ARTICLE IN PRESS

Phytomedicine 17 (2010) 23–27

Contents lists available at ScienceDirect

Phytomedicine

0944-71

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.de/phymed

Effect of green tea extract (catechins) in reducing oxidative stress seen inpatients of pulmonary tuberculosis on DOTS Cat I regimen

Astha Agarwal a, Rajendra Prasad b, Amita Jain a,n

a Department of Microbiology, Chhatrapati Shahuji Maharaj Medical University, Lucknow, UP 226003, Indiab Department of Pulmonary Medicine, Chhatrapati Shahuji Maharaj Medical University, Lucknow, UP 226003, India

a r t i c l e i n f o

Keywords:

Pulmonary tuberculosis

Oxidative stress

Antioxidants

Catechin

13/$ - see front matter & 2009 Elsevier GmbH

016/j.phymed.2009.10.019

esponding author. Tel.: +09415023928.

ail address: amita602002@yahoo.com (A. Jain

a b s t r a c t

Background and Aim: The role played by free radicals in pathogenesis of pulmonary tuberculosis and

treatment mediated toxicity is well established. Hence, the present study was undertaken to assess the

effect of crude green tea catechin in reducing the oxidative stress seen in patients of AFB positive

pulmonary tuberculosis.

Methods: A total of 200 newly diagnosed cases of AFB positive pulmonary tuberculosis, who received

CAT I regimen were enrolled consecutively from DOTS center. Out of 200 patients, 100 randomly

selected patients received catechin (500 mg) with antitubercular treatment (ATT) (cases) and 100

received starch (500 mg) with ATT (control). Oxidative stress level in blood samples of cases and controls

as compared at the time of enrollment and after one and four months of treatment. Oxidative stress was

measured in terms of free radicals (lipid peroxidation, nitric oxide), enzymatic antioxidant (catalase,

superoxide dismutase, glutathione peroxidase) and non enzymatic antioxidant (total thiol, reduced

glutathione) levels.

Results: The results showed significant difference in all the parameters among cases and controls.

A significant decrease (pr0.001) in LPO level was observed in cases as compare to controls during the

follow up while the level of NO was significantly increased (pr0.001) in cases as compare to controls.

Significant decrease (pr0.001) in catalase and GPx level was observed in cases as compare to controls

while SOD levels significantly rose (pr0.001) in cases as compared to controls. Significant decrease

(pr0.001) in SH level was observed in cases as compared to controls while the level of GSH was

significantly increased (pr0.001) .

Conclusion: These findings suggest that crude catechin extract can play a definite role as adjuvant

therapy in management of oxidative stress seen in pulmonary tuberculosis patients. More detailed

studies are needed to document use of catechin in reducing the frequency and severity of side effects of

treatment.

& 2009 Elsevier GmbH. All rights reserved.

Introduction

Mycobacterium tuberculosis is an intracellular micro-organismmultiplying within macrophages, triggering the production of freeradicals, though avoiding killing by these radicals. Duringpulmonary inflammation, increased amount of reactive oxygenspecies and reactive nitrogen intermediates are produced as aconsequence of phagocytic respiratory burst, thus resulting inhigh oxidative stress (Reddy et al. 2004; Chelnokova et al. 1992).A patient undergoing treatment for tuberculosis develops hepa-totoxicity, fresh liver disease like acute viral hepatitis or other freeradical injury as adverse reaction to drugs (Dhingra 2006).

. All rights reserved.

).

Adjuvant therapy can play a role in prevention and managementof disease/therapy mediated oxidative stress.

Tea (Camellia sinensis) is consumed world wide and rankssecond to water beverage. Its main effectives are called ‘‘green teapolyphenols’’ (GTPs). GTPs can scavenge active oxygen freeradicals produced in many systems and protect cells from damageinduced by the free radicals (Guleria et al. 2002). The pharmaco-logical importance of the green tea extract is water solubility andthorough metabolism by liver cells. It has been found thatnoticeable amount of catechin metabolites in urine were presentafter 6-48 hours (h) of intake of catechin. Therefore, no residualamount of catechin accumulates in body (Guo et al. 1996).

Therefore, the present study was planned to assess the role ofgreen tea (catechins) in lowering the oxidative stress in AFBpositive cases of pulmonary tuberculosis, who are on CAT Iantitubercular treatment.

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Material and Methods

Plan of work

Present study is a double blinded clinical trial. A total of 200newly diagnosed cases of pulmonary Tuberculosis were enrolled.Inclusion criteria were: (1) newly diagnosed AFB positive cases ofpulmonary tuberculosis enrolled from DOTS centre at ChhatrapatiShahuji Maharaj Medical University (CSMMU), Lucknow, India (2)no past history of ATT intake (3) no co-existing morbidity likediabetes/HIV/AIDS and (4) consenting to participate. Exclusioncriterions were: (1) extra pulmonary TB (2) not consenting toparticipate.

All the Patients were receiving CAT I (category I) regimenwhich includes isoniazid, rifampicin, Pyrazinamide and ethambu-tol. Total 100 randomly selected individuals received catechin 500micro gram (500 mg) powder with ATT, 3 days a week, directlyobserved, (cases) while controls received placebo (500 mg starch)with ATT, 3 days a week, directly observed. Catechin powder andplacebo were packed in identical capsules.

After enrollment blood was collected from each patient at day0, after one month and after 4 months of treatment. Oxidativestress in cases and controls was evaluated in terms of the changein antioxidants level, before starting the treatment and at eachfollow up. During every follow-up visit, subjects were interviewedfor development of any sign and symptoms related to drugtoxicity like nausea, vomiting, tingling in ears, epilepsy andtingling in limbs etc

Catechin extract

The catechin extract was commercially procured from ‘‘TulsiAmrit Private Limited’’ (Indore, India). Preliminary screening ofextract for presence of various components was done by two-dimensional chromatography followed by HPLC.

Chromatographic analysis

Descending two- dimensional (2D) paper chromatographytechnique was done for the preliminary screening of the extractfor the presence of various components as described by Bhatiaand Ullah (1968). One gram of the extract was refluxed in 10 ml ofmethanol for 15 min. Twenty microliters of the filtrate wasspotted along with the catechin standards (1 mg/ml). Thechromatogram was developed in a solvent system consisting ofn-butanol: Acetic acid: Water (4:1:5, v/v/v) as the first solvent and2% acetic acid as the second solvent. The chromatogram was air-dried and the violet spots were located under ultraviolet light,eluted in water and absorbance was measured at 275 nm forquantitative estimations. For further confirmation of the percen-tage of catechins present in the extract it was analysed on highpressure liquid chromatography (HPLC).

HPLC method

The HPLC was done on Hewlett Packard system (AgilentModel) using RP- 18 column (0.5 m�25 cm, Lichrospher 100, E.Merck). Mobile phase consisted of 0.1% phosphoric acid inwater and acetonitrile 20% (isocratic system). The solvents weredegassed before use. 25 mg sample was dissolved in 70% ethanol(10 ml) filtered through 0.45 mm filter to avoid column blockage.The injection volume was 10 ml and the flow rate was 1.0ml/minute. The variable wavelength detector (VWD) was set at275 nm. The data obtained was recorded on recorders. The majorpercentage of catechins was constituted by epigallocatechin

gallate (45.05%), epicatechin gallate (23.60%), epicatechin (1.11%)followed by epigallocatechin (0.02%) and others (30.22%).

Collection of blood samples

Three ml of blood was collected from each patient. One mlblood was left in syringe for separating the serum and 2 ml waskept in EDTA vial for separation of plasma and preparation ofhaemolysate. All the samples were stored at �40 1C till used.

Preparation of haemolysate

Erythrocytes were separated from whole blood by centrifugedat 300 rpm for 15 min in cold centrifuge at 4 1C, plasma andBuffy coat were discarded. Erythrocytes were washed withchilled normal saline (0.85 NaCl) 3 times, then haemolysed byadding chilled water, and haemolysate made up to originalvolume fallowed by freezing and thawing. After centrifugationat 3000 rpm for 15 min at 4 1C in cold centrifuge, haemolysatewas colleted at -20 1C till used and used as a source of antioxidantenzyme.

Catalase: was estimated in erythrocytic lysate by using themethod of Aebi (1984) with modifications. The reaction system, ina total volume of 3 ml, comprised of 2.8 ml of phosphate buffer(50 mM, pH 7.0) and 100 ml of haemolysate. The reaction wasstarted by quickly pipetting 0.1 ml hydrogen peroxide (30 m mole)at 25 1C and the absorbance recorded at 240 nm at 12 sec intervalfor 1 min. One IU of catalase is the enzyme, which decompose onemM of hydrogen peroxide per min at 25 1C.

Glutathione Peroxidase (GPx): was estimated in erythrocyticlysate by the method of Paglia and Valentine (1967). The assaysystem contained 2.2 ml of phosphate buffer (50 mM, pH 7.0),0.1 ml EDTA (1.5 mM) 0.1 ml sodium azide (1 mm), 0.1 ml reducedglutathione (1 mM), 0.1 ml NADPH (0.2 mM), 0.1 ml glutathionereductase (1 U/ml) and sutable amount of lysate to make thevolume up to 2.8 ml. the reaction was started by adding 0.2 ml(1.5 mM) hydrogen peroxide. Change in absorbance at 12 secinterval for 2.5 min was recorded on a spectrophotometer at340 nm.

Thiol content: was estimated in RBC lysate by the method ofHu (1994). An aliquot of lysate (0.20 ml) was mixed in a 10 ml testtube with 0.6 ml Tris-EDTA buffer (Tris base 0.25 M-EDTA 20 mM,pH 8.2) followed by addition of 40 ml of 10 mM 5,50 dithionitrobis2-nitrobenzoic acid (DTNB) in methanol. The final volume of thereaction mixture was made up to 4.0 ml by adding 3.16 ml ofmethanol. The test tube was capped, and colour was developed for15–20 min, followed by centrifugation at 3,000g for 10 min atambient temperature. The absorbance of the supernatant wasmeasured at 412 nm. Reduced glutathione was used as standard.Molar extinction coefficient of 13,000 m�1 cm 1 was used forcalculation.

Glutathione (GSH): was estimated by the method of Ellman(1959). An aliquot of 0.5 ml lysate diluted with suitable amount ofwater was mixed with 0.25 ml disodium hydrogen phosphatesolution (0.3 M). A final volume of 3 ml was made by adding0.25 ml DTNB reagent (40 mg, 5, 50 dithhiobis-2 nitrobenzoic acidin 100 ml of 1% (w/v) sodium citrate) just before measuring theabsorbance of the sample at 412 nm. GSH solution of knownconcentrations (10–15 mg) was simultaneously processed toprepare a standard curve. Amount of GSH in the sample wasexpressed accounting to the standard curve in mM/ml.

Super oxide dismutase (SOD): was estimated in the eythro-cytic lysate by using the method of Mishra and Fridovich (1972).The assay system in a final volume of 3.0 ml, consisted of 2.45 mlcarbonate buffer (0.05 M, pH 10.2), suitable aliquot of the lysate

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and water to make up the volume. Adding 0.3 ml of epinephrine,to give final concentration of 2 mM started the reaction. Thereaction was followed at 12 sec interval for 1 min at 480 nmat 25 1C. Suitable control lacking enzyme was run simultaneously.The enzyme unit expressed was calculated as the amountof the enzyme, required to inhibit 50% auto-oxidation ofepinephrine.

Lipid peroxidation (LPO): Thiobarbituric acid reactive sub-stance (TBARS) formation as a product of lipid peroxidation wasestimated in the plasma of the patients, using the method ofOhkawa et al. (1979). To 200 ml of plasma, 8.1% sodium doedocylsulphate and 0.2 ml acetic acid (20% w/v) was mixed. Final volumewas made upto 3 ml with the help of 0.8% TBA. The mixture wasvortexed and kept in boiling water bath for 1 h and cooledimmediately under tap water. To each tube 1.0 ml water and5.0 ml solution of n- butanol and pyridine (15:1 v/v) were added,vortexed and centrifuged at 800g for 10 minutes. The upper layerwas aspirated and absorbance measured at 532 nm. A molecularextinction coefficient of 1.56�105 M-1 cm-1 was used to calculaten moles of MDA formed per mg of protein in plasma.

Nitric oxide (NO): was estimated by following the method ofGreen et al. (1982) in plasma. The plasma was diluted withdeionized water (1:4) and sample was deproteinized with 35%sulfosalicyclic acid (5:1). Treated sample was vortexed at aninterval of 5 min and was allowed to treat for 30 min at roomtemperature. The sample was centrifuged at 10,000g for 15 min-ute. The supernatant was aspirated or the analysis. The Griessreagent was prepared by mixing solution A (0.5% sulfanilamidedissolved in 25% phosphoric acid) and solution B (0.5% naphy-lethylene diamine dihydrochloric acid in distilled water) in equalvolume, just before analysis. The supernatant an the Griess regentwere mixed together in a ratio of 1:1 to make the final volume to3 ml. the absorbance was measured at 550 nm after 10 min. astandard curve with sodium nitrite was plotted and the nitritelevels were estimated against this curve and expressed in nM.

Protein estimation: was estimated in plasma according to themethod of Lowry et al. (1951).

Table 1Change in free radical and antioxidant level in cases and control during antitubercular

Free radical/Antioxidant parameters Duration after enrollment

LPO (Mm/ml) Day 0

One month

Fourth month

NO (lm/ml) Day 0

One month

Fourth month

CATALASE (IU/ml) Day 0

One month

Fourth month

GPx (n mole NADPH/min/mg of protein) Day 0

One month

Fourth month

SOD (IU/ml) Day 0

One month

Fourth month

GSH (mm/ml) Day 0

One month

Fourth month

SH (mM) Day 0

One month

Fourth month

Significant difference in cases and controls po0.01.

SN 1 & 2=free radicals.

SN 3, 4, &5=enzymatic antioxidant.

SN 6 & 7=non-enzymatic antioxidant.

Statistical analysis: The data was analyzed by using student’st-test. A p value of r0.05 was considered as significant.

Results

The parameters taken for the study of oxidative stress revealeda highly significant difference between the cases and controls(Table 1). The level of free radical mediated destruction of lipidswas estimated by concentration of malondialdehyde (MDA)formed. The level of LPO at day zero was 6.9570.15 and6.9470.17 in cases and controls respectively. The level ofLPO in cases was decreased to 6.6470.22 and 5.670.46after one and fourth month of treatment respectively while incontrols it was increased to 7.1870.14 after one month of followup and to 7.4370.38 after four months of follow up (pr0.001)(Table 1).

The level of NO production was estimated in terms of totalnitrite concentration in plasma. The nitrite concentration ob-served at day zero was 31.7970.60 and 31.7670.74 in cases andcontrols respectively. It was decreased to 29.9770.81 after onemonth and to 28.4071.99 after four months of ATT in cases whilein controls it was increased to 32.2170.02 and to 32.8071.69after one and four months follow up respectively (Table 1).

The effect of crude catechin extract on enzymatic antioxidantwas observed by measuring the parameters catalase, GPx andSOD. A significant decrease (pr0.001) in catalase and GPx levelwas observed in cases as compare to controls during the follow upwhile in SOD significant increase (pr0.001) was observed incases as compared to controls (Table 1).

The effect of crude catechin extract on non-enzymaticantioxidants was observed by measuring the level of reducedglutathione and total thiol concentration. A significant decrease(pr0.001) in SH level was observed in cases as compared tocontrols during the follow up while the level of GSH wassignificantly increased (pr0.001) in cases as compare to controls(Table 1).

treatment.

Cases (received catechin +ATT) Cases (received placebo +ATT)

6.95715 6.9470.17

6.6470.22 7.1870.14

5.670.46 7.4370.38

31.7970.60 31.7670.74

29.9770.81 32.2170.02

28.4071.99 32.8071.69

52.1771.96 52.5871.13

48.1177.3 61.1873.4

44.9773.2 65.2673.3

142.076.3 141.8479.3

112.01720.0 147.07716.0

99.9777.7 154.9276.4

59.9570.47 60.0670.46

65.3472.3 56.2072.1

70.3774.04 53.2873.7

0.4970.05 0.4270.04

0.5870.03 0.3670.06

0.6570.04 0.4970.05

0.4970.05 0.4970.05

0.4070.03 0.5570.05

0.3270.04 0.6170.06

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Discussion

Mycobacterium tuberculosis is an intracellular pathogenresiding in macrophages avoiding killing by phagocytes. Theprocess of phagocytosis results in an increased consumption ofoxygen, which triggers the NADPH (nicotinamide adenine dinu-cleotide phosphate) production via the hexose monophosphateshunt. The reduced NADP is oxidized at the expense of molecularoxygen, resulting in burst of oxygen consumption augmentedgeneration of oxygen free radicals and their derivatives (Halliwelland Gutteridge 1989).

The present study showed that the level of SOD increased inpatients who received catechin, probably due to the inhibition ofthe production of hydrogen peroxide through dismutation reac-tion by catechin. It has been reported that catechin has a stronganti-superoxide formation effect, by scavenging superoxide anion(Ho et al. 1999; Reddy et al. 2004). In a study by Chan et al.(2002), the efficacy of an antioxidant cocktail on SOD activity inaged rat brain and DNA damage was measured. They demon-strated that one month administration of beta-catechin solutionincreased SOD activity in the mitochondrial fraction of striatumand midbrain and increased thiobarbituric reactive substanceformation in the cortex and cerebellum of aged rats.

Catalase, present in phagocytes is effective only at highconcentration of hydrogen peroxide. For a given concentration ofcatalase, the initial rate of hydrogen peroxide removal isproportional to the hydrogen peroxide concentration (Halliwelland Gutteridge 1989).

RBC sulfhydryl (-SH) groups are mainly protein associated.Hence, the access of Ellman’s reagent to the hidden protein thiolgroups within non-denatured proteins is not possible in normalperson (Rice-Evans and Anthony 1991). In the patients ofpulmonary tuberculosis, due to oxidative activity there may besome structural protein modifications, which detach or expose theprotein linked–SH groups to the reagent and thus result in asignificantly higher total sulfhydryl level. Free sulfhdryl groups inprotein play the role of highly reactive functional groups inbiological system and participate in several different reactionssuch as alkylation, arylation, oxidation, thiol-dissulphideexchangeetc. the modification of protein thiol groups may result in severefunctional damage including loss of enzyme activity in biologicalsystem (Miyagawa et al. 1997).

The present study showed that the level of GPx was decreasedin patients who received catechin. The enzyme glutathioneperoxidase plays a role in the breakdown of hydrogen peroxide.The decrease might be due to a positive role played by crudecatechin extract in shielding the substrate (GSH) from free radicalmediated modifications. Since, GSH is a substrate for GPx,therefore, a gradual lowering of GPx was observed after the oraladministration of crude catechin extract. The efficacy of catechinsmight have increased in presence of glutathione Peroxidase,which might have synergistically enhanced their antioxidativeactivities as reported earlier by Reddy et al. (2004).

The MDA concentration in plasma was decreased in ourpatients who received catechin. It has been proposed thatpolyphenols react with peroxy radicals resulting in terminationof radical chain reaction (Chaston and Richardson 2003). The bulkof the cell membrane is constituted of lipids which are quitesensitive to highly reactive free radicals generated duringphagocytosis. The decrease in MDA levels in cases indicates aprotective effective offered by catechin extract against free radicalmediated destruction of lipids and plasma membranes.

It has been reported that tea catechins such as (-)- epicatechin,(+)- catechins and (-)- epigallocatechin gallte, inhibit lipidperoxidation in rat liver mitochondria stimulated by ADP andascorbic acid, as well as lipid peoxidation in animal liver

microsomes stimulated by NADPH (Okuda et al. 1983; Vijayamaliniand Manoharam 2004). It has been shown that tea catechinsterminate autoxidation of fatty acids by functioning as radicalscavengers. Further, catechins are chelators of Fe (III) (Chastonand Richardson 2003). The basic catechin structure with 5- and 7-hydroxyl groups in the A ring and the o- dihydroxy structures inthe B ring facilitate them to be a potent antioxidant. Substitutionin B ring is important for stabilizing the free radical formed andfor metal chelating activity as well. The protection belongs to the‘‘pull mechanism’’. According to such a mechanism, the damagecaused by iron-induced lipid peroxidation is reduced by theintroduction of specific chelators that pullout iron (Seeram andNair 2002). Therefore this could have also resulted in lowering ofMDA level in patients who received catechin. It has been reportedthat antioxidant/ lipid perodixadtion system can actively partici-pate in the pathogenesis of development of nourotoxic reactionsto abtibacterial preparation (Seeram and Nair 2002). Theantioxidants thus can reduce the oxidative stress and at the sametime can be lethal to M. tuberculosis.

It has also been observed that consumption of catechinsprevented the alpha-tocopherol, beta-carotene and lipid oxidationproducts formation in human plasma and single dose of green teainduced a significant rise in plasma antioxidant activity, in vivo

(Lotito and Frag 1999).

Conclusion

Crude catechin extract can play a definite role in pulmonarytuberculosis as adjuvant therapy.

Acknowledgement

The present research work was funded by UPCST and researchwork was done by Miss Astha Agarwal.

References

Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126.Bhatia, S.K., Ullah, J.B., 1968. Polyphenols of tea IV: Quantitative and quantitative

study of the polyphenols of different organs and some cultivated varities of teaplant. J. Sci Food Agric. 19, 535–542.

Chan, P., Cheng, J.T., Tsai, J.C., Lien, G.S., Chen, F.C., Kao, P.F., 2002. Effect of catechinon the activity and gene expression of superoxide dismutase in cultured ratbrain. astrocytes. Neurosci. Lett. 328, 281–284.

Chaston, T.B., Richardson, D.R., 2003. Iron chelators for the treatment of ironoverload disease: relationship between structure, redox activity and toxicity.Am. J. Hematol. 73, 200–210.

Chelnokova, N.V., Strarastnko, E.V., Salpagarov, A.M., Smirnova, N.A., Kruglova, E.G.,1992. Biochemical mechanism of neurotoxic reactions to antibacterialpreparations in patients with pulmonary tuberculosis. Probl. Tuberk. 1-2, 8–10.

Dhingra, V.K., 2006. Treatment of tuberculosis in liver disease. Indian J. Tuberc. 53,232–233.

Ellman, G.L., 1959. Plasma antioxidant. Arch. Biochem. Biophys. 82, 70–77.Guleria, R.S., Amita, J., Tiwari, V., Mishra, M.K., 2002. Protective effect of

green tea extract against the erythrocytic oxidative stress injury duringMycobacterium tuberculosis infection in mice. Mol. Cell Biochem. 236,173–181.

Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum,S.R., 1982. Analysis of nitrate, nitrite and (15N) nitrate in biological fluids. Anal.Biochem. 126, 131–138.

Guo, Q., Zhao, B., Li, M., Shen, S., Xin, W., 1996. Studies on prospective mechanismof four components of green tea polyphenols against lipid peroxidation insynaptosomes. Biochim. Biophys. 1304, 210–222.

Halliwell, B., Gutteridge, M.C.J., 1989. In: Free Radicals in Biology and Medicinesecond ed. Oxford University press, Oxford.

Ho, K.Y., Huang, J.S., Tsal, C.C., Lin, T.C., Hsu, Y.F., Lin, C.C., 1999. Antioxidant activityof Tannin components from Vaccinium Vitis-idaea L. J. Pharm. Pharmacol. 51,1075–1078.

Hu, M., 1994. Measurements of thiol groups and glutathione in plasma. MethodsEnzymol. 233, 380–382.

Lotito, S.B., Fraga, C.G., 1999. (+) -catechin as antioxidant mechanisms preventinghuman plasma oxidation and activity in rat wines. Biofactors 10, 125–130.

ARTICLE IN PRESS

A. Agarwal et al. / Phytomedicine 17 (2010) 23–27 27

Lowry, O.H., Rosebrough, N.J., Farr, A.C., Randell, R.J., 1951. Protein measurementwith the folin phenol reagent. J. Biol. Chem. 193, 265–275.

Mishra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autooxidation ofepinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247,3170–3175.

Miyagawa, W., Kennedy, D.O., Nakatani, T., Ohtani, S., Kim, M., Yuasa, I.M., 1997.Protective effect of green tea polyphenols against the cytotoxicity of 1-4 naptho-quinone in isolated rat hepatocytes. Biosci. Biotechnol. Biochem. 61, 1901–1905.

Ohkawa, H., Oshishi, N., Yag, K., 1979. Assay of lipid peroxidation in animal tissuesby thiobarbutaric acid reaction. Anal. Biochem. 95, 351–358.

Okuda, T., Kinura, Y., Yoshida, T., Hatano, T., Okuda, H., Arichi, S., 1983. Studies onthe activities of Tannin related compounds from medical plants and drug.Inhibitory effects on lipid peroxidation in mitochondria and microsomes ofliver. Chem. Pharm.Bull. 31, 1625–1631.

Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitativecharacterization of erythrocytes glutathione peroxidase. J. Lab. Clin. Med. 70,158–169.

Rice-Evans, C.A., Anthony, T.D., 1991. Symons MCR, Techniques in Free RadicalResearch. Elsvier publication, The Netherlands 227–229.

Reddy, Y.N., Murthy, S.V., Krishna, D.R., Prabhakar, M.C., 2004. Role of freeradicals and antioxidants in tuberculosis patients. Ind. J. Tuberc. 51,213–218.

Seeram, N.P., Nair, M.G., 2002. Inhibition of lipid peroxidation and structureactivity related studies of the dietary constituents anthocyanins anthocyani-dina and cathechin. J. Agic. Food Chem.. 50, 5308–5312.

Vijayamalini, M., Manoharam, S., 2004. Lipid peroxidation, vitamin C, E andreduced glutathione levels in patients with pulmonary tuberculosis. CellBiochem. Funct. 22, 19–22.