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In-Vitro Antioxidant activity of Isolated Tannins of alcoholic extract of dried leaves of Phyllanthus Amarusschonn and Thonn

Wasnik Ujwala*, Singh Vijender, Ali Mohammad
  1. Affilitaions: R.V.Northland Institute, U.P.TechnicalUniversity,Lucknow,India
  2. Head Research and Development Division, Aimil Pharmaceuticals (I),Ltd
Corresponding Author:Wasnik Ujwala, E.mail: pharmaujju@gmail.com
Received:03 February 2012 Accepted: 10 February 2012
Citation: Wasnik Ujwala*, Singh Vijender, Ali Mohammad “In-Vitro Antioxidant activity of Isolated Tannins of alcoholic extract of dried leaves of Phyllanthus Amarusschonn and Thonn”, Int. J. Drug Dev. & Res., Jan-March 2012, 4(1): 274-285 doi: doi number
Copyright: © 2010 IJDDR, Wasnik Ujwala et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Tannins are naturally occurring, high molecular weight polyphenols which can be divided into hydrolysable tannins and condensed tannins. Tannins are the most abundant antioxidants in the human diet and they exhibit many biologically important functions which include protection against oxidative stress and degenerative diseases.The tannin compounds are widely distributed in many species of plants, where they play a role in protection from predation, and perhaps also as pesticides, and in plant growth regulation. The stems, infusion of leaves and roots of Phyllanthusspecies are used in folk medicine for treating intestinal infections, diabetes, the hepatitis B virus and disturbances of the kidney and urinary bladder and it has antioxidants activity. The purpose of the present study was to isolate the tannins from alcoholic extract of P.amarus and to evaluate the in –vitro antioxidant activity of isolated tannins from the extract of dried leaves of P.amarus leaves. Dried leaves of P.amarus were defatted by using Pet.ether 60- 80o C for 48 hours. The extract was used to evaluate the presence /absence of various phytoconstituents. Comparative antioxidant potential of tannins isolated from alcoholic extract of P.amarus was evaluated for the various in-vitro antioxidant activity models. In-vitro anti-lipid peroxidation activity, nitric oxide, superoxide scavenging, DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity were evaluated against standard antioxidants such as Curcumin and ascorbic acid were carried out. Tannins were isolated from Alcoholic extract of P.amarusleaves by lead acetate method and to evaluate its in-vitro antioxidant activity by various models. Isolated tannins from the alcoholic extract of P.amrusshowed significant in-vitro antioxidant activity in all the test systems. The tannins isolated from alcoholic extract of P.amarus showed good in-vitro antioxidant activity for all the antioxidant assays. Tannins isolated from alcoholic extract of P.aramusshowed significant antioxidant activity in all the test systems. Thus the results indicate that the tannins isolated from alcoholic extract of P.amarusare a potential source of natural antioxidants.

Keywords

Anti-lipid peroxidation, anti-oxidant activity, DPPH, Superoxide radical, nitric oxide, Phyllanthusamarus, .isolation of tannins

Introduction

Tannins are naturally occurring, high molecular weight polyphenols which can be divided into hydrolysable tannins and condensed tannins. Tannins are the most abundant antioxidants in the human diet and they exhibit many biologically important functions which include protection against oxidative stress and degenerative diseases. The tannin compounds are widely distributed in many species of plants, where they play a role in protection from predation, and perhaps also as pesticides, and in plant growth regulation.
Phyllanthus has been used in Ayurvedic medicine for over 2000 years and has a wide number of traditional uses. The World Health Organization has compiled more than 20 000 medicinal plants used in different parts of the world[1]. Among the medicinal plants more than one hundred botanicals have larger potential for commercial exploitation and could be marketed in the world drug markets [2]. P. amarus, commonly known as Keelanelli (Tamil), Bhuiaonla (Hindi) belonging to the family Euphorbiaceae occupies a prime position among the commercially cultivated medicinal plants. It has a long history of folk use in drug industry for the treatment of dropsy, urogenital problem, dysentery, diabetes, skin ulcer, dyspepsia, fever, asthma, bronchial infections, tumors and Hepatitis B virus. P.amarus is highly valued in the treatment of liver aliments and kidney stones and has been shown to possess anti-hepatitis B-surface antigen activity, oxidative stress and antioxidant activity processes. P. amarus is believed to possess anti-diabetic, anti-nociceptive, antioxidant, antiseptic, antiviral,contraceptive, diuretic, hypotensive and stomachic properties. It is used accordingly in many countries. Herbal medicines are widely perceived by the public as being natural, healthful and free from side effects. However, plants contain hundreds of constituents and some of them may elicit toxic side effects [3] .PhyllanthusamarusSchum&Thonn is an important medicinal plant species due to its antiviral properties, and thus useful against hepatitis infection. The plant is also highly valuable in the Ayurvedic system of medicine [4] .P.amarus is a rich source of phytochemicals such as alkaloids, astragalin, brevifolin, carboxylic acids, corilagin, cymene, ellagic acid, ellagitannins, gallocatechins, geraniin, hypophyllanthin, phyllanthin, lignans, lintetralins, lupeols,methyl salicylate, phyllanthine, phyllanthenol, phyllochrysine, phyltetralin, repandusinic acids, quercetin, quercetol, quercitrin, rutin, saponins, triacontanal and tricontanol[5]. The tannin compounds are widely distributed in many species of plants, where they play a role in protection from predation, and perhaps also as pesticides, and in plant growth regulation. All of these chemical components have their useful as well as toxic effects. Hence safety of herbal medicines is still an issue worldwide. Based on growing attention in free radical biology and the lack of efficient therapies for most chronic diseases, the utility of antioxidants in fortification against neurodegenerative diseases is warranted. Oxidation is essential to many living organisms for production of energy to fuel biologics. Oxidative stress occurs in a cellular system when the production of reactive oxygen species (ROS) exceeds the antioxidant capacity of the system. Oxidative stress plays an important contributory role in the process of aging and pathogenesis of numerous diseases like diabetes, cancer, neurodegenerative diseases, and respiratory tract disorders. Improved antioxidant status helps to minimize the oxidative damage, and thus can delay or decrease the risk for developing many chronic age related, free radical induced diseases[6].Almost all organisms are well protected against free radical damage by antioxidant enzymes such as superoxide radical dismutase (SOD), catalase or chemical compounds such as ascorbic acid polyphenols and glutathione but uncontrolled production of reactive oxygen species (ROS) is responsible for several pathophysiological processes[7]. Tannins were found to be the major phytoconstituents in individual alcoholic extract of P.amarus. Hence isolation of tannins from this extract was undertaken in order to check whether they possess any in - vitro antioxidant activity or not. Majority of disease condition like atherosclerosis, hypertension, ischemic disease, Alzheimer disease, parkinsonism, cancer diabetes mellitus, and inflammatory conditions are being considered to be preliminary due to imbalance between prooxidant and antioxidant homeostasis[8]. Stress, smoking, drugs and diet generates excessive free radicals in the human body. However there are defence mechanisms, which scavenge the free radicals and protect the body from attack of free radicals. These free radicals are important factors for several pathological conditions such as cardiovascular diseases, inflammation, atherosclerosis and degenerative diseases [9] . Antioxidant acts as a major defence against radical – mediated toxicity by protecting the damages caused by free radicals[10] .In the present study, antioxidant properties of P.amarus including its reducing power. The antioxidant may reconcile its effect by unswervingly reacting with ROS, quenching them or chelating the catalytic metal ions. Certain plants show antioxidant activity because of their phenolic constituents [11]. Flavonoids are a broad class of secondary metabolites widely distributed in plants. Flavonoids basically have the antioxidant and the chelating abilities. Numerous synthetic antioxidants e.g. BHA and BHT are commercially available but are pretty perilous and noxious, restricting its use in foods. Therefore the importance of searching natural antioxidants has significantly increased in the current years. The present study was undertaken to investigate the invitro antioxidant activity of isolated tannins of leaves of Phyllanthusamarus.

Material and Methods

Standards and reagents: Ascorbic acid, Potassium ferricyanide, ferrous chloride, ferric chloride, NBT, DPPH, PMS ,napthyl ethylene diaminedihydrochloride, TCA, ,NADH,TBA, were obtained from sigma. All other chemicals and solvents are of analytical grade.
Sample collection and extraction
All the leaves of Phyllanthusamarus were procured from the local market of Mumbai and authenticated at NicholusPiramalPvt.Ltd. India. The leaves were shade-dried, coarsely powdered using a cutter mill and stored in an air-tight, light resistant container for further use. Air–dried leaves of Phyllanthusamarus were defatted with Pet.ether (60-800C) by using Soxhlet extraction apparatus for 48 hours. All the solvents were removed from the pet. ether extract under reduced pressure. All the defatted leaves were air dried and then used individually for alcoholic and aqueous extraction. Individual alcoholic extraction was carried out by using Soxhlet extraction apparatus for 48 hours. All the extracts were removed by using reduced pressure. All the extract was freeze dried and stored in air tight container. Tannins were found to be the major phytoconstituents in individual alcoholic extract of P.amarus. Hence isolation of tannins from this extract was undertaken in order to check whether they possess any in - vitro antioxidant activity.
Isolation of Tannins
1g of individual alcoholic extract of P. amarus was shaken with (100ml) of ethanol on mechanical shaker for 30 minutes. The solution of extract was treated with lead acetate solution (10%) to precipitate tannins as a lead tannate. The suspension was centrifuged for 10 minute to allow the solvent to separate out. The supernatant was decanted off and the precipitate was washed with distilled water. The water was removed by centrifugation followed by decantation. The precipitate was suspended in ethanol and hydrogen sulphide gas was passed through it to remove excess of lead. The lead sulphide precipitate formed was filtered off and the resultant light yellowish coloured filtrate was evaporated to dryness on the water bath to constant weight. The total tannin content extract was determined.
Preliminary phytochemical evaluation:
Alcoholic extract of P. amarus was evaluated qualitatively for the presence or absence of various phytoconstituents[12] .
Assay of in-vitro antioxidant activity
In-vitro antioxidant activity by DPPH method
To determine the In-vitro antioxidant activity of isolated tannins from alcoholic extract of P.amarus , a method based on reduction of methanolic solution of colored free radical DPPH was used. Hydrogen atom or electron donating abilities of corresponding extract like isolated tannins from alcoholic extract of P.amarus and pure compound (synthetic compound like ascorbic acid) were measured from the bleaching of purple colored methanol solution of DPPH. This spectroscopic assay uses the stable radical DPPH as a reagent. The decrease in absorbance of DPPH is proportional to concentration of free radical scavenger added to DPPH reagent solution. Absorbance was taken at 517nm and ascorbic acid was used as a standard solution. The percent inhibition activity was calculated as (A0 – A1)/A0*100, where A0 is the absorbance without sample. And A1 was the absorbance with sample [13].
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Assay of Nitric Oxide Scavenging Activity
Griess reagent containing 1% sulphanilamide, 2% Phosphoric acid and 0.1% Napthyl-ethylene diaminedihydrochloride. Nitric oxide was generated from sodium nitroprusside and measured by Griess reaction. Sodium nitroprusside in aqueous solution, at physiological pH spontaneously generate nitric oxide, which interact with oxygen to produce nitrite ions that can be estimated by use of the Griess reagent. Scavenger of nitric oxide (isolated tannins from alcoholic extract of P. amarus ) compete with oxygen leading to reduced production of nitric oxide. The absorbance of chromophore created during diazotization of nitrite with sulphanilamide following coupling with naphthyl ethylene diaminedihydrochloride was read at 540 nm and compared to the absorbance of standard. The decrease in absorbance of Griess reagent at its absorption maximum of 540nm is proportional to the concentration of free radical scavenger added to the Griess reagent solution [14]. Ascorbic acid was used as a standard solution. (NO radical scavenging activity was calculated by using following equation:
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Lipid peroxidation methods
Mice liver homogenate was used to check the inhibition of lipid Peroxidation. During the aerobic incubation of tissue homogenate with in-vitro free radicals generated by ferrous ascorbate system, malondialdehyde was formed which on reaction with thiobarbituric acid produces a pink colour. Formation of this thiobarbituric acid reacting substance (TBARS) was monitored at 532 nm. Curcumin was used as a reference standard[15]. The method was based on the formation of malondialdehyde during lipid peroxidation. The content of malondialdehydes was estimated as thiobarbituric acid reacting substances (TBARs) by monitoring their colour intensity at 532 nm. Materials: - (AR Grade) 0.15 M Potassium chloride Tries buffer Ferrous sulphate (10 uM) Ascorbic acid (100 uM) Thiobarbituric acid reagent (TBA)
Method:
The liver was quickly removed and chilled in ice-cold saline. After washing with 0.9 % ice-cold saline, the liver was homogenised in 0.15 M Potassium chloride to get 10% (w/v) liver homogenate. Fresh liver homogenate (0.2ml) was mixed with 0.15 mM Potassium chloride (0.1ml) and Tries buffer (0.4 ml).The test extract was then added in various concentrations. In - vitro lipid peroxidation was initiated by addition ferrous sulphate (10 uM) and ascorbic acid (100 uM), 0.1 ml each. After incubation for 150mins. at 37 o C reaction was terminated by addition 2ml of ice cold 0.25N, HCL containing 15% TCA, 0.38% TBA and 0.2 ml of 0.05% (BHT) butylated hydroxyl toluene. These reaction mixtures were heated for 60 min at 80 oC . They were also cooled and centrifuged at 5000 rpm for 15mins.The absorbance of the supernatant was measured at 532nmined all reagent except liver homogenate and drug. Identical experiments were performed to determine the normal (without drug and ferric chloride) and induced (without drug) lipid peroxidation. Curcumin was used as a reference standard.
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Assay of Superoxide Radical Scavenging Activity
The effect of superoxide radical production was evaluated using nitrobluetetrazolium reduction method (Nishikimi et al., 1972). The reaction mixture consisted of 1ml NBT solution (156 μM),1ml NADH( 468μM) and 1ml of sample extracts .the reaction was started by adding 100 μl. Phenazinemethosulphate (60 μM PMS) in phosphate buffer pH 7.4) to the reaction mixture. The reaction was incubated at 250C for 5 min. and the absorbance was measured at 590 nm was measured against blank. Decreased absorbance of reaction mixture indicates increased superoxide anion scavenging activity. Mixture without sample was used as a control and mixture without PMS was used as a blank. The scavenging activity was calculated as follows: Scavenging activity (%) = (1- Abs of sample –Abs of Blank) / Abs of Control[16].
Hydroxyl radical scavenging activity
Hydroxyl radical (OH- ) are generally formFe2+ - ascorbate, EDTA – H2O2 system (Fenton’s reaction) which attack the deoxyribose and set off a series of reaction that eventually results in the formation of malondialdehyde (MDA), measured as a pink MDATBA chromogen at 535nm. Reaction mixture (1ml) contained deoxyribose (2.8 mM), KH2PO4-KOH (20mM) pH 7.4, FeCl3 (100mM), EDTA (104 μM), H2O2 (1mM) and ascorbate (100μM) reaction mixture was incubated at 370C for 1 hour and colored developed as degradation by the isolated tannins from alcoholic extract of Phyllanthusamarus over the control was measured. Curcumin was used as a positive control [17].The % inhibition was measured by the formula.The OH¯ radical scavenging activity of extract is reported as % inhibition of deoxyribose degradation and is calculated as:
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Determination of H2O2 radical scavenging activity
A solution of H2O2 was prepared in PBS (pH 7.4). H2O2 concentration was determined Spectrophotometrically, by measuring absorption with extinction coefficient for H2O2 of 81 m-1cm-1. Extracts (100-500 mg/ml) in distilled water were added to H2O2 solution (0.6 ml, 40 mM). Absorbance of H2O2 at 230 nm was determined 10 min later against a blank solution containing PBS without H2O2 [18].The percentage of H2O2 scavenging of isolated tannins from alcoholic extract and standard compounds were calculated as follows:
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Statistical analysis:
The results are presented as Mean ± SEM one –way analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons, was used for Statistical evaluation. The p-value less than were considered as significant.
Results
From the preliminary phytochemical evaluation it was proved that isolated tannins from alcoholic extract of P.amarusleaves has Alkaloids as confirmed by using the Mayer’s reagent and Dragendorff’s reagent. Glycosides were found in alcoholic tannins of P.amarusby Fehlings and Legal test. Flavonoids (in tannins isolated from alcoholic extract.) Fixed oils were also presents (in tannin isolated from alcoholic extract.) Carbohydrates were found in alcoholic tannin of P.amarus. Amino-acids were found in alcoholic tannin of P.amarus extract.
DPPH radical scavenging activity:
DPPH radical scavenging activities of isolated tannins from alcoholic extract of P.amaruswas presented in Fig 1.Results showed that the radical scavenging activity of isolated tannins of alcoholic extract of P.amarusincreased with increasing concentration. Isolated tannins of alcoholic extract of P.amarus shows IC50 14.35 at a concentration of 0.5mg/ml respectively and R2 were found to be 0.9946. However Ascorbic acid is used as a standard and its radical scavenging activity was found to be more potent. The IC50 Values of standard ascorbic acid was found to be 2.49 at the concentration of 100 μg/ml. Alcoholic extract of P.amarus was found to be more potent and it has good in-vitro antioxidant activity.
Nitric oxide scavenging activity
In the present study, the nitrate produced by the incubation of solution of sodium nitroprusside in standard phosphate buffer at 250C, was reduced by the isolated tannins from alcoholic extract of P.amarusand all exhibited a NO – Scavenging dose response curve. Percentage inhibition was calculated by comparing the results of the test with those of the control that i.e. ascorbic acid. The corresponding IC50 value for NO-Scavenging abilities was calculated from the linear range to concisely compare the relative NO-Scavenging potencies of isolated tannins from alcoholic extract of P.amarus. An IC50 value for the isolated tannins of alcoholic extract of P.amarus was found to be 17.31 at a concentration range of 0.5mg/ml and R2 was found to be 0.9989. Ascorbic acid was used as a standard and its IC50 value was found to be 10.28 at a concentration of 100 μg/ml. Alcoholic extract of P.amarus showed good nitric oxide scavenging activity.
Lipid peroxidation method
Decomposition of lipid membrane in the body leads to the formation of malondialdehyde (MDA) along with other aldehydes and enals as the end product. These react with thiobarbituric acid to form coloured complexes. Hence, these are called as the Thiobarbituric Acid Reactive Substances (TBARS). The complex of TBA-MDA is selectively detected at 532 nm using UV spectrophotometer. Lipid peroxidation involves series of free radical mediated chain reaction processes, which also associated with several biological damages. Lipid peroxidation mainly affects biological membranes of especially liver, brain, spinal cord, containing highly oxidizable PUFA (polyunsaturated fatty acids). Malondialdehyde (MDA) is volatile β-scission product formed as major product of lipid peroxidation along with other aldehydes and ketones. These substances are mainly measured by (TBA) thiobarbituric acid, so called as thiobarbituric acid reactive substances (TBARS). Measurements of MDA levels in biological fluids have been extensively used as measure of lipid peroxidation. Inhibitory effects of ascorbic acid, P.amarus extracts on TBARS formed in mice liver induced by FeCl3 in - vitro were studied. Mice liver homogenate was used to check the inhibition of lipid peroxidation. During the aerobic incubation of tissue homogenate with in-vitro free radicals generated by ferrous ascorbate system, malondialdehyde was formed which on reaction with thiobarbituric acid produces a pink colour. Formation of this thiobarbituric acid reacting substance (TBARS) was monitored at 532 nm. Curcumin was used as a reference standard. The IC50 value of Curcumin was found to be 2.40 at a concentration of 100μg/ml. IC50 value of tannins isolated from alcoholic extract of P.amarus was found to be 30.09 at a concentration range of 0.5 mg/ml and R2 was found to be 0.9934. Hence, Alcoholic extract of P.amarus shows good lipid peroxidation activity. Curcumin was used as a standard and it has good potency and good lipid peroxidation activity against the tannins isolated from alcoholic extract of P.amarus. The results showed inhibition of TBARS formation in mice liver homogenate increased by increasing concentrations.
Assay of Superoxide Radical Scavenging Activity
In the PMS-NADH-NBT system, superoxide anion derived from dissolved oxygen, by PMS-NADH coupling reaction, reduces NBT (yellow dye) to bluecolored product called formazon. Drugs possessing superoxide scavenging activity decreases the reduction of NBT, which is a measure of superoxide anion scavenging activity that is indicated by reduction in absorbance at 560 nm. Superoxide radical is known to be very harmful to cellular components as a precursor of more reactive species. One risk of the superoxide generation is related to its interaction with nitric oxide to form peroxinitrite which is a potent antioxidant that causes nitrosative stress in the organ systems. Superoxide radical scavenging activities of tannin isolated from alcoholic extract ofP.amarus was expressed as IC50 value. An IC50 value of isolated tannins from alcoholic extract of P.amaruswas found to be 84.36 at a concentration range of 0.5mg/ml and R2 was found to be 0.9926. The extracts show dose dependent free radical scavenging activity. The scavenging activity of isolated tannin from P.amarus and standard ascorbic acid IC50 values were found to be 49.32, 67.16 respectively at a concentration range of 0.5 mg/ml and 100μg/ml respectively. Alcoholic extract of P.amarus showed good superoxide radical scavenging activity. Ascorbic acid used as a standard and it is more potent.
Hydroxyl radical scavenging activity
Isolated tannins from alcoholic extract of P.amarusshowed potent hydroxyl radical scavenging activity. The scavenging ability of hydroxyl radical was effective in order of their IC50 values. IC50 value of standard vitamin C was found to be 49.63 at a concentration range of 100μg/ml. IC50 Value of isolated tannins from alcoholic extract of P.amaruswas found to be 25.56 at a concentration range of 0.5mg/ml. It shows moderate hydroxyl radical scavenging activity of the tannins.
Hydrogen peroxide radical scavenging activity
The results of the hydrogen peroxide scavenging activity of tannins isolated from alcoholic extract and Vitamin E showed good potency respectively. An IC50 value of vitamin E used as a standard was found to be 473.48 at a concentration of 100μg/ml. The activity was found to be very less concentration so that the standard Vitamin E was found to be more potent as compared to the alcoholic tannin extract of P.amarus. IC50 values of isolated tannins from alcoholic extract of P.amarus was found to be 5.92 at a concentration of 0.5mg/ml. Alcoholic tannin extract was found to be good potency because it gave results at 0.5mg/ml. Alcoholic tannin extract are good H2O2 scavenging activity.

Discussion

The present study indicates the in-vitro antioxidant activity ofisolated tannins from alcoholic extract ofP.amarus. Phyllanthusamarusis one of the widely used drugs in various Ayurvedic herbal formulations. Traditionally this plant has been recommended mainly for gastric liver damage. Phytochemical analysis were confirmed the presence of phenolic like flavonol glycosides and the glycosides, lignan. The results from thecurrent study clearly justified the use of isolated tannins from alcoholic extract of P.amarusleaves extract in various pathologic conditions. It gives protection against various free radicals by inhibiting the DPPH radical, superoxide anion, nitric oxide, decreasing the lipid peroxidation level. Different models used to evaluate the antioxidant activity suggest that P.amarus alcoholic tannins showed good source of natural antioxidants. Hence, on the basis of the present study it may be concluded that there is corroboration of traditional claims of P.amarus.
DPPH radical scavenging activities of alcoholic tannin extract was presented. Results showed that the radical scavenging activity of isolated tannins from alcoholic extract of P.amarusincreased with increasing concentration and were high for alcoholic extracts. Griess reagent method, all the extracts of P.amarusexhibited a NO – Scavenging dose response curve. Absorbance of the chromophore formed by the diazotization of nitrite with sulphanilamide with subsequent coupling with napthyl ethylene diaminedihydrochloride was read at 546 nm. Percentage inhibition was calculated by comparing the results of the test with those of the control that i.e. ascorbic acid. The corresponding IC50 value for NO-Scavenging abilities was calculated from the linear range to concisely compare the relative NOScavenginging potencies of alcoholic tannin extract of P.amarus. Ascorbic acid used as a standard and it showed good potency. Mice liver homogenate was used to check the inhibition of lipid peroxidation. During the aerobic incubation of tissue homogenate with in-vitro free radicals generated by ferrous ascorbate system, malondialdehyde was formed which on reaction with thiobarbituric acid produces a pink color. Formation of this thiobarbituric acid reacting substance (TBARS) was monitored at 532 nm. Curcumin was used as a reference standard. Hence, Alcoholic tannin extract of P.amarusshows good lipid peroxidation activity. It has good potency. Curcumin was used as a standard and it has good potency and good lipid peroxidation activity against the alcoholic tannin extract of P.amarus. The results showed inhibition of TBARS formation in mice liver homogenate increased by increasing concentrations. The highly reactive OH¯ radical can cause oxidative damage to DNA, lipid and proteins. The effect of extract on the inhibition of free radical mediated deoxyribose damage was assessed by means of iron (II) dependent DNA damage assay. The Fenton reaction generate OH- radical, which degrade deoxyribose sugar of DNA using Fe2+ salt as an important catalytic component. Ferric –EDTA was incubated with H2O2 and ascorbic acid at pH 7.4. Hydroxyls radical was formed in free solution and were detected by their ability to degrade 2- decoy-2- ribose into fragments that formed a pink chromogen upon heating with TBA at low pH. Alcoholic tannin extract of P.amarus showed potent hydroxyl radical scavenging activity. The scavenging ability of hydroxyl radicals, alcoholic tannin extract of P.amarus was effective in order of their IC50 values. IC50 value of standard vitamin C was found to be 49.63 at a concentration range of 100μg/ml. An IC50 Value of alcoholic tannin from P.amarus extract was found to be 27.42 at a concentration range of 0.5mg/ml. It shows moderate hydroxyl radical scavenging activity. In the PMS-NADH-NBT system, superoxide anion derived from dissolved oxygen, by PMS-NADH coupling reaction, reduces NBT (yellow dye) to blue-colored product called formazon. Drugs possessing superoxide scavenging activity decreases the reduction of NBT, which is a measure of superoxide anion scavenging activity that is indicated by reduction in absorbance at 560 nm. One risk of the superoxide generation is related to its interaction with nitric oxide to form peroxinitrite which is a potent antioxidant that causes nitrosative stress in the organ systems. Superoxide radical scavenging activities of alcoholic tannin extract from P.amaruswas expressed as IC50 value. The extract showed dose dependent free radical scavenging activity. Alcoholic extract of P.amarusshowed good superoxide radical scavenging activity. Ascorbic acid used as a standard and it is more potent for the extract. H2O2 radical it not very reactive but it can sometimes be toxic to cell because it generates OH¯ radical in the cell. The results of the hydrogen peroxide scavenging activity of alcoholic tannin extract and Vitamin E showed good potency respectively. Tannin exact was good H2O2 scavenging activity. The activity was found to be very less concentration so that the standard Vitamin E was found to be more potent as compared to the tannin extract of P.amarus. The results from thecurrent study clearly justified the use of isolated tannin from alcoholic extract of P.amarusleaves in various pathologic conditions. It gives protection against various free radicals by inhibiting the DPPH radical, griess reagent, lipid peroxidation method, hydroxyl radical, superoxide anion, hydrogen peroxide method, Different models used to evaluate the antioxidant activity suggest that P.amarusalcoholic tannin extract showedgood source of natural antioxidants.. Hence, on the basis of the present study it may be concluded that there is corroboration of traditional claims of P.amarus. These results suggest that the higher levels of antioxidant activity were due to presence of phenolic components. These findings are in accordance with the earlier report on total phenolic and antioxidant activity in isolated tannins from alcoholic extract of P.amarus. Tannins are very important plant constituents because of their scavenging ability due to their hydroxyl groups. The phenolic compounds may contribute directly to antioxidative actions. It is known that polyphenolic compounds have inhibitor effects on mutagenesis and carcinogenesis in humans. Phenolic compounds from plants are known to be good natural antioxidant. However, the activity of synthetic antioxidants was often observed to be higher than that of natural antioxidant.

Conclusion

The present investigation on the in-vitro antioxidant activities of isolated tannins from alcoholic extract of P.amarus leaves was found to be more potent antioxidants. The DPPH free radical assay, nitric oxide, in-vitro lipid peroxidation method, superoxide radical scavenging method ,hydroxyl radical , hydrogen peroxide methods were found to be good antioxidant activity for the extract. Different models used to evaluate the antioxidant activity suggest that isolated tannins of alcoholic extract of P.amarusshowed good source of natural antioxidants. Hence, on the basis of the present study it may be concluded that there is corroboration of traditional claims of isolated tannins from alcoholic extract ofP.amarus.
In the present study, there exist a positive correlation between total phenolic content and the antioxidant activity which is in accordance where the earlier findings. We found higher in-vitro antioxidant activity with higher polyphenolics compounds. The higher radical scavenging efficacy of isolated tannins from alcoholic extract of P.amarus may be due to their retention of antioxidant phytochemicals in this extract. A strong evidence support these findings that the isolated tannins from alcoholic extract of P.amarus enhances the antioxidants effects in–vitro models. These results suggest that isolated tannins from alcoholic extract of P.amarus may offer effective protection from free radicals and support that is a promising source of natural antioxidants.

Conflict of Interest

NIL

Source of Support

NONE

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