British Journal of Pharmaceutical Research

3(2): 202-216, 2013
SCIENCEDOMAIN international

Determination of Antioxidant Activity and Phytoconstituent Screening of Euphorbia heterophylla Linn
Muhammad Athar Abbasi1*, Hina Saleem1, Aziz-ur-Rehman1, Tauheeda Riaz1 and Muhammad Ajaib2

Department of Chemistry, Government College University, Lahore-54000, Pakistan. 2 Department of Botany, Government College University, Lahore-54000, Pakistan. Authors’ contributions

This work was carried out in collaboration between all authors. Author MAA designed and supervised the present study. Author HS performed the experimental study and statistical analysis, reviewed the literature and wrote the manuscript. Author AR co-supervised the present study. Author TR guided during experimental analysis and checked the manuscript. Author MA collected and identified the studies plant and issued its voucher number. All authors read and approved the final manuscript.

Research Article

Received 30 July 2012 st Accepted 1 December 2012 th Published 4 March 2013


Aim: To carry out qualitative determination of phytochemicals and evaluate antioxidant potential of Euphorbia heterophylla Linn. Place and Duration of Study: Department of Chemistry, Government College University Lahore, Pakistan, between October, 2011 and February, 2012. Material and Method: The methanolic extract of the plant was dissolved in distilled water and partitioned with n-hexane, chloroform, ethyl acetate and n-butanol sequentially. The antioxidant potential of all these fractions and remaining aqueous fraction was analyzed by these methods: 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging activity, total antioxidant activity, Ferric Reducing Antioxidant Power (FRAP) assay, Ferric Thiocyanate (FTC) assay while Folin-Ciocalteu colorimetric method was used to analyse total phenolic content. Phytochemical analysis were performed on the plant extracts to detect the presence of secondary metabolites. Results: Phytochemical screening revealed phenolics and flavonoids in abundance in
____________________________________________________________________________________________ *Corresponding author: Email:;

Antioxidant assays revealed that Ethyl acetate soluble fraction exhibited highest percent inhibition of DPPH radical i.01±0. propyl gallate (PG) and tertbutylhydroquinone (TBHQ) have been added to foodstuffs. Epidemiological and in vitro studies on 203 . FRAP value. Also the ethyl acetate soluble fraction.08 as well as highest FRAP value 200.53% percent inhibition of lipid peroxidation. Currently there is growing interest towards natural antioxidants of herbal resources. 3(2): 202-216. Antioxidants. IC50 value of ethyl acetate fraction was found to be 36.15±0. 0. highest amount of total phenolic compounds (190. phytochemicals.87% at a concentration of 120 μg/ml as compared to other fractions. as well as reactive nitrogen species (RNS).21 GAE mg/g) and highest percentage of inhibition of lipid peroxidation (54. n-Butanol soluble fraction showed IC50 value of 117.8% percent inhibition of lipid peroxidation. there are some disadvantages because they are suspected of having some toxic properties. It also showed the highest value of total antioxidant activity i. Keywords: Euphorbia heterophylla Linn. DPPH assay.85±1. Chloroform soluble fraction showed IC50 value of 149. total phenolic content 36.British Journal of Pharmaceutical Research.09±0. Reactive oxygen species (ROS). total antioxidant activity 0.03.57%). hypertension and several other disorders are becoming increasingly recognized [1]. Number of synthetic antioxidants such as butylated hydroxyl anisole (BHA).e. FRAP value127.5±1. total antioxidant activity 0.9 GAE mg/g and 25. FRAP value115.532±0. Therefore search for natural antioxidants has received much attention and efforts have been made to identify natural compounds that can act as suitable antioxidants to replace synthetic ones [4].152±0.15±0.7±0.3±0. Aqueous fraction showed IC50 value of 669. inhibition of lipid peroxidation (%).9% percent inhibition of lipid peroxidation. Terpenoids were detected in all other fractions except the aqueous fraction.9 μM/ml.02.8±0. Alkaloids were determined in ethyl acetate and n-butanol soluble fraction only while tannins and cardiac glycosides were present in n-butanol soluble fraction and ethyl acetate soluble fraction respectively. total phenolic content 19. INTRODUCTION The role of free radicals and tissue damage in disease such as atherosclerosis. FRAP value 98. 2013 chloroform soluble fraction.174±0.23±0. and UV irradiation and they are well recognized for playing a dual role as both deleterious and beneficial species.5±0. n-butanol soluble fraction and remaining aqueous fraction contained saponins and sugars. 1.09±0.2 μM/ml. Conclusion: Ethyl acetate soluble fraction was found to be rich in natural antioxidants and a good source of phytochemicals.3±1.4 TE μM/ml. total phenolic content 137. diabetes mellitus. antioxidant activity 0.5.06. total antioxidant activity.041. heart failure. total phenolics.8 μM/ml.1±1.05±0.5±0.3 μM/ml. FRAP value 68.918±0.1±1. total phenolic content 93. since they can be either harmful or beneficial to living systems [2].67±0.23 GAE mg/g and 12.8 μg/ml relative to ascorbic acid having IC50 value 58. 80. butylated hydroxyl toluene (BHT).7. cancer. both exogenous and endogenous whether synthetic or natural can be effective in preventing free radical formation by scavenging them or promoting their decomposition and suppressing many disorders [3].7±1. neurodegenerative disorders. environmental stresses.07.4 GAE mg/g and 41. ethyl acetate soluble fraction and n-butanol soluble fraction. aging.3 GAE mg/g and 32.89 μg/ml.26±0. n-Hexane soluble fraction showed IC50 value of 769. Although these synthetic antioxidants are efficient and cheap.e. antioxidant activity 0.96% percent inhibition of lipid peroxidation.31±0.04.84±1. are products of normal cell metabolism.739±0.

Medicinal plants are of great importance to the health of individuals and communities in general. traditionally as snake bite remedy [14]. Euphorbia prolifera is used for the treatment of inflammation and tumors [15]. antiinflammatory. Muhammad Ajaib (Taxonomist). therefore. The leaves of E. triangularis are possible sources of rubber [12]. E. stalks. MATERIALS AND METHODS 2. co-carcinogenic. fruits. tirucalli and E. sedative. hepatoprotective. In the family Euphorbiaceae genus Euphorbia is a giant genus of flowering plants consisting of 2000 known species varying from annuals to trees having unique flower structure and contain latex. Euphorbia heterophylla leaf is used in traditional medical practices as laxative. prostrata is used as anti-hemorrhoidal. trees. heterophylla leaf Linn [20]. The plant latex has been used as fish poison and insecticide and ordeal poisons [16. immunostimulant. 2013 medicinal plants and vegetables strongly supported this idea that plants are capable of exerting protective effects against oxidative stress in biological systems [3]. E.British Journal of Pharmaceutical Research. sticky sap. The use of plants and their extracts in treatment of diseases dates back to 460-370 BC when Hippocrates practiced the art of healing by the use of plant based drugs [5](Soforowa. antigonorrheal. E.17]. mild CNS depressant. hypolipidemic. anti-spasmodic. leaves. E. th 2. wound healing and radioprotective characteristics [13]. anti-fungal and anti-malarial properties. 2004). ingens. The use of plants as spices and herbs indicates that the oxidative and antimicrobial constituents are present in all parts of the plants including tree barks. heterophylla [19]. heterophylla have been reported to contain quercetin [18] (Falodun and Agbakwuru. anti-fertility. The most important of these bioactive constituents of plants are alkaloids. Diterpenoids have also been reported in the root of E. herbs or rarely lianas [9]. The plant was identified by Mr. The present work was aimed to investigate the phytoconstituents and evaluate the antioxidant potency of Euphorbia heterophylla plant. flavonoids and phenolic compounds [6]. anti-asthmatic. anti-asthmatic and for various skin diseases. Euphorbia heterophylla Linn is medicinal plant with the common name “spurge weed”. Department of 204 . tannins. mey. 3(2): 202-216. The Euphorbiaceae family is the 4 largest family of the angiosperms comprising over 300 genera and about 7500 species distributed widely in tropical Africa [8]. The Euphorbiaceae plants are shrubs. The skin irritant. nerrifolia is reported to have analgesic. anti-dirroheal. anthelmintic. antidiabetic. roots and so on [7]. severe skin irritant and toxic to live stock and humans [11].1 Plant Collection The fresh plant Euphorbia heterophylla was collected from Azad Kashmir (Pakistan) in August 2011. tumor-promoting and antitumor/anti-cancer and recently anti-HIV activities of Euphorbia species have also been reported in E. Euphorbia hirta possesses anti-bacterial. anti-inflammatory. Generally they have characteristic milky latex [10]. migraine and wart cures. analgesic. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. 1982). also. Latex of E. justifying the use of plant in ethno-medicine for treatment of various ailments.

chloroform. TLC card was heated on a TLC heater.2 Extraction and Fractionation The shade-dried ground whole plant was exhaustively extracted with methanol on the soxhlet apparatus. ferrous chloride.1-diphenyl-2-picryl hydrazyl).C University. TPTZ (2.4. ferric chloride. chloroform (400ml x 4). The extract was evaporated in rotary evaporator (Laborta 4000-efficient Heidolph) at 45ºC under vaccum to yield the residue which was dissolved in distilled water and partitioned with n-hexane (400ml x 4). hydrochloric acid. Trolox.   First.4. to 0. The frothing was mixed with 3 drops of olive oil and shaken vigorously after which it was observed for the formation of an emulsion.Bot. (n-hexane at 35ºC. lead acetate. ceric sulphate. 2. 2013 Botany.3 Test for saponins To 0. ammonium molybdate. ethyl acetate (400ml x 4). Second. A voucher specimen (GC. aluminium chloride. n-butanol).) Ltd. 2. n-butanol at 70ºC and water at 60ºC under vacuum) and the residues thus obtained were used to evaluate their phytoconstituents and in vitro antioxidant potential. ammonia from Merck (Pvt. 3(2): 202-216. 3 ml of concentrated H2SO4 was carefully added to form a layer. n-butanol (400ml x 4) respectively. 2. G.5 g of each of the extract was added 2 ml of chloroform. These organic fractions and remaining aqueous fractions were concentrated separately on rotary evaporator.British Journal of Pharmaceutical Research. Ceric sulphate solution was sprayed on TLC card having spots of samples.1 Test for alkaloids For the test of alkaloids the TLC card having spots of the studied samples was sprayed with Draggendroff’s reagent. The solution was shaken vigorously and observed for a stable persistent froth.2 Test for terpenoids Two methods were used to test presence of terpenoids. Lahore. 2.4.22. A reddish brown colouration of the interface indicated the presence of terpenoids.4 Phytochemical Screening The phytochemical screening testing for the presence of phytoconstituents was performed using the standard procedures [21. Follin-Ciocalteu’s phenol reagent and BHT (Butylated hydroxytoluene) were obtained from Sigma Chemical Company Ltd. ethyl acetate 45ºC. tween-20. Appearance of orange colour indicated the presence of alkaloids.6 Tripyridyl-s-triazine). sulphuric acid. 2. 2. linoleic acid. Appearance of brown colour indicated the presence of terpenoids. ethyl acetate.23]. copper sulphate.580) is kept in the Herbarium of the Botany Department of the same university. (USA) and organic solvents (n-hexane. 205 .4. acetic acid. Gallic acid.5 g of extract was added 5 ml of distilled water in a test tube. (Germany).3 Chemicals and Standards DPPH (1. sodium phosphate.Herb. chloroform at 42ºC.

Green flourescence in UV light indicated the presence of flavonoids.5 g in 5 ml of water) were added to boiling Fehling’s solution (A and B) in a test tube. various concentrations of the samples (1000 µg/ml. This was underplayed with 1 ml of concentrated sulphuric acid.  Third. dilute ammonia (5 ml) was added to a portion of sample solution in water.  Second. 15 µg/ml) were mixed with 3 ml of methanolic solution of DPPH (0.British Journal of Pharmaceutical Research. 500 µg/ml. a few drops of 1% aluminium chloride solution were added to sample solution.1 DPPH radical scavenging activity The DPPH radical scavenging activities of various fractions of plant were examined by comparison with that of known antioxidant. ascorbic acid using the reported method [24].5 g of each sample diluted to 5 ml in water was added 2 ml of glacial acetic acid containing one drop of ferric chloride solution.5 Antioxidant Assays Following analytical antioxidant assays were performed on all the studied fractions.8 Test for cardiac glycosides (keller-killiyani test) To 0. Briefly. A yellow colouration that disappears on standing indicated the presence of flavonoids.1mM). The mixture was shaken vigorously and allowed to stand at room temperature for one hour.  Fourth. Concentrated sulphuric acid (1 ml) was added.4. the TLC card having spots of sample was sprayed with lead acetate solution.5. Appearance of red colour indicated the presence of tannins. 2.4.4. 2.6 Test for phenolics Neutral ferric chloride was added to each fraction. Green flourescence in UV light indicated the presence of flavonoids. A yellow colouration indicated the presence of flavonoids. 250 µg/ml. 125 µg/ml. Mixture was warmed for 1 hour at 95ºC in a water bath.4 Test for tannins 2 ml of sample was taken in test tube and 5 ml of n-butanol-HCl solution was added. 3(2): 202-216.4. Then absorbance was measured at 517 nm against methanol as a blank in the UV–visible 206 . 2. the TLC card having spots of samples was sprayed with Benedict’s reagent.4. 2. A violet ring may appear below the brown ring while in the acetic acid layer a greenish ring may form just above the brown ring and gradually spread throughout the layer. 2013 2. 2. Formation of red precipitates indicated the presence of sugars.5 Test for sugars Sample solutions (0.7 Test for flavonoids Four methods were used to test for flavonoids. 30 µg/ml. 60 µg/ml.  First. Appearance of bluish green colour indicated presence of phenolics. 2. A brown ring at the interface indicated the presence of deoxysugar characteristic of cardenolides.

3 Ferric reducing antioxidant power (FRAP) assay The FRAP assay was done according to Benzie and Strain [26] with some modifications.5. Briefly.5 mg/ml) was mixed with 2. Results were expressed in TE µM/ml.0).5. Total phenolic contents were expressed as milligrams of gallic acid equivalents (GAE) per gram of sample using the standard calibration curve constructed for different concentrations of gallic acid. Reading of the coloured product [ferrous tripyridyltriazine complex] was then taken at 593 nm by UV–visible spectrophotometer. The fresh working solution was prepared by mixing 25ml acetate buffer.5 ml TPTZ solution and 2. The solution of plant samples 500 µg/mL and that of trolox were formed in methanol. 2. 28 mM sodium phosphate and 4 mM ammonium molybdate) in sample vials.6 M sulphuric acid.5ml of linoleic acid emulsion (0.1ml of 2N Folin-Ciocalteu’s phenol reagent. 2. After the samples had been cooled to room temperature.2 Total antioxidant activity by phosphomolybdenum complex method The total antioxidant activities of various fractions of plant were evaluated by phosphomolybdenum complex formation method [25].0ml of phosphate buffer (0.1ml of each of sample solution (0.1ml (0.6H2O solution and then warmed at 37ºC before using. The 0.02 M. 10 µl of each of sample solution and BHT solution were taken in separate test tubes and 2990 µl of FRAP solution was added in each to make total volume up to 3 ml. 2. The antioxidant activity was expressed relative to that of BHT. All determinations were assayed in triplicate and mean values were calculated.5.5mg/ml) of sample was combined with 2. The curve was linear between 50 mg/mL to 500 mg/mL of gallic acid. The blank solution contained 4 ml of reagent solution. 10 mM Hydrochloric acid and 20 mM Ferric chloride hexahydrate solution. The plant samples were allowed to react with FRAP solution in the dark for 30 minutes. 2013 spectrophotometer (CECIL instruments CE 7200 Cambridge England).5 ml FeCl3. 3(2): 202-216.3H2O and 16 ml CH3COOH).British Journal of Pharmaceutical Research. pH 7.6. The 0.0) and 2. The stock solutions included 300 mM acetate buffer (3.4 Total phenolic contents The total phenolics of various fractions of plant were determined by reported method [27].5 Ferric thiocyanate (FTC) assay The antioxidant activities of various fractions of plant on inhibition of linoleic acid peroxidation were assayed by thiocyanate method [28].8ml of 10% Sodium carbonate and 0. The percent of DPPH decoloration of the samples was calculated according to the formula: Antiradical activity = Acontrol – Asample / Acontrol x 100 Each sample was assayed in triplicate and mean values were calculated. the absorbance of mixture was measured at 695 nm against blank. pH 3. 2. Results were expressed in GAE mg/g. pH 7. 2. The vials were capped and incubated in water bath at 95ºC for 90 minutes. The linoleic acid emulsion was prepared by 207 .5. 500 µg/ml of each sample was mixed with 4 ml of reagent solution (0. The FRAP values were determined as micromoles of trolox equivalents per ml of sample by computing with standard calibration curve constructed for different concentrations of trolox. Lower absorbance of spectrophotometer indicated higher free radical scavenging activity.1g CH3COONa.02 M. After 40 minutes absorbance at 725 nm was measured by UV-visible spectrophotometer.

Preliminary phytochemical studies revealed the presence of flavonoids. Ethyl acetate soluble fraction.1ml of 20 mM ferrous chloride in 3. 2013 mixing 0.British Journal of Pharmaceutical Research. Phytochemical constituents of various fractions of Euphorbia heterophylla Linn Test n-Hexane soluble fraction Alkaloids Terpenoids ++ Saponins Tannins Sugars Phenolics Flavonoids Cardiac glycosides Chloroform soluble fraction ++ ++ + Ethyl acetate soluble fraction + +++ + + +++ ++ + n-Butanol soluble fraction + +++ + + ++ + + Remaining aqueous fraction + +++ - ‘+’ represents presence and ‘–’ represents absence.. It was observed from the preliminary phytochemical investigation that phenolics and flavonoids were present in chloroform soluble fraction. heterophylla Linn plant samples revealed the presence of medicinally active constituents (Table 1). of control)} × 100].28 g of Tween-20 as emulsifier and 50.[29] who carried phytochemical analysis on leaf extract of the plant. 0. choline and shikmic acid [12]. saponins. 3. cardic glycosides and balsam (gum) [30]. nerrifola predominantly contains sugar. The presence of these phytoconstitents in Euphorbia heterophylla plant also confirmed the reports of James et al. sterols. Ethyl acetate soluble fraction contained phenolics and flavonoids in more amounts as compared to other fractions. RESULTS AND DISCUSSION 3. The reaction mixture was incubated for 5 days at 40ºC. The mixture (0. E. The antioxidant activity of BHT was assayed for comparison as reference standard. steroid. tannins. flavonoids.1ml) was taken and mixed with 5. Terpenoids were detected in all fractions except the aqueous fraction.1ml of 30% ammonium thiocyanate and 0. tannins. glycosides and saponins in the alcoholic extract of the E. hirta include triterpenoids.5% HCl and allowed to stand at room temperature. flavonoids. Table 1. 208 . alkaloids and triterpenoidal saponins [13]. 0. tannins. 3(2): 202-216. Also the reported phytoconstituents of E.1 Phytochemical Investigation The present study carried out on the E. balsamifera revealed the presence of tannins. Cardiac glycosides were only present in ethyl acetate soluble fraction. glycosides. Alkaloids were present in ethyl acetate soluble fraction and n-butanol soluble fraction while absent in all other fractions.0ml of 75% ethanol. Phytochemical analysis of the crude extracts of the E.0ml of phosphate buffer. prostrata [14]. Tannins were only present in n-butanol soluble fraction. [IP% = {1-(abs. The mixture without extract was used as control. Precisely 3 minutes after addition of ferrous chloride to the reaction mixture. alkaloids.28 g of linoleic acid. phenols. absorbance was recorded at 500nm. The antioxidant activity was expressed as percentage inhibition of peroxidation (IP %). of sample) / (abs. n-butanol soluble fraction and remaining aqueous fraction contained saponins and sugars while n-hexane soluble fraction and chloroform soluble fraction showed absence of these compounds. terpenoid. ethyl acetate soluble fraction and n-butanol soluble fraction while n-hexane soluble fraction and remaining aqueous fraction showed absence of these compounds. flavonoids.

05 is taken as significant).8 11. which will disappear after the acceptance of an electron or hydrogen radical from an antioxidant in the solution to become a stable diamagnetic molecule [32]. 2013 3. 1-Diphenyl-2-picryl hydrazyl radical (DPPH) Sr.55 2 Chloroform soluble fraction 3 Ethyl acetate soluble fraction n-Butanol soluble fraction 4 5 Remaining aqueous fraction Ascorbic acid b) 6 standard mean error of three assays.No.76* 35. such as metal ion chelation and enzyme inhibition. a) It can be clearly seen that the scavenging activity of Euphorbia heterophylla extracts were concentration-dependent. In the radical form.09 ±0. resulting in the scavenging of the radical by hydrogen donation and is visualized as a discoloration from purple to yellow [33].09±0.5±0. The various concentrations of ethyl 209 . 3(2): 202-216.4 4.( µg / mL) 1000 500 250 120 250 120 60 30 120 60 30 15 120 60 30 15 1000 500 250 120 125 60 30 % Scavening of DPPH radical a) ± S.34±1.86 29. DPPH radical has the advantage of being unaffected by certain side reactions of polyphenols.16±0.97± 0.45±0.12 13.05 when compared with negative control i.British Journal of Pharmaceutical Research.88±1.01 80.82±0. b) a reference standard antioxidant.87 72.35±0.44 27. Free radical scavenging activity of various fractions of Euphorbia heterophylla Linn. In this present study. blank/solvent (p<0.98* 31.99±1. *p< 0.32±0.M 57.34 79.23 53.29±0. The relationship between the scavenging activity and concentrations are shown in Table 2.31±0. using 1.05* 27.2 DPPH Radical Scavenging Activity Free radical scavenging is one of the known mechanisms by which antioxidants inhibit cellular damage. Besides.89* 16.22 22. It is reported that the decrease in the absorbance of DPPH radical caused by phenolic compound is due to the reaction between antioxidant molecules and radicals. the molecule of DPPH has an absorbance at 517nm.E.2* 53. different concentrations of extracts were used. 1 Sample n-Hexane soluble fraction Conc.79 16.23±1.14± 0.93 58. Table 2.25±1. With the increase of the concentration of the samples.8±0.34 69.86±0.48* 44.99±0. The DPPH free radical scavenging method is colorimetric assay and can be used to evaluate the radical scavenging capacity of specific compounds or extract in a short time [31].99±0.e.56 5.75±1.34* 51. the scavenging activity of the extracts increased accordingly.87* 63.21±0.

acting possibly as primary antioxidants [34].05) while that of remaining aqueous fraction was found to be non significant (p > 0.04 relative to Ascorbic acid having IC50 of 58. Chloroform soluble fraction. The phosphomolybdenum method usually detects antioxidants such as ascorbic acid. n-butanol soluble fraction and remaining aqueous fraction showed IC50 value 149.8μg/ml compared to other studied fractions.8±0. The ferric reducing antioxidant power (FRAP) assay measures the reducing ability of antioxidants against oxidative effects of reactive oxygen species.3 Total Antioxidant Activity by Phosphomolybdenum Method The total antioxidant capacity of all fractions was measured spectrophotometrically by the phosphomolybdenum method. The various concentrations of the fractions which showed percent inhibition greater than 50% were found to be significant (p < 0.174±0.7±1. Ethyl acetate soluble fraction exhibited lowest IC50 36.05) when compared with negative control i. It was revealed that the antioxidant activity of the extract exhibited increasing trend with the increasing concentration of the plant extract [36]. neriifolia was found to have higher activity.4 Ferric Reducing Antioxidant Power (FRAP) Assay The reducing capacity of compounds may serve as a significant indicator of their potential antioxidant activity.925±0. The total antioxidant activity shown by chloroform soluble fraction. 2013 acetate soluble fraction exhibited highest percent inhibition of DPPH radical as compared to other fractions.3±1. some phenolics. The extract of Euphorbia neriifolia possess efficient scavenging character when compared with the standards and the study reveals that the extract of Euphorbia neriifolia exhibits the proton. It showed 80. Electron 210 . The total antioxidant activities of these fractions were compared with the standard antioxidant BHT and the results shown in Table 3. ethyl acetate soluble fraction and n-butanol soluble fraction were found to be significant (p < 0. 117. tocopherols and carotenoids.89 μg/ml.7. 769.67±0. ethyl acetate soluble fraction and nbutanol soluble fraction was found to be significant (p < 0.87% inhibition of DPPH radical at the concentration of 120 μg/ml. Also the extract of E.06) > nbutanol soluble fraction (0.08.84±1.08). The IC50 values of chloroform soluble fraction. which is based on the reduction of Mo(VI) to Mo(V) by the sample analyte and the subsequent formation of green phosphate/Mo(V) compounds with a maximum absorption at 695 nm [35].07) > aqueous fraction (0.5. blank.03) > n-hexane soluble fraction (0. as compared to the standard (gallic acid).041).05) when compared with ascorbic acid. IC50 is defined as the concentration of the extracts to quench 50% of DPPH in the solution under the chosen experimental conditions and IC50 values given in Table 3.85±1. The antioxidant activity of the various plant fractions decreased in the following order: chloroform soluble fraction (0.918±0. IC50 value is inversely related to the activity as it is the measure of inhibitory concentration and a lower value would reflect greater antioxidant activity of the fraction.British Journal of Pharmaceutical Research. Electron transfer occurs in this assay which depends upon the structure of the antioxidant [25].532±0. 3. 3(2): 202-216.09 ± 0. n-hexane soluble fraction. The results indicated that ethyl acetate soluble fraction had highest total antioxidant activity (0.152±0.739±0. a reference standard.donating ability and could serve as free radical inhibitors or scavengers. 3. 669.05) while that of n-hexane and aqueous fraction were found to be non significant (p > 0.05) when compared with BHT.e. BHT showed total antioxidant activity 0.02.

nerrifolia extract was found to be significantly higher as compared to the standards i.9 μM/ml respectively while n-hexane soluble fraction and remaining aqueous fraction showed very less FRAP values i. are prominent free radical scavengers and also responsible for exhibiting multiple medicinal and physiological functions in animals as well as in plants [40].05) when compared with blank. This assay is based on the ability of 3+ 2+ antioxidants to reduce Fe to Fe in the presence of tripyridyltriazine [TPTZ] forming an 2+ intense blue Fe -TPTZ complex with an absorbance maximum at 593 nm [26]. Phenols are one of the major groups of nonessential inhibition of atherosclerosis that have been associated with the inhibition of atherosclerosis and cancer.26±0. phenolic acid. it is well known that plant phenolics are highly effective free radical scavengers and antioxidants. 211 .4 GAE mg/g.British Journal of Pharmaceutical Research. These compounds exhibit antioxidant activity by inactivating lipid free radicals or by preventing the decomposition of hydroperoxides into free radicals [38]. the investigation on the antioxidant activity of E.1±1.5±0.23. quercetin and BHT and thus the extract of E. capable of transforming reactive free radical species into stable non radical products [36].5±0. 3(2): 202-216. a well-known group of plant secondary metabolites. Table 3 shows the total phenol content (TPC) expressed as gallic acid equivalents (GAE) achieved by FC method.4 μM/ml. The FRAP values of E. as well as for age-related degenerative brain disorders [31]. Phenolics. 3. High FRAP values obtained for polar fractions may be ascribed partially to the phenolic and flavonoid contents. The results for total phenolic contents of chloroform soluble fraction.7±0.05) while that of n-hexane soluble fraction and remaining aqueous fraction were found to be non significant (p > 0. Moreover. and n-butanol soluble fraction were found to be significant (p < 0.3.05±0. n-butanol soluble fraction and remaining aqueous fraction were found to be 19.8 μM/ml. 98. ethyl acetate soluble fraction. Therefore.5±1. nerrifolia extract was found to be significantly higher as compared to the reference standard [34]. The total phenolic contents of n-hexane soluble fraction. 68. and n-butanol soluble fraction were found to be significant (p < 0.15±0. and the activity is derived largely from the phenolic and polyphenolic compounds. The total phenolic content of E.9 GAE mg/g respectively. nerrifolia act as free radical scavenger.e. diterpenes and tannins [37]. From the results it was revealed that ethyl acetate soluble fraction showed highest FRAP value of 200. The ethyl acetate soluble fraction showed the highest amount of total phenolic compounds 190.2 μM/ml.05) when compared with blank. proanthocyanidins. These potential mechanisms make the diverse group of phenolic compounds an interesting target in the search for beneficial phytochemicals [39].3±0. ethyl acetate soluble fraction. Heterophylla extracts was of great importance.3 μM/ml respectively. Phenolic compounds are known to inhibit various types of oxidizing enzymes. Increasing absorbance indicates an increase in reductive ability.1±1. The FRAP values of chloroform soluble fraction.05) while that of n-hexane soluble fraction and remaining aqueous fraction were found to be non significant (p > 0. 127. 93. 36.5 Total Phenolic Content The therapeutic effects derived from several medicinal plants have been attributed to the presence of phenolic compounds such as flavonoids.e.21 GAE mg/g while that of chloroform soluble fraction was found to be 137. 2013 donating antioxidants can be described as reductants and inactivation of oxidants by reductants can be described as redox reactions. Chloroform soluble fraction and n-butanol soluble fraction also showed good FRAP values 115.

Ferric ions will then unite with ammonium thiocyanate and produce ferric thiocyanate. a reddish pigment [42].09±0. in which peroxide will react with ferrous chloride and form ferric ions.53%) > n-butanol (32.E. neriifolia extract also exhibit good effect in inhibiting linoleic acid oxidation compared to control (BHT).15±0. particularly hydroxyl radicals thereby terminating the radical-chained reaction and retarding the formation of hydroperoxides [43]. Statistical analysis were determined using one way analysis of variance (ANNOVA) followed by post-hoc Tukey’s test. Lipid peroxidation contains a series of free radical mediated chain reaction processes and is also associated with several types of biological damages [41].01±0.48±1. The ferric thiocyanate method is used to measure the amount of peroxide at the beginning of lipid peroxidation. The data revealed.9%) > aqueous fraction (25.8%). the inhibition of peroxidation by the plant extracts indicates its antioxidant potency.05) when compared with BHT. The phenolic compounds present donate hydrogen and terminate the free radical reaction chain by changing it to the stable compounds [36]. The high peroxidation scavenging effect may be due to the high contents of phenolic compounds or radical scavengers involved in the extracts [44] which can terminate the peroxidation chain reactions easily [45] and quench reactive oxygen or nitrogen species. 2013 3. Hydrogen donating antioxidants can react with lipid peroxyl radicals and break the cycle of generation of new radicals.96%) > n-Hexane soluble fraction (12. The antioxidant activities also increased with increasing concentration of the E. the peroxidation inhibition activity of the E.7 Statistical Analysis All the measurements were done in triplicate and statistical analysis was performed by Statistical software. 212 .British Journal of Pharmaceutical Research. The data revealed that the ethyl acetate fraction and chloroform soluble fractions showed a good inhibition of lipid peroxidation formed during linoleic acid system. Therefore. 3.M.57%) > chloroform soluble fraction (41. The E. The inhibition of lipid peroxidation by BHT (standard) was 62. In this study.6 Ferric Thiocyanate (FTC) Assay It has been reported that the lipid peroxidation is one of the causes of the occurrence of cardiovascular disease and cancer. All the data were expressed as ± S. thereby inhibiting the oxidation of lipids and other biological molecules [46]. The results for percent inhibition of lipid peroxidation of chloroform soluble fraction. neriifolia extract. heterophylla extracts was measured and shown in Table 3. ethyl acetate soluble fraction and n-butanol soluble fraction were found to be significant (p < 0.07%. The fractions which showed greater values of percent inhibition of lipid peroxidation might contain primary antioxidant compounds which are able to react aggressively with free radicals.05) while that of n-hexane soluble fraction and aqueous fraction were found to be non significant (p > 0.31±0. inhibition percentage of lipid peroxidation was high in the order of ethyl acetate soluble fraction (54.23±0. 3(2): 202-216.

total antioxidant activity.4* 190.48±1. total phenolics and lipid peroxidation of different fractions of Euphorbia heterophylla Linn Sr.532±0. FRAP values. blank/solvent (p<0.8 ± 0.09±0.26±0.2* 200.03** 0.95 Total phenolics (GAE mg/g) 19.67±0. 213 .08 FRAP value TE (μM/ml) 98.05±0.5±0.85±1.9* 68.3 24.7±0. a) Standard antioxidants.739±0.4* 127.7** 669.1±1.7±1. No 1 2 3 4 5 6 6 7 Sample n-Hexane soluble fraction Chloroform soluble fraction Ethyl acetate soluble fraction n-Butanol soluble fraction Remaining aqueous fraction a) Ascorbic acid a) BHT Blank IC50 (µg/mL) 769.04 58.15±0.56 Inhibition of lipid peroxidation (%) 12.96 62. 2013 Table 3.21* 93.07 - All results are presented as mean ± standard mean error of three assays.e.5±1.31±0.02** 36.041 0.96±0.08** 0.23±0.8 115. IC50.07 0.174±0.5 149.8** 117.918±0.925±0.9** 25.01±0.9 14.3±1.05 when compared with reference standards (BHT/Ascorbic acid).152±0.89 Total antioxidant activity 0.11 ± 0.5±0.British Journal of Pharmaceutical Research. 3(2): 202-216.1±1.05 is taken as significant).23 137.8 41.06** 0.84±1.53** 54. **p < 0.3* 36.3±0. *p< 0.05 when compared with negative control i.57** 32.15±0.

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