INTRODUCTION:

Ayurveda is a time tested empherical medicinal system developed by dhanvantris of ancient India who have continuously screened the plants of India for their therapeutic effects in an empherical manner. This led to the development of ayurveda. The present multinational companies are also screening these same plants but in a more scientific manner to obtain lead molecules for certain target receptors. To compete with allopathy, ayurveda needs standardization of drugs and dosages with animal and clinical studies for explanation of its superiority. This study is a step to establish the antioxidant activity of Terminalia chebula fruit.

Naturally occurring antioxidants such as ascorbic acid, vitamins E and phenolic compounds, possess the ability to reduce the oxidative damage associated with many diseases¹.recent studies indicate that carotenoids,² tocopherols, ascorbates and phenols are strong natural antioxidants, generally found in fruits and vegetables³ Phenols, a major group of phytochemicals with antioxidant properties has profound importance as preventive medicine⁴. Plants with a high total phenolic content are Cassia fistula, Azadirachta indica, Trewia nudiflora, Casuarina equisetifolia, Averrhoa carambola, Lawsonia inermis, Indigofera tinctoria, Terminalia chebula, molinga oleifera.⁴

Terminalia chebula belongs to the family Combretaceae and has classical names Haritaki, Abhaya, Pathya, kayastha, Putana, Haimavati, Avyatha, chetaki, Shiva, Vayastha, Rohini⁵.Fruit is a hard stony drupe, greenish yellow in color, odorless, ovate, longitudinally wrinkled. The major chemical constituents in Terminalia chebula fruit are tanins (20 to 40 percent) which on hydrolysis gives chebulic acid and D- gallolyl glucose. Other chemical constuents present are anthraquinone glycosides, chebulic acid, chebulagic acid, tannic acid, terchebin, tetrachebulin, vitamin C (fruits); arachidic, behenic, linoleic, oleic, palmitic and stearic acid (fruit kernels); Chebulin (flowers); 2-α- hydroxylmicromeric acid, maslinic acid and 2-α- hydroxy ursolic acid (leaves).⁶ Fruits are astringent, sweet, acrid, bitter, sour, thermogenic, anodyne, anti-inflammatory, vulnerary, stomachic, laxative, purgative, carminative, digestive, anthelminthic, dentifrice, cardiotonic, aphrodisiac, antiseptic, diuretic, febrifuge, depurative and tonic. In combination with Phylanthus emblica and Terminalia bellirica under the name triphala, fruits of Terminalia chebula are extensively used as an adjunct to other medicine in almost all disease.^5,6,7

MATERIAL AND METHODS:

Collection of Fruits:

Dry fruits of Terminalia chebula retz were purchased from a wholesale dealer named Batliwala, Dhan Mandi, Udaipur. A specimen copy of the sample was kept in the Pharmacognosy laboratory. The fruits of the plants were then authenticated by Dr. Yuvraj Singh Sarangdeot, Professor and Mr. .Bhupendra Vyas Associate Professor, Pharmacognosy Department, B.N. College of Pharmacy, Udaipur.

Extraction:

Thousand grams of the fruit were ground in a mixer-grinder until a coarse powder was obtained. The coarse powder was transferred to four glass jars each of 1litre capacity. Each jar contained 250gm of powdered fruit and 400ml of 70% ethanol was added to it. The jars were kept in a room with their lids tightly closed. The mixture was stirred 3 to 4 times a day. This type of cold maceration was carried out for 15 days. The ethanolic extract was then filtered through a muslin cloth.⁸

Drying:

The ethanolic extract was then vaccum evaporated and then spread in a very thin layer in steel plates in a dust free room and allowed to dry at room temperature. The dried ethanolic extract was transferred to air tight containers and kept in desiccators for further use.

In vitro Studies for antioxidant activity:

Various models are used for determining the antioxidant activity in plant extract.

DPPH Radical Scavenging Activity:

Chemicals:

DPPH, Ascorbic acid, methanol, distilled water, extract.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Pipettes, Volumetric flasks.

Procedure:

The free radical scavenging capacity of the ethanolic extracts of Terminalia chebula was deter-mined using DPPH. The DPPH solution (0.1mM) was prepared in methanol. Ethanolic extract of Terminalia chebula were mixed with distill water to prepare the stock solution (1mg/1mL). From stock solutions 50 µg/ml, 100µg/ml, 150µg/ml, 200 µg/ml and 250 µg/ml dilutions of the extract were made in 5 ml containers. To 0.3ml of each dilution of the extract was added freshly prepared DPPH solution (0.1mM). The mixture was shaken and kept for 10 minutes and absorbance was measured at 517nm wave-length using a spectrophotometer (UV-17000 Pharmaspec, UV- visible-spectrophotometer, Shimazdu). Ascorbic acid was used as a reference standard and dissolved in distilled water to make the stock solution with the same concentrations. Control sample was pre-pared containing the same volume without any extract and reference ascorbic acid. Methanol (95%) was used as blank.^{9, 10, 11, 12} Scavenging of the DPPH free radical was measured using the following equation:

% DPPH radical-scavenging = [(Absorbance of control−Absorbance of test Sample) / (Absorbance of control)] x 100.

Total antioxidant activity:

Chemicals:

Sulfuric acid, Sodium phosphate, Ammonium molybdate, Ascorbic acid, methanol, distilled water, extract.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Pipettes, Volumetric flasks.

Procedure:

The total antioxidant capacity of the extract was assessed by phosphomolybdenum method. The assay is based on the reduction of Mo (vi) – Mo (v) by the extract and subsequent formation of green phosphate / Mo (v) complex at acidic pH. Ethanolic extract of Terminalia chebula and ascorbic acid were mixed with distill water to prepare the stock solution (1 mg/1mL). From stock solution of Ascorbic acid 50 µg/ml, 100µg/ml, 200µg/ml, 250µg/ml, 400 µg/ml dilutions were made in 5 ml containers. 0.2ml of extract and 0.2ml of all dilutions of ascorbic acid were pipated in separate small tubes. To each tube was added 3ml of reagent solution (0.6M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). The tubes containing the mixture were incubated at 95ºC for 90 minutes. The absorbancre of the solution was measured at 695nm using a spectrophotometer (UV-17000 Pharmaspec, UV- visible-spectrophotometer, Shimazdu) against blank after cooling at room temperature. Methanol (0.2ml) in place of extract is used as the blank. The antioxidant activity is expressed as the number of equivalent of ascorbic acid calculated from linear equation.^{13, 11,12,14}

Total Flavanoid content:

Chemicals:

Aluminium Chloride, Sodium Nitrite, Sodium hydroxide, Rutin, methanol, distilled water, extract.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Pipettes, Volumetric flasks.

Procedure:

Ethanolic extract of Terminalia chebula and Rutin were mixed with distill water to prepare the stock solution (1mg/1mL). From stock solution of rutin 50 µg/ml, 100 g/ml, 200µg/ml, 250µg/ml, 400 µg/ml dilutions were made in 5 ml containers. To 0.5ml of each dilution prepared, add 1.5ml methanol followed by 0.15ml of Sodium Nitrite (1:20). After 5 minutes add 1.5ml of AlCl₃ (1:10). After another 6 minutes add 1ml of 1M sodium Hydroxide. Make final volume by adding 0.35 methanol. The solutions are mixed well and absorbance was measured against blank at 510nm using UV –visible spectrophotometer. Rutin was used as standard. Total flavonoid content was measured using linear equation.^{9, 18,15,10}

Total Phenolic content:

Chemicals:

Gallic acid, Folin Ciocalteu reagent, Sodium carbonate, methanol, distilled water, extract.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Pippetes, Volumetric flasks.

Procedure:

Stock solutions (1mg/ml) of Ethanolic extracts of Terminalia chebula and Gallic acid were prepared. From stock solutions of Gallic acid 50 µg/ml, 100 µg/ml, 200 µg/ml, 400µg/ml and 500µg/ml dilutions were made in 5 ml containers. To 100 µg/ml of the extract was added 2ml of methanol and 0.2ml 2N Folin Ciocalteu reagent. After 3 minutes, 1ml of 15% sodium carbonate was added. The tubes were mixed thoroughly, kept for 2 hrs at room temperature and read at 735nm using UV spectrophotometer. The same procedure was repeated with all dilutions of Gallic acid (50µg/ml-500µg/ml) and a standard curve was obtained. The Total Phenolic content was expressed as GAE and calculated from linear equation.^16,17,18,19

Reducing Power:

Chemicals:

Potassium dihydrogen phosphate, Sodium hydroxide, Potassium ferricyanide, TCA, Ferric chloride, distilled water, extract.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Pipettes, Volumetric flasks, and research centrifuge.

Procedure:

The reducing power of the extract was evaluated according to Oyaizu (1986). Different dilutions (50µg/ml, 100 µg/ml, 200 µg/ml, 400µg/ml and 500µg/ml) of the extracts of Terminalia chebula and ascorbic acid were prepared. One ml of the extract and ascorbic acid were mixed with 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% Potassium Ferricyanide. The mixture was incubated at 50˚C for 20 minutes, 2.5ml of 10% TCA was added to the mixture and centrifuged at 3000 rpm for 10 minutes. The upper layer of the solution (2.5ml) was mixed with distill water (2.5ml) and Ferric Chloride (0.5ml, 0.1%) and absorbance was measured at 700nm. Increase in the absorbance of the reaction mixture indicated reducing power. Ascorbic acid was used as standard and reducing power was expressed as Ascorbic acid equivalent.^{11, 14,12,20}

Scavenging of Super oxide:

Chemicals:

Potassium dihydrogen phosphate, Sodium hydroxide, EDTA, Riboflavin, NBT, Sodium cyanide, methanol, ascorbic acid, distilled water and extracts.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Pipettes, Volumetric flasks. Illuminating chamber.

Procedure:

The super oxide was generated in riboflavin-light NBT system. Stock solutions (1mg/1mg) of the extract and ascorbic acid were prepared. Dilution of 50 µg/ml, 100 µg/ml, 200 µg/ml, and 400µg/ml of the extracts and ascorbic acid were made. 100 µl Riboflavin (20 µg), 200 µl EDTA solution(12mM), 200 µl methanol and 100 µl methanol and 100 µl NBT(Nitro-blue tetrazolium) solutions(0.1mg) were mixed in test tubes and reaction mixture was diluted up to 3ml with phosphate buffer (50mM). The absorbance of the solution was measured at 590nm on UV-17000 Pharmaspec, UV- visible-spectrophotometer, Shimazdu, using phosphate as blank after illumination for 5 minutes. This is taken as control. Repeat the above procedure using 0.1ml of extract and ascorbic acid. Ascorbic acid was used as positive control and IC₅₀value of the extract was determined using linear equation. ^{21, 22, 23, 16}

Scavanging of H₂O₂

Chemicals:

Potassium dihydrogen phosphate, Disodium hydrogen phosphate, Sodium chloride, Hydrogen peroxide, ascorbic acid, distilled water and extracts.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Pipettes, Volumetric flasks, test tubes.

Procedure:

Scavanging of H₂O₂by the extracts was determined by the method of Ruch et al (1989). Stock solutions (1mg/1mg) of the extract and ascorbic acid were prepared. Dilution of 50 µg/ml, 100 µg/ml, 200 µg/ml, and 400µg/ml of the extract and ascorbic acid were made using Phosphate buffer saline (PBS). Hydrogen Peroxide (4mM) was also prepared in PBS. To 2.4ml of PBS was added 0.6ml of hydrogen peroxide (4mM) and the mixture was kept for 10 minutes at room temperature. The absorbance of the solution was measured at 230nm against a blank of PBS on a UV-17000 Pharmaspec, UV- visible-spectrophotometer, Shimazdu. This was taken as control. To 1ml of each dilution of extract and standard ascorbic acid was added 1.4 ml of PBA and 0.6ml of hydrogen Peroxide (4mM). The solutions were kept for 10minutes and absorbance was measured at 230nm. Percentage scavenging of the extract and ascorbic acid was calculated from the following equation.^{16, 24, 14}

% scavenging =100 ×[(Absorbance of control –Absorbance of sample) / (Absorbance of control)].

Scavanging of Hydroxyl radical:

Chemicals:

Ferrous ammonium sulhate, EDTA, DMSO, Potassium dihydrogen phosphate, Sodium hydroxide, Ascorbic acid, ammonium acetate, glacial acetic acid, acetyl acetone, Trichloroacetic acid (TCA), Rutin, distilled water and extract.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Incubator, Pipettes, Volumetric flasks, test tubes.

Procedure:

Scavanging of Hydroxyl radical by extracts was determined using Halliwel method. Stock solutions (1mg/1mg) of the extract and rutin were prepared. Dilution of 50 µg/ml, 100 µg/ml, 200 µg/ml, and 400µg/ml of the extract and rutin were made. A quantity of 0.1ml of each the above dilutions of the extract and rutin were taken in separate vials, to which 1.0 ml of Iron EDTA solution (0.13% ferrous ammonium sulphate and 0.26% EDTA), 0.5ml of EDTA solution(0.018%) and 1ml of DMSO (0.85% v/v in 0.1M phosphate buffer, pH 7.4). The reaction was initiated by adding 0.5ml of ascorbic acid (0.22%) and incubated at 80-90˚C for 15 minutes in a water bath. After incubation the reaction was terminated by the addition of ice-cold TCA (17.5% w/v). Three milliliters of Nash Reagent (75g of ammonium acetate, 3.0 ml of glacial acetic acid and 2ml of acetyl acetone were mixed and raised to 1L with distilled water) was added and left at room temperature for 15 minutes. The reaction mixture without sample was used as control. The intensity of the colour was measured at 412nm against a blank using a UV-17000 Pharmaspec, UV- visible-spectrophotometer, Shimazdu. The percentage inhibition was calculated by the formula as follows.^{11, 12, 25, 26}

% scavenging =100 ×[(Absorbance of control –Absorbance of sample) / (Absorbance of control)].

IC₅₀value for the extract was calculated from curve fitting method.

Nitric Oxide Radical Scavenging activity:

Chemicals:

Potassium dihydrogen phosphate, Disodium hydrogen phosphate Sodium chloride, Sodium Nitroprusside, sulphanilic acid reagent, Naphthylethylene diamine dihydrochloride., glacial acetic acid. Ascorbic acid, distilled water and extract.

Instruments used:

UV Spectrophotometer, Electronic weighing Balance, Incubator, Pipettes, Volumetric flasks, test tubes.

Procedure:

Nitric oxide radical inhibition is estimated by use of Griess Illosvoy reaction. Here Griess Illosvoy reagent was modified by using naphthyl ethylene diamine dihydrochloride (0.1%w/v) instead of 1-napthylamine (5%). Stock solutions (1mg/1mg) of the extract and ascorbic acid were prepared. Dilution of 50 µg/ml, 100 µg/ml, 200 µg/ml, and 400µg/ml of the extract and ascorbic acid were made. The reaction mixture (3ml) containing sodium nitroprusside (10mM, 2ml), phosphate buffer saline(0.5ml), 0.5ml of each dilution of Terminalia chebula extracts and ascorbic acid was incubated at 25ºC for 150 minutes. After incubation 0.5ml of the reaction mixture was mixed with 1ml of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to atand for 5 minutes for completing diazotization. Then naphthlethylene diamine dihydrochloride (1ml) was added, mixed and allowed to stand for 30 minutes at 25˚C. A pink coloured chromophore is formed in diffused light. The absorbance of these solutions was measured at 540nm against corresponding blank solutions. The entire procedure was repeated without extract or ascorbic acid which gave the control readings. Ascorbic acid was used as positive control. The percentage inhibition was calculated by the formula as follows.^{19, 18, 21, 12}

% scavenging =100 ×[(Absorbance of control –Absorbance of sample) / (Absorbance of control)].

IC₅₀value for the extract was calculated from curve fitting method.

Protein Oxidation

Chemicals

Bovine Serum Albumin(BSA), Hydrogen peroxide, Ferric chloride, Ascorbate, EDTA, Tris buffer pH 8.2, 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) Potassium dihydrogen phosphate, Sodium hydroxide, Ascorbic acid, Trichloroacetic acid (TCA), Rutin, distilled water and extract.

Instruments used

UV Spectrophotometer, Electronic weighing Balance, Incubator, Pipettes, Volumetric flasks, test tubes.

Procedure

The protein (BSA) was oxidized by a Fenton type reaction. BSA (1mg/ml) was incubated at 25ºC in solution with 2.5mM H2O2_, 1.0 mM FeCl₃, 1.0 mM ascorbate and 3.0mM EDTA in presence or absence of extract (control). After incubation for 45 minutes, proteins were precipitated with 10% ice cold TCA, centrifuged (5000rpm, room temperature, 10 minutes) and the supernatant was decanted . Proteins pellets were dissolved in 50mM potassium phosphate buffer, pH 7.5.

Total sulfydryl (-SH) group determination was performed according to the method of Sedlak and Lindsay (1968) using Ellmans reagent. Aliquots of 0.5ml of the above solution were mixed in a 15 ml test tube with 1.5ml of 0.52M Tris buffer, pH 8.2 and 0.01M DTNB (Ellmans reagent). The mixture was brought to 10 ml with 7.9ml of absolute methanol or water or SDS. A reagent blank (control) and a sample blank (without DTNB) were prepared in a similar manner. Colour was developed for 15 to 30minutes and the reaction mixture was centrifuged at room temperature for 15 minutes. The absorbance of the supernatant was measured at 412 nm in 1 cm quartz cell.^{16, 27,
28}

Lipid Peroxidation

Chemicals

Hydrochloric acid, Sodium dodecyl sulphate (SDS), Thiobarbituric acid (TBA), n- Butanol, Ferric chloride, Tris HCl buffer pH 7.2 Ascorbic acid, Rutin, distilled water and extracts.

Instruments used

UV Spectrophotometer, Electronic weighing Balance, Incubator, Research centrifuge, Homogenizer, Pipettes, Volumetric flasks, test tubes.

Procedure

Preparation of liver homogenate: Liver homogenate was prepared by collecting fresh chickens liver from poultry. The liver was immediately dipped in ice cold 150mM of Tris HCL (pH 7.2). The liver was immediately cut into pieces and homogenized using a homogenizer with Tris HCL to give 20% w/v liver homogenate. The liver homogenate was then centrifuged at 3000 rpm for 10 minutes to get a clear supernatant.

Anti-lipid peroxidation activity was determines by Ohkawa et al method (1979).

A Reaction mixture was prepared containing 250µl of liver homogenate, 100 µl of Tris HCL buffer(pH 7.2), 50 µl of 0.1mM ascorbic acid, 50 µl of 4mM FeCl₃and 50 µl of extracts or standard. The mixture was prepared in a capped tube, shaken well and incubated at 37ºC for 1 hour. After incubation, 500 µl of 0.1N HCl, 200 µl of 9.8 % SDS, 900 µl distill water and 2 ml of 0.6% Thiobarbituric acid (TBA) was successively added. The mixture was vigorously shaken before it was placed into a boiling water bath (95ºC) for 30 minutes. After cooling, 5 ml n-Butanol was added. The mixture was then centrifuged at 5000 rpm at a temperature of 25ºC for 25 minutes to remove flocculent precipitate. The lipid peroxide concentration was determined by MDA-TBA adduct (conjugation of malondialdehyde with thiobarbituric acid) at 532 nm using a UV-17000 Pharmaspec, UV- visible-spectrophotometer, Shimazdu. The entire procedure was repeated without extracts which gives the control readings. Percentage inhibition was calculated and compared with standard rutin using the formula as follows. ^{29, 22, 30, 26, 31}

% scavenging =100 ×[(Absorbance of control –Absorbance of sample) / (Absorbance of control)].

RESULTS:

Table No. 01. Total Phenolic content, Total antioxidant activity and Total flavanoid contents of Terminalia chebula fruit extract.

S. No.	Extract Name	TPC (gm GAE /kg ext.)	TAA (µg Ascorbic acid Eq./mg ext.)	TFC (µg rutin Eq/mg ext.)
1	Terminalia chebula fruit extract.	241± 1.57	295.73± 27.86	305.96± 47.23

Table No. 02. 1,1 -diphenyl-2-picryl-hydrazol (DPPH) free radical scavanging activity of ascorbic acid and Terminalia chebula fruit extract.

Terminalia chebula extract(µg/ml)		Ascorbic acid
Conc µg/ml	% DPPH scavanging	Conc µg/ml	% DPPH scavanging
2	61.99±1.43	10	87.13±0.33
4	65.31±0.16	20	87.78±0.38
6	66.72±0.23	30	90.98±0.23
8	68.56±0.248	40	92.89±0.24
10	70.19±0.09	50	95.76±0.24
EC90	30.52±2.85	EC90	25.80±1.11

Figure No. 01: 1,1 -diphenyl-2-picryl-hydrazol (DPPH) free radical scavanging activity of Terminalia chebula fruit extract.

Figure No. 02: 1,1 -diphenyl-2-picryl-hydrazol (DPPH) free radical scavanging activity of ascorbic acid.

Table No.03. Scavaning of Nitric oxide radical by Terminalia chebula fruit extract and Ascorbic acid

Conc. (µg/ml)	Terminalia chebula extract.	Ascorbic acid extract.
50	19.23±7.65	9.42±6.40
100	31.32±11.09	16.25±10.21
200	50.52±10.60	31.31±8.83
250	59.03±8.05	36.22±9
400	69.39±0.228	63.29±1.63
IC50	210.380±58.22 µg/ml	311.656±36.94 µg/ml

Data are Mean ± S.E.M for three replicates.

Figure No. 03: Scavanging of Nitric oxide radical by Terminalia chebula fruit extract.

Figure No. 04: Scavanging of Nitric oxide radical by Ascorbic acid.

Table No.4: In vitro Superoxide radical scavanging activity of Terminalia chebula fruit extract and ascorbic acid.

Conc. (µg/ml)	% Scavanging of Supreoxide radical
Conc. (µg/ml)	Terminalia chebula extract.	Ascorbic acid.
50	22.09±12.95	3.83±2.21
100	33.64±10.95	5.73±1.82
200	59.22±1.32	8.72±1.11
250	66.76±0.90	12.5±1.48
400	84.94±5.40	18.10±2.82
IC50	161.99±33.04 µg/ml	1206.41±172.78µg/ml

Data are Mean ± S.E.M for three replicates.

Figure No. 05: Superoxide radical scavanging activity of Terminalia chebula fruit extract.

Figure No. 06: Superoxide radical scavanging activity of ascorbic acid.

Table No.05: Inhibitory effect of extracts and standards on BSA oxidation induced by Fenton type reaction.

Conc. (µg/ml)	% Inhibition of BSA oxidation
Conc. (µg/ml)	Terminalia chebula extract.	Ascorbic acid	Rutin
50	75.09±4.80	68.154±5.63	83.677±.506
100	78.54±10.17	80.53±13.08	84.917±.506
200	78.54±12.43	70.385±2.158	85.330±.843
250	77.16±9.04	74.645±5.63	86.983±.168
400	74.91±8.33	74.441±1.158	87.809±.506

Data are Mean ± S.E.M for three replicates.

Figure No. 07: Inhibitory effect of various concentration of Terminalia chebula fruit extracts on BSA oxidation induced by Fenton type reaction.

Figure No. 08: Inhibitory effect of various concentration of Ascorbic acid on BSA oxidation produced by Fenton type reaction.

Figure No. 09: Inhibitory effect of various concentration of Rutin on BSA oxidation induced by Fenton type reaction.

Table. No.6: Reducing power of Terminalia chebula fruit extract and ascorbic acid

Ab. At 700nm
conc(µg/ml)	Terminalia chebula extract	Ascorbic acid
50	0.24	0.38
100	0.29	0.515
250	0.924	0.71
400	0.944	1.32
500	1.772	2.44

Table No.07.: Percentage inhibition of lipid peroxidation by Terminalia chebula fruit extract and rutin

Conc. (µg/ml)	% inhibition of lipid peroxidation
Conc. (µg/ml)	Terminalia chebula extract.	Rutin
50	21.31±2.70	18.81±3.17
100	23.02±2.36	21.31±2.70
200	24.74± 2.17	23.15±2.70
250	26.93±2.16	25.37±2.18
400	28.49±2.43	27.71±2.36
IC50	1424.54±70.74 µg/ml	1323±149.26 µg/ml
Data are Mean ± S.E.M for three replicates.

Figure No. 10: Reducing power of Terminalia chebula fruit extract.

Figure No. 11: Inhibition % of lipid peroxidation by Terminalia chebula fruit extract.

Figure No. 12: Inhibition % of lipid peroxidation by rutin.

Table. No.08: Scavenging of Hydrogen Peroxide by Terminalia chebula fruit extract and ascorbic acid

Ab. At 700nm
Conc (µg/ml)	Terminalia chebula extract	Ascorbic acid
50	0.766	0.373
100	1.117	0.49
250	1.851	0.785
400	3.214	1.303
500	3.983	1.57

Figure No. 13: Scavanging of Hydrogen Peroxide by Terminalia chebula fruit extract.

Table No. 09 Scavanging of Hydroxyl radiclal by Terminalia chebula fruit extract and ascorbic acid

Ab. At 700nm
Conc (µg/ml)	*Terminalia chebula* extract	Rutin
50	17.36±9.86	34.82±16.84
100	20.25±5.12	24.58±7.26
250	16.63±4.63	36.47±5.16
400	19.69±4.15	24.82±8.62
500	6.75±2.13	17.29±4.55

Figure No. 14: Scavanging of Hydroxyl radical by Terminalia chebula fruit extract.

RESULT AND DISCUSSION:

Reactive oxygen species (ROS) such as superoxide radicals, hydroxyl radicals, iron –oxygen complexes, hydrogen peroxide and lipid peroxides are generated by several oxidative reactions. Although ROS can help the immune system to clear out extrusive microorganisms, excessive ROS can also react with biological molecules such as DNA, proteins and phospholipids, and eventually cause oxidative damage in tissues and free radical related diseases such as inflammation, heart disease, diabetes, gout, cancer, etc. For aerobic organisms, the major system of defense against oxidative damage is the use of “antioxidants” enzymes to convert excessive ROS into non toxic compounds. An imbalance between the amount of ROS and antioxidant enzymes is a problem for our health. This is why the daily intake of foods with antioxidant activity is necessary.³² The Plant extracts property of scavenging these ROS species has been evaluated using separate assays for the each type of Reactive oxygen species.

The Total Phenolic content, Total antioxidant activity and Total flavanoid contents of Terminalia chebula fruit extract were 241±1.57, 295.73±27.86 and 305.96± 47.23 respectively which was appreciably high and is responsible for the high medicinal value of terminalia chebula fruits.

1, 1 -diphenyl-2-picryl-hydrazol is a relatively stable radical. The DPPH assay is based on the measurement of the scavenging ability of antioxidants towards the stable radical, DPPH which reacts with suitable reducing agents. The electrons become paired off and the solution loses colour stoichiometrically depending on the number of electrons taken up. Ascorbic acid has a strong DPPH scavenging property. Terminalia chebula fruit extracts show results comparable with ascorbic acid. The EC₉₀ of Terminalia chebula and Ascorbic acid as presented in Table. No.2 were calculated as 30.52±2.85 and 25.80±1.11 respectively from regression as shown in figure. No. 1 and 2.

Nitric oxide is a free radical generated by the endothelial cells, macrophages, neurons etc. and is involved in the regulation of various physiological processes. Excess concentration of NO is associated with several diseases.^{33, 12, 18,
15, 34} Oxygen reacts with excess nitric oxide to generate nitrite and peroxynitrite anions, which act as free radicals. In the assay carried out, which measures the NO scavenging property, the ethanolic extracts of terminalia Chebula showed Nitric oxide scavenging activity whose IC₅₀ was 380±58.22 µg/ml and ascorbic acid had IC₅₀ -311.656±36.94 µg/ml. figure No. 3 and 4 show regression. Thus Terminalia chebula fruit extracts have the potential to quench excess Nitric oxide released by endothelium cells and macrophages.

Superoxides are produced from molecular oxygen due to oxidatives enzymes of the body. The scavenging activity towards the superoxide radical is measured in terms of inhibition of generation of superoxide radical. The superoxide radical reduces NBT to a blue coloured Formosan that is measured at 560 nm.^{9,14,20,30,31,35} The results show that Terminalia chebula extract has a potent scavenging activity whose IC50 as shown in Table. No. 4 was 161.99±33.04 µg/ml. and ascorbic acid has a IC50 equal to 1206.41±172.78µg/ml µg/ml and were calculated from Figure No. 5 and 6.

Proteins are oxidized by Fenton type reactions in which the –SH groups are removed. In our study we have observed that Terminalia chebula protects these –SH groups as effectively as ascorbic acid. Rutin provides higher protection as shown in table. No.05 and Figure No. 7,8 and 9.

A substance may act as an antioxidant due to its ability to reduce ROS by donating hydrogen atom.^{12, 14} The reducing assay is used to measure this property of extract. Terminalia chebula fruit extract shows a good reducing power at 250µg/ml and 400µg/ml as shown in Table no. 06 and Figure no. 10, which implies that it is capable of donating hydrogen atom in a dose dependent manner. The high content of phenolic compounds in the Terminalia chebula fruit extracts may be a contributing factor towards the antioxidant activity because the phenolic compounds contain hydroxyl groups which can functions as hydrogen donors.¹⁴

The scavenging of H₂O₂ by ascorbic acid and the extract after 10 minutes increased with increase in concentration. The Terminalia chebula fruit extract has exhibited higher H₂O₂ scavenging property than ascorbic acid as shown in table no. 08. and Figure no.13. Removal of H₂O₂ is important for antioxidant defense in cell and food systems. Dietary polyphenols are known to prevent bacterial cells from cytotoxicity induced by H₂O₂, especially those compounds with orthodihydroxy phenolic structure e.g. quercetin, catechin, gallic acid, esters, caffeic acid esters.^{14, 16, 24} Therefore the phenolic compounds of the test extract might be responsible for removing the H₂O₂.

The peroxidation of membrane lipids initiated by oxygen radicals may lead to cell injury. initiation of lipid peroxidation takes place through ferryl-perferyl complex or through hydroxyl generated by Fenton’s reaction, thereby initiating a cascade of oxidative reaction. The inhibition of lipid peroxidation may be attributed to one of the several reasons viz., inhibition of ferryl-preferryl complex formation; scavenging of hydroxyl or superoxide radical or changing the ratio of Fe³⁺/ Fe²⁺; reducing the rate of conversion of ferrous to ferric or by chelation of iron itself. In the present in vitro study lipid peroxidation was induced in the liver homogenate by using FeSO4 and ascorbic acid. Ther lipid peroxides are measured as TBARS. Increase in levels of TBARS indicates enhanced lipid peroxidation leading to tissue injury and failure of the defense mechanism to prevent formation of free radicals.^36,37,38

Terminalia chebula shows inhibition of lipid peroxidation. The IC₅₀ is 1424.54±70.74 µg/ml and that of rutin is 1323±149.26 µg/ml which is calculated from the graph in figure no. 11 and 12.

The hydroxyl radical scavenging activity is measured as the percentage of inhibition of hydroxyl radicals generated in the fenton’s reaction mixture. The hydroxyl radicals are generated by halliwel method.^21,25,30 Terminlia chebula also shows some hydroxyl radical scavanging activity as shown in table no. 9 and Figure no.14.

CONCLUSION:

In the in vitro studies terminalia chebula has high Phenolic contents, Flavanoid content, total antioxidant activity, high DPPH scavenging, NO scavenging, superoxide scavenging, better hydrogen peroxide scavenging, protein oxidation and reducing power. These results clearly indicate that Terminalia chebula has a good antioxidant agent and supports the practice of use of Terminalia chebula in ayurveda in various formulations. One of the popular formulations is triphala.

ACKNOWLEDGEMENT:

Authors are thankful to the members of B.N. College of Pharmacy, Udaipur for providing the necessary facilities for completion of study.

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Received on 19.10.2011 Modified on 25.10.2011

Research J. Pharm. and Tech. 4(12): Dec. 2011; Page 1835-1843