Ashwagandha Root Extract Inhibits Acetylcholine Esterase , Protein Modification and Ameliorates H 2 O 2-Induced Oxidative Stress in Rat Lymphocytes

Background: Herbs have long been the basis of medical treatment but the practice of herbalism is not strictly based on evidences gathered using scientific methods. Ashwagandha (Withania somnifera L.) is a traditional medicinal herb used in the treatment of various ailments. Objective: In current study we have evaluated the acetylcholine esterase (AChE) inhibitory and antioxidant potential of Ashwagandha root extract (ARE) in in-vitro and ex-vivo models to give a scientific base to its use in herbal medicine. Methodology: Simplest extraction e.g. maceration method was performed for preparation of extracts using methanol as solvent. Both in vitro and in vivo experimental trial were executed to evaluate the efficacy of root extract. Result and Discussion: ARE substantially scavenged free radicals and effectively prevented protein degradation as well as modification as studied by SDS-PAGE and Western blotting. Pre-treatment with ARE protected rat lymphocytes against H2O2-induced oxidative damage. H2O2 treatment resulted in DNA fragmentation and significantly decreased the activities of key antioxidant enzymes namely superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase while decreased and increased the content of glutathione and malondialdehyde, respectively. ARE pre-treatment almost reversed these changes indicating its efficiency to suppress hydrogen peroxide-induced oxidative stress. Conclusion: The study provides the scientific basis of pleiotropic functions of Ashwagandha.


INTRODUCTION
Reactive oxygen species (ROS) like superoxide anion, hydroxyl radicals, hydrogen peroxide, singlet oxygen etc. are routinely formed in the animal body as a result of normal metabolic activity 1 and/or as by-products of various oxidative reactions including metabolism of xenobiotics. 2These species are detoxified by endogenous enzymatic and non-enzymatic antioxidative defences so as to preserve the optimal cellular functions.Weakening of these detoxifying mechanisms leads to an accumulation of ROS thereby resulting in pathological conditions.The oxidative stress caused by accumulated free radicals may contribute to degradation of cellular components like protein oxidation, DNA damage, lipid peroxidation, enzyme inactivation, irreversible cellular dysfunction and ultimately cell death if the pro-oxidant-antioxidant balance is not the loss of cholinergic innervation, reduction of choline acetyltransferase (ChAT) and enhanced acetylcholine esterase (AChE) activity.AChE terminates the interaction between neurotransmitter acetylcholine (ACh) and the corresponding receptor protein (acetylcholine receptor, nAChR), which is the basis of the intercellular communication in brain.Pharmacological and analytical data from human tissue and body fluids have implicated oxidation products of fatty acids in the pathogenesis of AD. 7 Antioxidants can act by scavenging the reactive products of lipid peroxidation and may be useful in prevention and treatment of AD.The modulation of AChE is presently the most accepted and recognized therapeutic marker for development of cognitive enhancers. 8Currently used AChE inhibitors, like tacrine, produce side effects such as hepatotoxicity. 9So, the use of natural bioactive compounds, like antioxidants, finds their possible application in the prevention and treatment of AD.Dastmalchia and his associates have summarized that interest in the discovery of natural antioxidants has risen exponentially, principally for three reasons: Determination of antioxidant capacity using ABTS + (2, 2'-azinobis 3-ethylbenzothiazoline-6-sulfonate) assay The method used was the ABTS + (cation radical) decolorization assay which is based on the ability of an antioxidant compound to quench the ABTS + free radical. 16A stock solution of ABTS + radical cation was prepared by mixing ABTS and potassium persulfate solution at 7 mM and 2.45 mM final concentration, respectively.The mixture was maintained in the dark at room temperature for 12-16 h before use.For experimental purpose, the ABTS + stock solution was diluted with phosphate-buffered saline (pH 7.4) to an absorbance of 0.70 at 734 nm.Different amounts of ARE (50 to 250 µg) was added to ABTS + working solution (2 mL).Absorbance was measured by means of a UV-visible spectrophotometer at 734 nm after 20 min of mixing and percent inhibition was calculated.The results were calculated in terms of IC 50 .

Protein oxidation
Protein oxidation was assayed as described previously by Mayo et al. with minor modifications. 17Degradation of BSA (5 μg) in PBS was initiated by AAPH (20 mM) and inhibited by different concentrations of ARE (50 to 150 µg).After incubation for 2 h at 37 ºC, 0.02% BHT was added to the reaction mixture to prevent the further formation of peroxyl radical.The samples were then assayed with normal SDS-PAGE. 18

Protein nitration
In this experiment, the method of Ippoushi et al. was employed with slight modifications. 19Briefly, BSA (30 µg) was incubated in the absence (control) and presence of 3-morpholinosydnonimine (SIN-1) (final concentration 37.5 mM) with (50, 100 150 µg) or without addition of ARE.After 3 h incubation at room temperature, the samples were run on 10% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, MA, USA).The membrane was blocked overnight at 4°C with 5% (v/v) non-fat dry milk in Tris-buffered saline with Tween-20 (TBS-T) (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20, pH 7.5).Further the membrane was incubated for 3 h with 3-nitrotyrosine antibody (3-NT, N5538, Sigma, St. Louis, MO, USA) and washed with TBS-T buffer followed by incubation with goat anti-mouse peroxidase-labelled secondary antibody (Dako, Denmark) and washing with TBS-T buffer.Finally the immunoreactivity was detected by chemiluminescent peroxidase substrate (Sigma, USA).

Inhibitory activity of ARE on protein carbonyl (PCO) formation
The efficacy of ARE to prevent PCO formation was carried out according to the slightly modified method of Wang et al.where BSA was oxidized by a Fenton-type reaction. 20The reaction mixture (1.2 mL) containing extract, potassium phosphate buffer (20 mM, pH 7.4), BSA (4 mg), FeCl 3 (50 µM), H 2 O 2 (1 mM) and ascorbic acid (100 µM) were incubated for 60 min at 37 ºC.For determination of PCO content in samples, 1 mL of 10 mM DNPH (2, 4-Dinitrophenylhydrazine) in 2 N HCl was added to the reaction mixture.Samples were incubated for 60 min at room temperature.Then, 1 mL of 10 % TCA was added to reaction mixture and centrifuged at 3,000 x g for 10 min.The protein pellet was washed thrice with 2 mL of ethanol/ethyl acetate (1:1, v/v) and dissolved in 1 mL of guanidine hydrochloride (6 M, pH 2.3) and incubated for 10 min at 37 o C. The absorbance of the sample was read at 370 nm.The data were expressed in terms of percentage inhibition, calculated from a control measurement of the reaction mixture without extract.

Amelioration of H 2 O 2 -induced stress in rat lymphocytes
Isolation of rat lymphocytes Lymphocytes were isolated from fresh whole blood using HiSep TM LSM (HiMedia).Briefly, anti-coagulated blood was diluted with an equal (i) the overwhelming epidemiological and clinical evidence suggesting an inverse relationship between the consumption of fruits and vegetables and the risk of developing chronic disease like can cer; (ii) concerns regarding the safety of the chronic consumption of synthetic compounds traditionally used as preservatives in foods and beverages; and (iii) the public's belief that phytochemicals are inherently safer than synthetic chemicals.The principal candidates in this exploratory process have always been herbs, spices, and medicinal plants. 10e Indian medicinal plant Withania somnifera (L.dunal) (family Solanaceae), commonly known as Ashwagandha, is widely used in herbal medicines for stress, arthritis, inflammations, conjunctivitis and tuberculosis.Its active principles, sitoindisides VII-X and withaferin-A, have been shown to exhibit significant antistress and antioxidant effects. 11,12he medicinal properties of Ashwagandha are attributed mostly to its antioxidant compounds but the detailed report on its antioxidant array is lacking.3][14] In continuation, an attempt has been made to delineate the AChE inhibitory, protein modification preventive and H 2 O 2 -induced oxidative stress ameliorating properties of the Ashwagandha root extract.

Preparation of sample extract
The methanolic extract of Ashwagandha, used in present study, was prepared as described in our earlier reports. 13,14aluation of AChE inhibitory activity AChE inhibitory activity was measured by slightly modifying the spectrophotometric method developed by Ellman and his associates. 15lectric eel AChE was used, while S-acetylthiocholine iodide was employed as substrate of the reaction.DTNB [5,5'-dithio-bis(2-nitrobenzoic) acid] was used for the measurement of the AChE activity.Briefly, 1.25 mL of 100 mM sodium phosphate buffer (pH 8.0), 50 µL of Ashwagandha root extract (ARE) of different concentrations (1 to 5 mg), 5 µL of appropriately diluted AChE and 20 µL of acetylthiocholine iodide (75 mM) were mixed and incubated for 15 min at room temperature.The hydrolysis of substrate was monitored spectrophotometric ally by the estimation of yellow 5-thio-2-nitrobenzoate anion, after adding 100 µL of 10 mM DTNB at a wavelength of 412 nm.The per cent of inhibition (% I) was calculated as following: where, A control is the absorbance of the control and A sample is the absorbance of the test sample.The experiments were carried out in triplicate and the IC 50 (concentration of sample required to inhibit 50% of enzyme activity) was calculated by linear regression analysis.
volume of PBS, gently overlaid on HiSep TM LSM and centrifuged at 400g for 15 min at 4ºC.Lymphocytes were separated as a pink layer at the top of the HiSep TM LSM.Cells were washed twice with PBS and centrifuged at 400×g for 15 min at 4ºC.Cells were rinsed with PBS and diluted to ~6 × 10 6 cells/mL.

Evaluation of cytotoxicity of ARE
Cell number and viability (Trypan blue exclusion) were determined using a Haemocytometer before treatment.Rat lymphocytes were incubated at a density of ~5 ×10 5 /mL and viability was over 95%.Cell suspensions were incubated with different concentrations of ARE (the final concentration was 10-50 µg/mL) for 30 min at 37 °C in a dark incubator together with untreated control samples.Samples were then centrifuged at 1000×g, the lymphocytes were resuspended in RPMI 1640 and 0.4% trypan blue, and viable and dead cell were scored.

Grouping of lymphocyte
The lymphocytes were divided into 3 groups: Group I: Treated with PBS and served as control.
Group III: Pre-treated with ARE followed by 100 µM H 2 O 2 .
The cells were pre-treated with ARE at a concentration of 50 µg/mL for 2 h at 37ºC, together with control group, which contained PBS alone.These samples were then centrifuged at 1,000×g for 5 min at 4ºC.The ARE treated cells were re-suspended in PBS containing 100 µM H 2 O 2 for 30 min at 4ºC in dark condition.The samples were then centrifuged at 1,000×g for 5 min at 4ºC.After H 2 O 2 treatment, cells were centrifuged and washed twice with PBS at 1,000×g for 5 min at 4ºC.The cells were then lysed in RIPA buffer.

Biochemical assays
Superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) activities were assessed using commercially available kits as per manufacture instructions (RANDOX, Canada).Catalase activity was assayed by noting the decrease in absorbance at 240 nm caused by decomposition of H 2 O 2. 21 Concentration of malondialdehyde was estimated using thiobarbituric acid, forming a red-coloured compound absorbing at 535 nm. 22Glutathione content was estimated using Ellaman reagent. 24Protein content was estimated using the method of Lowry et al. 24

Determination of DNA damage by single cell gel electrophoresis (SCGE)
The integrity of DNA under H 2 O 2 induced oxidative stress was evaluated by SCGE method with minor modifications. 25Briefly, ~2.5 × 10 4 lymphocytes from each group (in 85 µL of 0.75% LMP agarose) were spread on a base layer of 100 µL of 1% NMP agarose placed on frosted microscopic slides and covered with a cover slip.Once the agarose set, slides were covered with another 100 µL of 0.75% LMP agar and to lyse the cells and nuclear membranes, slides were immersed in freshly prepared cold lysis solution buffer (2.5 mol/L NaCl, 0.1 mol/L Na 2 EDTA, 10 mmol/L Tris, pH 10.0 and 1% Triton X-100) for 90 min.After lysis the slides were transferred to an electrophoresis buffer (0.3 mol/L NaOH, 1 mmol/L Na 2 EDTA, pH > 13.0) for unwinding (40 min, 4°C) and then subjected to electrophoresis at 25 V (current adjusted to 0.3 A) for 30 min at 4°C.After electrophoresis, the slides were neutralized with Tris-HCl (0.4 mol/L, pH 7.5) twice for 10 min and stained with ethidium bromide (EtBr, 20 µg/mL).Finally the DNA damage was evaluated with fluorescence microscope (Olympus microscope, Germany) by measuring the percentage of fluorescence in tail using RS image software and the results were expressed as per cent inhibition of tail length.

Statistical analysis
All the data were expressed as mean±standard deviation of three experiments.IC 50 values were calculated using linear regression equation.Statistical analysis was performed using Student's t-test and the p-values less than 0.05 were considered as significantly different.

AChE inhibitory activity
In the present study, we observed that ARE inhibited the AChE activity in a dose-dependent manner with a R 2 value of 0.97.The IC 50 value which is inversely related to the activity, as calculated from linear regression equation, was found to be 4.44 mg/mL (Table 1).

Radical scavenging competence of ARE
This blue-green radical discoloration assay is widely used to assess the antioxidant activity of plant extracts.The ARE exhibited ABTS + radicalscavenging activities to different extents in a concentration dependent manner (Table 2).The R 2 value between concentration and per cent inhibition was 0.96 and the IC 50 value, calculated using linear regression equation, was found to be 127.49µg/mL.

Protein oxidation
In the present study, protection against protein degradation damage was determined by the oxidation of BSA initiated by AAPH.After incubation with ARE, the samples were assayed with normal SDS-PAGE.The results (Figure1) demonstrate that ARE exhibited significant protective effect against oxidation of BSA which was augmented by increasing concentrations of extract.At a concentration of 150 µg, the extract showed almost complete inhibition of BSA degradation.

Inhibitory activity of ARE on protein nitration
In the present study, SIN-1 was used to induce protein nitration and the extent of nitration was detected by immune-reactivity with 3-NT antibody.Pre-treatment of BSA with 50, 100 and 150 µg of ARE dose dependently inhibited the protein nitration, while no immunoreactivity was observed in BSA alone group which served as a control (Figure 2).

Inhibitory activity of ARE on PCO formation
Protein oxidation was determined in terms of PCO formation.As shown in Table 3, ARE exhibit dose-dependent inhibitory effects on PCO formation by 15.4, 39.3, 50.6, 70.4 and 74.1% at the extract concentrations of 100, 200, 300, 400 and 500 µg/mL, respectively.The IC 50 calculated using linear regression equation of R 2 = 0.97 was found to be 303.61µg/mL.

Cytotoxicity of ARE
The cytotoxicity of ARE towards rat lymphocytes, induced at room temperature for 30 min, is shown in Table 4.As a whole, the cell viability was greater than 95% at the concentrations tested, indicating no cytotoxicity toward selected lymphocytes.

Biochemical assays
Results of the experiment describe the onset of oxidative stress in rat lymphocytes by the treatment of H 2 O 2 as evidenced by enhanced TBARS/MDA.A significant decrease in the activities of all the five antioxidant enzymes namely SOD, CAT, GPx, GR and G6PDH was observed in H 2 O 2 -treated (group II) lymphocytes as compared to normal control group (group I) (Figure 3 and its sub figure ).But, the activities of antioxidant enzymes were significantly restored almost to their normal levels in ARE pre-treated lymphocytes (group III).Similarly, concentration of    glutathione showed a significant decrease after H 2 O 2 treatment in comparison to normal group (Figure 4a).However, pre-treatment with ARE caused a significant increase in the concentration of glutathione and it was almost restored to normal values.When compared with the normal group, there was a significant increase in the concentration of MDA in the H 2 O 2 treated group while a substantial decrease in MDA concentration was brought by pre-treatment of ARE (Figure 4b).

Effect of ARE on DNA damage
The effect of ARE on DNA damage in each group of lymphocytes was investigated and the results were expressed in terms of tail length.The value of tail length in H 2 O 2 challenged lymphocyte was 135 µm.However, the DNA damage in rat lymphocytes induced by H 2 O 2 was decreased by ARE supplementation to an extent of ~88 % (Figure 5).The values of tail lengths in group I and III were not significantly different (P > 0.05) indicating that ARE almost completely prevented the DNA damage in rat lymphocytes.

DISCUSSION
7][28] The antioxidants defence system of an organism comprising of different antioxidant enzymes and metabolites plays an important role in cellular defense against oxidative abuse.Natural antioxidants present in herbs are responsible for inhibiting and/or preventing the deleterious consequences of oxidative stress.Herbs contain free radical scavengers like polyphenols, flavonoids and phenolic compounds.Reports have shown that there is   lesser likelihood of drug and herb interaction when subjects are concomitantly administered with CY3PA substrates. 29This suggests that herbal formulation have their own effect without counteracting with other drugs.Apart from it, the sub-effective doses of withania somnifera enhance the anticataleptic action of L-DOPA and this adjunctive therapy reduces the doses and the adverse effects of dopamine precursors in Parkinson disease. 30The novel herbomineral test formulation based on Ashwagandha might act as an effective anti-inflammatory and immunomodulatory product, and this can be used as a complementary and alternative treatment for the prevention of various types of inflammatory and auto-immune disorders. 31In continuation with our previous work 13,14 we have examined ex-vivo model of stressed rat lymphocytes and observed AChE inhibitory and pro-antioxidant activities of ARE.
According to Atta-ur-Rahman and Choudhary, 32 the use of antioxidants may slow the progression of Alzheimer's disease (AD) and minimize neuronal degeneration.Currently there is no cure for AD.To date, the most promising target for the symptomatic treatment and slowing of AD progression is cholinesterase inhibitors. 33In AD, the destruction of cholinergic neurons causes the depletion of the neurotransmitter, acetylcholine (ACh).By inhibiting, AChE, the enzyme which catalyses breakdown of ACh, the levels of this transmitter can be elevated and function improved.The compounds that exhibit AChE inhibitory activity are also related to radical-scavenging activity. 34The potentiation of cholinergic activity through the inhibition of AChE is one of the major strategies of pharmacotherapy for AD disease.Its inhibitors like tacrine, donepezil, rivastigmine, and galanthamine are generally used in treating neurodegenerative disorders but because of the side effects associated with these drugs, it is worthwhile to explore the herbal remedies as an alternative and safe medicine to the existing pharmacological agents.
In order to verify these approaches, we have evaluated the AChE inhibitory activity of ARE and found it to substantially inhibit AChE activity which is in line with an earlier study by Mukherjee et al. who reported the AChE inhibitory activity of various plant extracts. 35Amorpha fruticosa fruit having acetylcholinesterase inhibitory activity, 48.86 ± 0.55% (2 mg/mL) could be useful in therapy of free radical pathologies and neurodegenerative disorders. 36The results of the present work showed that ARE can effectively be used to increase the level of ACh.
Antioxidants are closely related to their multifaceted biofunctionalities like reduction of chronic diseases by inhibiting mutagenesis and carcinogenesis; inhibition of pathogenic bacterial growth etc. 37 Thus, antioxidant capacity is widely used as a parameter for medicinal bioactive components.9] ABTS + radicals are more reactive than DPPH radicals and unlike the reactions with DPPH radical which involve H atom transfer, the reactions with ABTS + radicals involve electron transfer process.Phenolic compounds have been ascribed as an important contributor to the antioxidant activity of various plant extracts. 26In our study, the ARE was found effective in ABTS + radical scavenging assay in a concentration dependent manner.This ability of ARE to scavenge ABTS + radical may contribute to its significant antioxidant potential.
It is also a well known fact that the progressive accumulation of macromolecular oxidative damage is the root cause of senescence-associated cellular changes.Oxidation of cellular proteins has been described under many pathological conditions.AAPH, a water-soluble initiator, decomposes at physiological temperature and produces alkyl radicals followed by fast reaction with oxygen to give alkyl peroxyl radicals to initiate protein oxidation. 40The free radical provoked oxidative protein damages have been demonstrated to play a significant role in aging and several pathological events. 41Radical mediated damages to proteins might be initiated by electron leakage, metal-ion dependent reactions, and autooxidation of lipids and sugars. 42In our study it was found that ARE has the capability to almost block the AAPH induced protein degradation.Nitric oxide (NO) or reactive nitrogen species (RNS) have been found to alter the structural and functional behavior of many cellular components particularly proteins.In biological system, NO is synthesized by nitric oxide synthase (NOS) as a by-product during conversion of L-arginine to L-citrulline.The NO-mediated peroxynitrite formation results in protein nitration and its effects have also been implicated in the pathophysiology of neurodegenerative disorders. 43Peroxynitrite is not only a powerful oxidant but also a strong nitrating agent. 44Protein tyrosine residues are especially susceptible to peroxynitrite-mediated nitration reactions producing 3-nitrotyrosine. 45To test the protective effect of ARE against peroxynitrite-induced nitration of tyrosine, we selected BSA as a model target.Nitration reaction of BSA was carried out in the presence of various dosages of ARE and peroxynitrite-induced nitration of tyrosine residues in BSA was examined by Western blotting using anti-3-nitrotyrosine antibody.Exposure of BSA to in-situ generated peroxynitrite resulted in nitrotyrosine immune-reactivity.The intensity of the SDS-PAGE gel band derived from 3-nitrotyrosine residues in BSA decreased with increasing amounts of ARE confirming the protection of BSA by ARE against peroxynitrite induced tyrosine nitration.The present investigation shows the potential antioxidant activity of ARE which can therefore play a key role in arresting the chain of free radical reactions initiated by excess generation of NO.Our results are in line with the recently published report published for NO scavenging activities of Lycopodium species. 47roteins have also been identified to be damaged by free radicals directly and to be the targets of secondary modifications by aldehydic products of lipid peroxidation or ascorbate auto-oxidation resulting in PCO formation.The assessment of PCO is a widely used marker for oxidative protein modification and it is reported to be a sensitive and early marker of oxidative stress.3][14] Earlier reports also advocate that flavonoids exert a stimulatory action on transcription and gene expression of certain antioxidant enzymes. 47GPx catalyses the reduction of peroxides using reduced glutathione and converting into oxidized glutathione.Hence, increased activities of the enzymes, GPx and GR, were correlated with increased glutathione levels.
The comet assay also called as 'single-cell gel electrophoresis assay' , is a sensitive method for detecting DNA strand breaks.

CONCLUSION
The current study indicates that ARE has AChE inhibitory activity and is equally effective in preventing the oxidative stress induced by free radicals.The extract showed substantial eukaryotic DNA damage protection induced by H 2 O 2 and also exhibited a significant protection against protein oxidation and nitration induced by AAPH and SIN-1.Therefore ARE can be used as a source of antioxidant for dietary supplementation to alleviate oxidative stress induced cellular injuries.

Figure 5 (
Figure 5 (A-B): Effect of ARE (Ashwagandha root extract) on DNA through fluorescence microscopy and quantitative estimation of chromosomal tail length damage.

Table 1 : AChE inhibition activity of Ashwagandha root extract (ARE) Extract (mg/ml) Percent inhibition Linear regression equation (correlation coefficient) IC 50 (mg/ ml)
Our study shows that ARE is fully equipped to inhibit PCO formation.The inhibitory effect of extracts might operate by scavenging the peroxyl radical generated in the reaction mixture.H 2 O 2 has been found to induce oxidative stress in isolated rat lymphocytes as shown by decreased activities of antioxidant enzymes (Figure3and subfigure).The levels of reduced glutathione and MDA approached the normal levels in ARE pre-treated H 2 O 2 exposed lymphocytes.The restoration of MDA to almost normal values by ARE may be due to an enhancement of antioxidant enzymes, such as SOD, catalase, GPx, GR and G6PDH.ROS, such as superoxide anions and H 2 O 2 , and RNS are produced throughout cells during normal aerobic metabolism.The intracellular concentration of ROS depends both on their production as well as removal by various antioxidants.A major component of the antioxidant system in mammalian cells consists of three enzymes, namely SOD, CAT and GPx.These enzymes work in concert to detoxify superoxide anion and H 2 O 2 in cells.Our results indicated that pre-treatment with ARE caused an increase in the activities of antioxidant enzymes in H 2 O 2 -challenged lymphocytes.
51,14fore, an increasing number of laboratories have begun to use this extremely versatile assay to detect DNA damage.In present work, this assay was used to determine the effect of ARE on DNA damage in selected lymphocyte exposed to H 2 O 2 and protection of DNA damage by ARE clearly indicates its prophylactic potential at molecular level.As proved earlier by us,13,14ARE exhibited a scavenging effect on H 2 O 2 which might have contributed to inhibition of DNA damage in rat lymphocytes.Our results corroborate with previous work where Aherne and O'Brien showed that pre-treatment with quercetin and rutin had reduced DNA damage induced in Caco-2 and Hep G2 cells by a subsequent exposure to H 2 O 2 .48Gleietal.reported that β-carotene reduces H 2 O 2 -induced genetic damage in human lymphocytes when pre-treated with β-carotene.49AnotherreportbyLeanetal.suggested that dietary flavonols protect diabetic human lymphocytes against oxidative damage to DNA.50Docosahexaenoic acid was also reported to protect human lymphocytes against H 2 O 2 -induced oxidative stress.51