Na2-EDTA, 0.5 ml of 20 mM FeCl2 solution, 3.8 ml distilled water, 0.2 ml of E. ganitrus (100-500 ?g/ml) and 1 ml of 10 mM H2O2 in the order given, and the mixture (total volume of 10 ml) was incubated for 4 h at 37?C in a water bath. At the end of the period, the reaction was arrested by adding 5 ml of 2.8% TCA. To this added 5 ml of 1% TBA and the reaction mixture was kept in a boiling water bath for 10 minutes. The mixture was cooled under running tap water, and the absorbance at 520 nm was recorded. Percentage inhibition was calculated from the formula (1).
Quantitative analysis of antioxidative components
Total phenolics (TPC)
Total phenolics were quantified and expressed as gallic acid equivalents according to a method proposed by Singleton et al. (15), 1999. To 0.1 ml of E. ganitrus, added 3.9 ml of distilled water and 0.5 ml of Folin-Ciocalteau reagent. The tube was incubated at room temperature for 3 minutes. To this added 2 ml of 20% sodium carbonate and kept at boiling water bath for 1 minute. The blue color formed was read at 650 nm.
Total flavonoids (TFC)
TFC was estimated colorimetrically based on the method modified by Zhishen et al. (16), 1999. To 0.1 ml of E. ganitrus in a 10 ml volumetric flask, distilled water was added to make the volume to 5 ml and 0.3 ml 5% NaNO2 was added to this. 3 ml of 10% AlCl3 was added 5 minutes later. After 6 minutes, 2 ml of 1 M NaOH was added and the absorbance was measured at 510 nm. Rutin was used as a standard for constructing a calibration curve.
The results were expressed as means?SD. Linear regression analysis was used to calculate IC50 values whenever needed. Correlation analysis of total antioxidant capacity versus TPC and TFC were carried out using the correlation programme in the EXCEL program.
Results and Discussion
Phosphomolybdenum assay used to determine the total antioxidant capacity is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH (9). Figure 1 illustrates the antioxidative capacities of various concentrations of E. ganitrus (100-500 ?g/ml). Total antioxidant capacity of E. ganitrus was found to be 24.18 mg ascorbic acid equivalents at 500 ?g/ml extract concentration. This good antioxidant activity might be attributed to the presence of phytochemicals, such as flavonoids and biflavones (17). This assay has been successful in the quantification of vitamin E antioxidant activity (9) and it was efficient to extend its application to plants polyphenols (18).
Fe3+/Fe2+ transformation was investigated in the presence of samples for the measurements of the reductive ability. The reducing power of E. ganitrus ranged from 1.112 to 1.973 Abs for 100 ?g/ml to 400 ?g/ml of extract (Figure 2). Okuda et al reported that the reducing power of tannins prevents liver injury by inhibiting the formation of lipid peroxides. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity (19).
The chelation of Fe2+ ions was estimated by the method of Decker and Welch in which ferrozine quantitatively forms complexes with Fe2+. In the presence of chelating agents, the formation of this complex is disrupted, thereby impeding the formation of red color imparted by the complex as well. Measurement of this color change therefore allows for the estimation of the chelating activity of the coexisting chelator. As shown in Figure 3, the formation of Fe2+-ferrozine complex is not complete in the presence of extract, indicating that E. ganitrus chelate the iron. 76.70 % inhibition was noted with 500 ?g/ml of E. ganitrus. IC50 value was found to be 211.73 ?g/ml E. ganitrus. Metal chelating agents reduce the concentration of catalyzing transition metal in lipid peroxidation by forming sigma bonds with metals, reducing the redox potential, thereby stabilizing the oxidized form of the metal ion (20).
The decolorization of ABTS+ radical is an unambiguous way to measure the antioxidant activity of phenolic compounds. Recently Awika et al. (21), 2003 found positive correlations between the determination of phenolic antioxidant using the oxygen radical absorbance capacity (ORAC), ABTS+, and the 1, 1-diphenyl-2, 2picrylhydrazyl (DPPH) assays. Thus monitoring the antioxidant activity by ABTS+ radical scavenging assay gives good prediction of their ORAC and DPPH radical scavenging capacity. E. ganitrus showed potential activity in ABTS+ decolorization. 55.77 % inhibition was noted with 500 ?g/ml of E. ganitrus. The dose-dependent results observed were shown in Figure 3. IC50 value was found to be 297.12 ?g/ml E. ganitrus. Decolorization of ABTS+ in the present study reflects the capacity of an antioxidant species to donate electrons or hydrogen atoms to inactivate this radical cation.
Hydroxyl radical is the most reactive free radical and can be formed from superoxide anion and hydrogen peroxide, in the presence of metal ions, such as copper or iron. Hydroxyl radicals have the highest 1-electron reduction potential (2310 mV) and can react with everything in living organisms at the second-order rate constants of 109-1010 mol/s. Hydroxyl radicals react with lipid, polypeptides, proteins, and DNA, especially thiamine and guanosine. When a hydroxyl radical reacts with aromatic compounds, it can add across a double bond, resulting in hydroxycyclohexadienyl radical. The resulting radical can undergo further reactions, such as reaction with oxygen, to give peroxyl radical, or decompose to phenoxyl-type radicals by water elimination (22). The ability of the extracts to scavenge these radicals was evaluated by the Fenton-mediated 2-deoxyribose assay. The result of present study does not show any promising hydroxyl radical scavenging property. Only 13.43% inhibition was noted with 500 ?g/ml of E. ganitrus (Figure 4). Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation.
Recent studies have shown that many flavonoids and related polyphenols contribute significantly to the total antioxidant activity of many plants (23). Total phenolic content in E. ganitrus was found to be 56.79?1.6 mg gallic acid equivalents/g of dry material. Total flavonoids in E. ganitrus were found to be 18.58? 0.3 mg rutin equivalents/g of dry material. There was a positive correlation between the total phenolic content and antioxidant capacity, R=0.8501, whereas the correlation between the total flavonoids and antioxidant capacity was determined to be R2=0.8413. These results suggest 85% of the antioxidant capacity of E. ganitrus is due to the contribution of phenolics and flavonoid components. In addition, the antioxidant activity may be due to enzymatic and other non-enzymatic antioxidants, which needs further analysis.
(1) Yu BP. Cellular defenses against damage from reactive oxygen
species. Physiol. Rev. (1994) 74: 139-162
(2) Halliwell B and Gutteridge JMC. In: Free Radicals in Biology and Medicine. 2nd ed. Clarendon Press, Oxford (1989) 28-31
(3) Lata H and Ahuja GK. Role of free radicals in health and disease. Ind. J. Physiol. Allied Sci. (2003) 57: 124-130
(4) Cotran RS, Kumar V and Collins T. In: Robbin?s Pathological Basis of Diseases. 6th ed. Thomson Press (I) Ltd, Noida, India (1999) 1
(5) Yu BP, Suescun EA and Yang SY. Effect of age-related lipid peroxidation on membrane fluidity and phospholipase A2: modulation by dietary restriction. Mech. Ageing Dev. (1992) 65: 17-33
(6) Campbell IC and Abdulla EM. Strategic approaches to in vitro neurotoxicology. In: Approaches and Methods: Neurotoxicology. Academic Press, London (1995) 495
(7) Pratt DE. Natural antioxidants from plant material. In: Hang M, Ho C and Lee C. (eds.) Phenolic Compounds in Food and Their Effects on Health II: Antioxidants and Cancer Prevention (ACS Symposium Series 507). American Chemical Society, Washington DC (1992) 54
(8) Dasgupta A, Agarwal SS and Basu DK. Anticonvulsant activity of the mixed fatty acids of Elaeocarpus ganitrus Roxb. (Rudraksh). Indian J. Physiol. Pharmacol. (1984) 28: 245-246
(9) Prieto P, Pineda M and Aguilar M. Spectrophotometric quantification of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application of vitamin E. Anal. Biochem. (1999) 269: 337-341
(10) Oyaizu M. Studies on product of browning reaction prepared from glucosamine. Jpn. J. Nut. (1986) 44: 307-315
(11) Decker EA and Welch B. Role of ferritin as a lipid oxidation catalyst in muscle food. J. Agric. Food Chem. (1990) 38: 674-677
(12) Mondal SK, Chakraborty G, Gupta M and Mazumder UK. In vitro antioxidant activity of Diospyros malabarica Kostel bark. Indian J. Exp. Biol. (2006) 44: 39-44
(13) Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice- Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. (1999) 26: 1231-1237
(14) Bektaşoğlu B, Esin Celik S, Ozy?rek M, G??l? K and Apak R. Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method. Biochem. Biophys. Res. Commun. (2006) 345: 1194-1200
(15) Singleton VL, Orthofer R and Lamuela-Raventos RM. Analysis of total phenols and oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent. Methods Enzymol. (1999) 299: 152-177
(16) Zhishen J, Mengcheng T and Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. (1999) 64: 555-559
(17) Wang C and Wixon R. Phytochemicals in soybeans: their potential health benefits. Inform. (1999) 10: 315-321
(18) Lu Y and Foo LY. Antioxidant activities of polyphenols from sage (Salvia offincialis). Food Chem. (2001) 75: 197-202
(19) Meir S, Kanner J, Akiri B and Hadas SP. Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. J. Agric. Food Chem. (1995) 43: 1813-1817
(20) Elmastas M, G?l?in İ, Işildak ?, K?frevioğlu ?İ, İbaoğlu K and Aboul-Eneinc HY. Radical scavenging activity and antioxidant capacity of Bay leaf extracts. J. Iranian Chem. Soc. (2006) 3: 258-266
(21) Awika JM, Rooney LM, Wu X, Prior RL and Cisneros-Zevallos L. Screening methods to measure antioxidant activity of Sorghum bicolor and Sorghum products. J. Agric. Food Chem. (2003) 51: 6657-6662
(22) Lee J, Koo N and Min DB. Reactive oxygen species, aging, and antioxidative nutraceuticals. Comprehensive Reviews in Food Science and Food Safety (2004) 3: 21-27
(23) Luo XD, Basile MJ and Kennelly EJ. Polyphenolic antioxidants from the fruits of Chrysophyllum cainito L. (star apple). J. Agric. Food Chem. (2002) 50: 1379-1382