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Phytomedicine 11 (2004) 530538

www.elsevier.de/phymed

Studies on the aqueous extract of Terminalia chebula as a potent antioxidant and a probable radioprotector
G.H. Naika, K.I. Priyadarsinia,*, D.B. Naika, R. Gangabhagirathib, H. Mohana
a b

Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India

Received 14 June 2003; accepted 17 August 2003

Abstract
Aqueous extract of a natural herb, Terminalia chebula was tested for potential antioxidant activity by examining its ability to inhibit g-radiation-induced lipid peroxidation in rat liver microsomes and damage to superoxide dismutase enzyme in rat liver mitochondria. The antimutagenic activity of the extract has been examined by following the inhibition of g-radiation-induced strand breaks formation in plasmid pBR322 DNA. In order to understand the phytochemicals responsible for this, HPLC analysis of the extract was carried out, which showed the presence of compounds such as ascorbate, gallic acid and ellagic acid. This was also conrmed by cyclic voltammetry. The extract inhibits xanthine/xanthine oxidase activity and is also an excellent scavenger of DPPH radicals. The rate at which the extract and its constituents scavenge the DPPH radical was studied by using stopped-ow kinetic spectrometer. Based on all these results it is concluded that the aqueous extract of T. chebula acts as a potent antioxidant and since it is able to protect cellular organelles from the radiation-induced damage, it may be considered as a probable radioprotector. r 2004 Elsevier GmbH. All rights reserved.
Keywords: Herbal extract; Terminalia chebula; g-radiation; Free radicals; Antioxidant; Radioprotector

Introduction
In the last few decades, there has been a considerable growth in the eld of herbal medicine. It is getting popularized in developing and developed countries due to its natural origin and lesser side effects (Chopra et al., 1956; Khopde et al., 2001; Haramaki and Packer, 1995; Scartezzini and Speroni, 2000; Naik et al., 2003; Ahmad et al., 1998). T. chebula also popularly known as Harde in India is one such medicinal herb, used commonly in many ayurvedic preparations. The medicinal properties of T. chebula and several other herbal plants have been documented in the ancient Indian literature (Jagtap and Karkera, 1999; Kaur et al., 1998; Trease and Evans,
*Corresponding author. Fax: +91-22-25505151. E-mail address: kindira@apsara.barc.ernet.in (K.I. Priyadarsini). 0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2003.08.001

1983; Inamdar and Rajarama, 1954; Saleem et al., 2002). T. chebula belongs to the family Combretaceae and is found throughout India especially in deciduous forests and areas of light rainfall. Its yellowish-brown fruits are included in the Indian pharmacopoeia under the category astringent. It possesses laxative, diuretic, cardiotonic and hypoglycemic properties. A combination drug Triphala, a composite mixture of T. chebula, T. bellerica, and Emblica ofcinalis, is a very popular traditional medicine used for the treatment of many chronic diseases such as ageing, heart ailments and hepatic diseases, etc. (Kaur et al., 2002; Jagetia et al., 2002; Sabu and Kuttan, 2002). Oxidative stress or excessive production of reactive oxygen species (ROS) is being implicated in many diseases such as cancer, atherosclerosis, ageing, diabetes, etc. (Finkel and Holbrook, 2000; Halliwell, 1997;

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Halliwell and Gutteridge, 1993). The potential targets for the ROS in cells are membrane lipids, DNA and proteins. External supplementation through antioxidants is recommended to protect cells from the deleterious effects of such oxidative stress conditions. Earlier we studied a number of medicinal plant extracts for their potential as antioxidants (Khopde et al., 2001; Naik et al., 2002, 2003). Among these extracts, the aqueous extract of T. chebula showed maximum activity. In this paper, we present in detail, different in vitro experiments, through which the antioxidant activity of the extract of T. chebula is tested. The experiments include its ability to protect membrane lipids, antioxidant enzymes and DNA from g-radiation induced damage. To understand the active principles responsible for its activity, HPLC analysis and cyclic voltammetry was carried out. The total radical scavenging activity of the extract was also tested by using 2,20 diphenyl-1-picrylhydrazyl (DPPH) radicals employing stopped ow spectrometer. Of late, there is also a considerable attention on the possibility of using some antioxidants as radio protectors, since exposure to highenergy radiation such as g-rays can lead to the damage of cellular organelles by generating excessive ROS. Based on these results, possibility of using T. chebula extract as a potent radio protector is also discussed.

get the dry powder of the extract. The concentration is expressed as mg/ml.

Isolation of microsomes and mitochondria

Rat liver mitochondria and microsomes were isolated from the liver of male albino wistar strain rats as described elsewhere (Satav et al., 1976; Khopde et al., 2000). Lipid peroxidation (LPO) studies were carried out using radiation from a 60Co g-source. The detailed methodology used in the lipid peroxidation is given in our earlier work (Khopde et al., 2000). The dose rate (8.4 Gy/min) was determined by standard Fricke dosimetry. N2O/O2-purged microsomal solutions were exposed to a dose of 270 Gy in the absence and in the presence of different concentrations of the extract at physiological pH 7.4 (phosphate buffer). The lipid peroxidation was estimated in terms of thiobarbituric acid reactive substances (TBARS) using 15% w/v trichloroacetic acid, 0.375% w/v TBA, 0.25 N hydrochloric acid, 0.05% w/v BHT as TBA reagent measuring the absorbance at 532 nm (e532=1.56 105/M/cm) (Buege and Aust, 1978). The protein was estimated according to the method reported by Lowry et al., 1951.

Estimation of SOD enzyme activity

Mitochondria (isolated from rat liver) was diluted with oxygenated phosphate buffer by adjusting its protein concentration to 2 mg/ml. It was taken in a glass vial and was exposed to a total g-dose of 270 Gy both in the presence and in the absence of the extract. SOD levels in the control and the irradiated samples were estimated. Briey 1 ml solution contains sodium carbonate buffer (50 mM, pH 10), 5 mM Epinephrine and 40 mg mitochondrial protein. Monitoring its absorbance at 320 nm spectro-photometrically initially followed the rate of auto-oxidation of only epinephrine standard. Similarly the absorbance at 320 nm was also monitored in unirradiated and irradiated mitochondria samples under identical condition. The difference in the absorbance of epinephrine standard and that in mitochondria sample was used to calculate the enzyme activity. A difference in the absorbance of 0.033 at 320 nm is dened as 1 unit of SOD (Sum and Zigman, 1978).

Materials and methods

Chemicals
2,20 -diphenyl-1-picrylhydrazyl (DPPH), gallic acid, and ellagic acid were purchased from Aldrich Chemicals, USA. Xanthine, xanthine oxidase, 2,6- dicholrophenol-indophenol (DCIP), thiobarbituric acid (TBA), butylated hydroxytoluene (BHT), ascorbic acid and epinephrine were obtained from Sigma Chemicals, USA. All the other reagents used were of highest available purity. Nitrous oxide (N2O) and oxygen (O2) gases obtained from Indian Oxygen Ltd., Mumbai, were of IOLAR grade purity. Nanopure water was obtained from a Millipore Elix/A-10 water purication system. Freshly prepared solutions were used for each experiment.

Preparation of aqueous extract from dry fruit of T :chebula

The dry fruits of T. chebula were nely powdered (mesh size 20 #) and stirred with eight parts of distilled water at about 7080 C for 2 h. The liquid extract was ltered through sieve (mesh size 200 #). The ltrate was concentrated up to two parts on a rotary vacuum evaporator. The concentrated liquid was spray dried to

Studies on plasmid DNA

Agarose gel (1%) was prepared in 89 mM Tris-borate/ 2 mM EDTA (TBE) buffer. Ethidium bromide was added in the gel preparation at a concentration of 0.5 mg/ml to enable the visualization of the DNA bands in a UV transilluminator (Devasagayam et al., 1995; Kumar et al., 1999). The gel was submerged in an

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electrophoresis tank lled with TBE buffer. About 2 mg of pBR322 DNA was suspended in 50 ml of 10 mM sodium phosphate buffer, pH 7.4 and exposed to a gdose of 6 Gy in the absence and in the presence of varying concentration of aqueous extract of T. chebula (25200 mg/ml). Control and samples were mixed with the dye (0.25% bromophenol blue and 30% glycerol) and loaded into the wells. Electrophoresis was carried out at 60 V for about 2.53 h till the open circular and the supercoiled form of DNA were well separated. The movement of the DNA bands was visualized on a UV transilluminator. The intensity of the bands was determined using syngene gel documentation system (Devasagayam et al., 1995; Kumar et al., 1999).

DPPH assay
1 ml of 500 mM DPPH in methanol was mixed with equal volume of the extract solution in phosphate buffer (pH=7.4). The mixture was slightly shaken and kept in dark for 20 min. The absorbance at 517 nm was monitored in the presence and in the absence of different concentrations of the extract.

Stopped-ow studies
Kinetics of DPPH reaction with the extract and its constituents was studied using stopped-ow kinetic spectrometer (model SX 18 MV from Applied Photophysics, UK) in single mixing mode. In this experiment, syringe I contained 100 mM DPPH in methanol and syringe II contained varying concentration of the extract, ascorbic acid, gallic acid and ellagic acid (separately). After mixing, the rate of disappearance of DPPH radical was followed by monitoring the absorbance at 517 nm as a function of time at 25 C. Analysis of the kinetic traces was carried out with an exponential function using built in software. At least three independent runs were used to determine the rate constant at any concentration.

HPLC analysis
HPLC analysis was carried out using C18 PCX 500 Dionex analytical column with 0.1 M KCl, 0.05 M HCl and 32% acetonitrile as the mobile phase. The detection was carried out at 260 nm using UV detector. Peak areas were quantied by using external standards.

Cyclic voltammetry
Cyclic voltammetric studies were performed on Ecochemie Auto Lab, PGSTAT 20 Model and by using a conventional three electrode system of glassy carbon working electrode, platinum wire as auxiliary electrode and AgAgCl as reference electrode. Glassy carbon electrode was resurfaced with alumina. The electrochemical cell containing aqueous solution of 100 mg/ml extract and 0.1 M KCl buffer pH 7.4 was thermostatted at 2570.1 C. The cyclic voltammograms were recorded in the voltage range of 0.25 to 1.2 V at a scan rate of 100 mV/s.

Results and discussion

Lipid peroxidation and SOD enzyme inhibition studies
Peroxidation of lipids is a measure of damage to the membrane lipids caused by the attack of reactive oxygen species. Inhibition of lipid peroxidation by any external agent is often used to evaluate its antioxidant capacity. Studies on inhibition of lipid peroxidation in presence of the extract were carried out in rat liver microsomesinduced by g-irradiation. Since water is the major constituent of all the living cells, exposure to g-radiation causes mainly radiolysis of water producing radical species like H, OH and e aq and some molecular products like H2O2 and H2 (Khopde et al., 2001; Naik et al., 2003). Purging this solution with nitrous oxide, e aq is converted to OH radicals (e aq+N2ON2+ OH+ OH) (Sonntag, 1987). Thus, when rat liver microsomes purged with N2O-air, were exposed to g-radiation, peroxidation of lipids takes place through the mediation of OH radicals. The lipid peroxidation, determined by TBARS method was found to increase with increasing radiation dose from 100 to 600 Gy. Under the same conditions, when microsomes containing the aqueous extract of T. chebula were exposed to gradiation, there is a considerable reduction in the extent of lipid peroxidation. At a xed radiation dose of

Xanthine-oxidase assay
The xanthine oxidase assay was carried out spectrophotometrically by two different methods. In the rst method, inhibition of superoxide is monitored by the DCIP method (Satav et al., 2000). Briey, 3 ml system consists of 38 mM tris-HCl buffer pH 7.4, 16 mM xanthine, 10 mM DCIP and about 0.02 units/ml of xanthine oxidase enzyme. The decrease in absorbency of DCIP was monitored at 600 nm in the presence and in the absence of the extract (25100 mg/ml). In the second method inhibition of xanthine oxidase enzyme activity was studied by monitoring the uric acid formation at 290 nm (Noro et al., 1983).

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absence and in the presence of the extract. In the absence of the extract, on irradiation, the activity of the enzyme decreased by 4475% and in presence of 12 and 75 mg/ml extract the decrease in activity was 1573% and 471%, respectively, thus restoring the activity of the enzyme to a great extent. These studies suggest that the extract of T. chebula protects the antioxidant enzyme from the reactive oxygen species produced by gradiation. The actual mechanism of protection may be due to direct scavenging of the free radicals produced during irradiation by the active constituents of the extract.

Inhibition of radiation-induced strand breaks in plasmid DNA

Fig. 1. Inhibition of lipid peroxidation assessed in terms of TBARS, when rat liver microsomes suspended in N2O/O2 purged buffer (pH 7.4) solution were exposed to g-dose of 270 Gy in the absence and in the presence of different concentration of extract.

270 Gy, the lipid peroxidation was studied at varying concentration (535 mg/ml) of the extract and the results are shown in Fig. 1. From the gure it can be seen that the extract shows protection at all the concentrations. We have also compared the inhibition of lipid peroxidation by the extract with its constituents gallic acid and ascorbic acid (see HPLC analysis). However, the comparisons could not be made under identical concentrations, as the extract contains a mixture of compounds and cannot be expressed as mol/l while the constituents are pure compounds. Therefore, for these comparative studies, the microsomes were irradiated at a xed radiation dose, keeping the concentration of the extract at 15 mg/ml and that of gallic acid and ascorbic acid, respectively, at 1.8 and 2.2 mg/ml. The amount of gallic acid and ascorbic acid has been decided according to their percentage estimated by HPLC analysis. Under these conditions, the percentage inhibition of lipid peroxidation was 82, 55 and 69, respectively. The results suggest that the activity of the extract is not due to any individual components and the overall activity is the combined result of all the constituents. SOD is an important cellular antioxidant enzyme, which converts superoxide radical into H2O2 and O2 (Halliwell and Gutteridge, 1993). During the irradiation by g-rays, the SOD activity initially increases to combat oxidative stress induced by g-radiation, and further irradiation causes decrease in its activity due to its own consumption and damage by the excessive reactive oxygen species production. These studies have been carried out using rat liver mitochondria as they are rich in SOD enzyme and the aqueous mitochondrial samples were exposed to a radiation dose of 270 Gy in the

The antimutagenic activity of the extract was studied using plasmid DNA as an in vitro model system. Exposure of plasmid DNA to g- radiation results in strand breaks formation, mainly due to the generation of hydroxyl radicals and the subsequent free radical induced reactions on DNA. Hydroxyl radicals, react with nucleic acid bases of DNA producing base radicals and sugar radicals. The base radicals in turn react with the sugar moiety causing breakage of sugar-phosphate back bone, resulting in strand break formation (Sonntag, 1987). For these studies, the samples of pBR322 DNA were exposed to an absorbed dose of 6 Gy and the conversion of the supercoiled form to open circular form was monitored using horizontal gel electrophoresis. Lanes 1 and 2 from left of Fig. 2 show the electrophoretic pattern of pBR322 DNA without irradiation. Increase in open circular form due to irradiation can be seen clearly in Lanes 34. The DNA samples were irradiated in presence of the extract to see its effect on inhibiting strand breaks formation. Lanes 612 of Fig. 2 give change in the relative yield of the open-circular

Fig. 2. Agrose gel electrophoretic pattern showing protection by the extract on g-radiation-induced strand breaks in plasmid pBR 322 DNA as a function of concentration of the extract. Lanes 1, 2: pBR 322 control; Lanes 3, 4: exposed to radiation dose of 6Gy; Lanes 512 exposed to radiation dose of 6 Gy in presence of 25, 50, 100 and 200 mg/ml of extract in duplicate.

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Table 1. Inhibition of g-radiation induced damage to pBR322 DNA by the aqueous extract of T. chebula Concentration of % O.Ca the extract Unirradiated 6 Gy 6 Gy+25 mg/ml 6 Gy+50 mg/ml 6 Gy+100 mg/ml 6 Gy+200 mg/ml
a

Average strand % protection breaks/DNA molecule 0.17070.051 0.85770.016 0.66670.039 0.53470.028 0.40470.034 0.32870.026 21.4 38.7 59.1 70.6

15.671.4 57.570.7 48.672.1 41.371.7 32.771.8 27.971.7

Open circular form of pBR322 DNA.

form in the presence of different concentrations of the extract from 25 to 200 mg/ml. The detailed analysis of the change in the supercoiled and open circular forms in absence and presence of the extract before and after irradiation are given in Table 1. It can be seen that the extract inhibits strand break formation in a concentration-dependent manner. These studies suggest that the extract shows antimutagenic activity and this can be attributed to its ability to scavenge free radicals generated by the irradiation and thus protecting DNA. Here also the ability of the extract in protecting DNA was compared with its constituents as in the lipid peroxidation studies. The results suggested that the extract is more effective as compared to the individual constituents gallic acid and ascorbic acid. For these comparisons, the concentrations of the constituents were taken according to their percentage estimated by HPLC analysis.

Fig. 3. HPLC chromatograms of (a) aqueous extract of T. chebula, (b) ascorbic acid, (c) gallic acid and (d) ellagic acid obtained by using C18 PCX500 analytical column, 0.1 M KCl, 0.05 M HCl, 32% acetonitrile as the mobile phase and UV detector set at 260 nm.

HPLC analysis
The HPLC analysis of the extract was carried out to determine the constituents, which are responsible for its antioxidant and antimutagenic activity. In general polyphenols present in several natural products exhibit antioxidant activity and radical scavenging ability (Hagerman et al., 1998; Hotta et al., 2002). We therefore made an attempt to look for the phenolic constituents in the extract, by HPLC analysis. Here the extract was dissolved in methanol or in aqueous buffer. The chromatograms were recorded by monitoring the absorbance of the individual constituents at 260 nm using UV detector. The HPLC analysis of the extract showed three prominent peaks with retention times of 3.76, 4.63 and 7.38 min (Fig. 3a) The three peaks have been identied to be ascorbic acid, gallic acid and ellagic acid respectively using standard solutions, under similar condition. The retention times for ascorbic acid, gallic acid and ellagic acid in standard solutions were found to be 3.54, 4.41 and 7.16 min, respectively (insets of Fig. 3).

Slight change in the retention times of these constituents in the extract may be either due to overlap of the peaks in the mixture or due to the fact that in the extract, these individual components are not in the free form but are bound as glycosides and other water soluble forms. Using the standard calibrations and the integrated peak areas, the relative concentrations of the three components in the extract were estimated to be 1572% ascorbate, 1172% gallic acid and 1.570.5% ellagic acid. All these three compounds are well-known antioxidants and are found in a number of natural herbs and fruits.

Cyclic voltammetric studies

Electrochemical methods such as cyclic voltammetric studies can be used to obtain the redox potential, which can be used to estimate the reducing power of an antioxidant, a key factor governing its antioxidant ability (Kilmartin et al., 2001; Chevion et al., 2000). Cyclic voltammetric studies of natural formulations have often been used to estimate the total antioxidant capacity, in terms of either ascorbate equivalents or

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Fig. 4. Cyclic voltammogramms of (a) aqueous extract of T. chebula, (b) ascorbic acid, (c) gallic acid and (d) ellagic acid recorded at pH 7.4 by using 0.1 M KCl as a supporting electrolyte and applied voltage range 0.25 to 1.2 V with scan rate of 100 mV/s.

Fig. 5. Effect of concentration of the extract on superoxide radical scavenging activity generated by xanthine (16 mM)/ xanthine oxidase (0.02 units/ml) system and assessed as decrease in reduction of DCIP(10 mM) dye at 600 nm.

trolox equivalents (Chevion et al., 2000). In the case of T. chebula extract similar attempt has been made to understand its antioxidant capacity. Fig. 4ad show the voltammograms for the extract, ascorbic acid, gallic acid and ellagic acid, respectively. The cyclic voltammogram for the extract showed no clear reversible peaks but only gave a shoulder and a monotonously increasing peak. The initial shoulder is at a potential of 0.303 V and another broad peak is at B0.55 V. Ascorbic acid (Fig. 4b) shows a reversible peak at 0.303 V. Gallic acid shows two peaks at 0.3 and 0.42 V (Fig. 4c), and ellagic acid has peak potentials at 0.29 V and 0.48 V (Fig. 4d). The rst strong peak in the extract may be due to the presence of ascorbic acid. The second continuously increasing broad peak at 0.55 V in the extract may be due to the overlap of oxidation peaks of other two components of the extract namely gallic acid and ellagic acid. Since the voltammogram does not show any clear reversibility, it was not possible to calculate the area of the voltammogram, which in turn could be used to estimate the total antioxidant capacity of the extract. However, the peak positions conrm the presence of the above determined products from HPLC analysis.

Xanthine oxidase assay

Xanthine oxidase is an enzyme widely distributed from unicellular species such as bacteria to multicellular species like human beings (Halliwell, 1997; Halliwell and Gutteridge, 1993; Satav et al., 2000; Noro et al., 1983). It is an important source for oxygen free radical. It produces superoxide radical during the last two steps of purine metabolism. In vitro xanthine oxidase assay

was carried out by using xanthine as substrate. During this process, xanthine is converted to uric acid and oxygen is reduced to superoxide. (Halliwell and Gutteridge, 1993). The studies with the extract were carried out to follow both the superoxide scavenging ability and also inhibition of xanthine oxidase activity. For the superoxide radical scavenging assay, reduction of DCIP by superoxide radical was followed at 600 nm. In the absence of the extract, reduction of DCIP dye takes place effectively. However, in the prescence of different concentrations (24100 mg/ml) of the extract a signicant decrease in the reduction of dye was observed due to the scavenging of superoxide radical by the extract. Fig. 5 shows percentage scavenging of superoxide radical by the extract at different concentrations. From the gure IC50 value i.e. the concentration of the extract at which the superoxide radical inhibited by 50% under the experimental conditions has been estimated to be 5075 mg/ml. We also measured the concentration of uric acid both in the presence and in the absence of the extract spectrophotometrically. The results qualitatively suggested that the extract reduced the production of uric acid. However, quantitative estimation could not be made due to the interference of the absorption by the extract at 290 nm. From these studies it is concluded that the extract is an efcient scavenger of superoxide radicals and is also an inhibitor of xanthine oxidase enzyme.

Free radical scavenging ability of the extract

Both HPLC analysis and cyclic voltammetry conrmed that the extract contains phenolic compounds.

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Fig. 6. Bar graph showing the scavenging of 250 mM methanolic solution of DPPH radical at different concentrations of the extract. The DPPH radical scavenging activity was measured as decrease in its absorbance at 517 nm.

Fig. 7. Absorption-time plot showing the decay of DPPH radical at 517 nm in presence of 44 mg/ml of the extract. Inset shows linear plot for the observed decay rate constant at 517 nm as a function of different concentrations of the extract.

Since these compounds are good scavengers of free radicals, we have studied the ability of the extract to neutralize the free radicals such as DPPH radicals. DPPH is a stable free radical having a maximum absorption at 517 nm (emax=9660/M/cm) (Yokozawa et al., 1998; Wang et al., 1999; Kumar et al., 2002). In the presence of compounds capable of donating an H atom or an electron, its free radical nature is lost; hence, a decrease in absorption at 517 nm is seen. As it is very convenient to follow DPPH reactions, it has often been used to estimate the antiradical activity of the natural products. The decrease in DPPH absorption in presence of varying concentration (3.523 mg/ml) of the extract has been monitored and it can be seen that the absorbance due to DPPH decreases continuously up to 23 mg/ml and further increase in the concentration did not change the absorbance (probably due to its own absorption). Fig. 6 shows the percentage scavenging of DPPH radical by the extract at different concentration. The IC50 value for the extract i.e. the concentration at which it scavenges 50% of the DPPH radical was found to be 1272 mg/ml.

and remained stable. However, in the presence of the extract the absorption due to the DPPH radical decayed completely in a few seconds (Fig. 7). This absorptiontime plot was tted to a single exponential function to get a rate constant, which was found to increase linearly with increasing extract concentration from 33 to 160 mg/ ml. (inset of Fig. 7). As discussed above, the reactions of the extract with DPPH is an indicator of the radical neutralizing activity of the extract, which has also been attributed to the presence of phenolic antioxidants. In order to further support these observations, the bimolecular rate constants of the constituents were determined independently by stopped-ow studies. In each case, the decay kinetics of DPPH radical was followed at varying concentration of the particular compound (ascorbic acid, or gallic acid or ellagic acid) and by tting this to a linear plot the bimolecular rate constants of these compounds were determined to be 1.470.3 102, 1.270.2 102 and 1.070.3 103/M/s for ascorbic acid, gallic acid and ellagic acid, respectively. This data conrms that all the three compounds present in the extract show very high reactivity towards DPPH radicals. This clearly shows that the phenolic constituents present in the extract are responsible for the overall reactivity of the extract towards DPPH radical.

Kinetics of DPPH reaction

The kinetics of reaction of the extract with DPPH radical was determined by using stopped-ow spectrometer. The decrease in the DPPH absorbance at 517 nm immediately after mixing DPPH with the extract solution was monitored as a function of time to determine the rate constant. For these studies, the concentration of DPPH is kept at 50 mM. In the absence of the extract, the DPPH signal did not show any decay

Conclusions
The fruits of Indian medicinal plant T. chebula are known for their pharmacological activity and in this paper it has been shown that the extract can be used as an effective antioxidant. The aqueous extract was found to be a very efcient inhibitor of radiation-induced lipid peroxidation and damage to the SOD enzyme. It can

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prevent strand break formation in supercoiled DNA. It is an excellent free radical scavenger, a property arising mainly from the presence of well-known antioxidants like ascorbate, gallic acid and ellagic acid, as conrmed by the HPLC analysis. Presence of these potent antioxidants in signicant levels either in free or bound form in the extract may therefore be responsible for the overall antiradical and antioxidant activity of T. chebula extract. Thus, our in vitro studies coupled with the phytochemical analysis conrm that the extract possesses potential antioxidant activity. Since the extract protects various cellular organelles from radiation induced damage, we feel that the extract can be used as a radioprotector, especially to protect humans from radiation induced damage. For such an activity, natural products with well established pharmacological history are best suited as they are gifted with lesser side effects.

Acknowledgements
The authors are thankful to M/S Ajanta Pharma Pvt. Ltd., Mumbai for supplying the extract and to Dr. J.G. Satav, RB & HS Division, BARC for providing the microsomes. Thanks are also due to Dr. J.P. Mittal Director Chemistry & Isotope Group and Dr. T. Mukherjee, Head, RC & CD Division, Dr. K.P. Mishra, Head, RB & HS Division, BARC for their support to this work.

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