Drug Discov Ther. 2009; 3(4): Pterocarpus marsupium extract reveals strong in vitro antioxidant activity

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1 Drug Discov Ther. 2009; 3(4): Originl Article Pterocrpus mrsupium extrct revels strong in vitro ntioxidnt ctivity Mhnz Mohmmdi 1, Swti Khole 1, Thoms Pul Asir Devsgym 2, Sroj S. Ghskdi 1, * 1 Deprtment of Zoology, University of Pune, Pune, Indi; 2 Rdition Biology nd Helth Sciences Division, Bhh Atomic Reserch Centre, Mumi, Indi. ABSTRACT: Dietes mellitus is complex chronic disese chrcterized y hyperglycemi, which vi severl mechnism leds to n increse in production of rective oxygen species (ROS) leding to vrious secondry complictions. Thus, drug hving oth ntidietic nd ntioxidnt properties would hve gret therpeutic vlue for overcoming the oxidtive lod in dietes. The present study ws imed t extensively evluting the ntioxidnt properties of n nti-dietic plnt extrct of stem rk of Pterocrpus mrsupium using vrious in vitro rdicl scvenging ssys s well s y using liver slice cultures s model system. Our results demonstrte tht the whole queous extrct showed high ntioxidnt ctivity in ll different ssys used nd lso protected mitochondri ginst oxidtive dmge. Ethnol ws used s n inducer of oxidtive stress in liver slice culture nd cytotoxicity ws estimted y quntitting relese of cytotoxicity mrker enzymes such s lctte dehydrogense (LDH). Additionlly, levels of ntioxidnt enzymes (AOEs) nmely superoxide dismutse, ctlse, glutthione peroxidse, nd glutthione reductse were lso estimted. The whole queous extrct significntly reduced LDH relese long with reduction of lipid peroxidtion compred to ethnol treted slices. These results indicte tht the P. mrsupium extrct my serve s potentil source of nturl ntioxidnt for tretment of dietes. Keywords: Antioxidnt ctivity, P. mrsupium, liver slice culture, in vitro ntioxidnt ssys 1. Introduction Dietes mellitus is metolic disorder chrcterized y hyperglycemi nd insufficiency of secretion or *Address correspondence to: Dr. Sroj Ghskdi, Deprtment of Zoology, University of Pune, Gneshkhind, Pune , Indi. e-mil: ssg@unipune.ernet.in ction of endogenous insulin. It is common disese ffecting over 124 million individuls worldwide. Persistent hyperglycemi during dietic conditions leds to production of free rdicls or impired ntioxidnt defenses vi severl mechnisms (1). Also there is strong evidence tht dietes induces chnges in the ctivity of ntioxidnt enzymes in vrious tissues (2). Mny studies revel tht ntioxidnts cple of neutrlizing free rdicls re effective in preventing experimentlly induced dietes in niml models (3,4) s well s reducing the severity of dietic complictions (5). Additionlly, supplementtion with ntioxidnts in dietic ptients provided greter protection ginst free rdicl induced dmge (6). Recent reviews suggest tht certin herl plnts possess oth ntidietic nd ntioxidtive ctivities nd tht their proper use in the diet my help decrese the oxidtive lod in dietes mellitus. Therefore, vrious plnt or her extrcts hving oth ntidietic nd ntioxidnt ctivity would hve etter therpeutic vlue thn other tretments for overcoming the oxidtive lod in dietes. Pterocrpus mrsupium Rox (from the fmily Leguminose) known in the vernculr s ''vijysr'' of ''Bijsr'' is lrge tree tht commonly grows in the centrl, western, nd southern prts of Indi nd in Sri Lnk. Vrious portions of the rk re used s stringent, nti-dirrhel, ntcid, for tretment of toothche nd for the mngement of dietes (7). The hertwood of P. mrsupium is known to e useful in rthritis, gout, ronchitis, skin infections sthm, dietes, etc. The flvonoids mrsupiun, pterosupin, nd liquiritegenin re reported to posses ntihyperglycemic (8) nd ntihyperlipidemic (9) ctivities. The queous extrct of stem rk ws found to reduce the lood glucose level in lloxn-induced dietic rts (10). In one lortory study, pncretic et-cell regenertion ws oserved in lloxn-induced dietic rts tht received the flvonoid frction from the rk of P. mrsupium (11). In view of this, the present study evluted the ntioxidnt ctivity of the stem rk of Pterocrpus mrsupium ginst ethnol induced oxidtive stress

2 152 Drug Discov Ther. 2009; 3(4): in liver slice cultures nd y using different in vitro iochemicl ssys. 2. Mterils nd Methods 2.1. Chemicls Chemicls were from one of the following compnies: SRL (Mumi, Indi), BDH (Mumi, Indi), Hi-medi (Mumi, Indi), Sigm-Aldrich (St Louis, MO, USA), Merck (Mumi, Indi), Accurex (Mumi, Indi), nd EMD (Mdison, WI, USA) ssy kits. Ethnol ws otined from Fluk, Buchs, Switzerlnd. Stem rk of P. mrsupium ws otined nd uthenticted from the phrmcy Biologicls Adult Swiss lino mice (6-8 weeks old) of either sex, red in the niml house of Deprtment of Zoology, University of Pune, were used for preprtions of liver slices. Prior pprovl for the protocols involving nimls during this work ws otined from Pune University Institutionl Animl Ethicl Committee. Animls were mintined t 12:12 h light/drk cycle nd were given food d liitum Preprtion of smple Stem rk of P. mrsupium ws used for prepring 10% queous extrct. Briefly, 10 g of powder ws dissolved in 100 ml of distilled wter nd stirred for 1 h. The suspension otined ws centrifuged nd the superntnt ws collected nd used s the whole queous extrct. Sequentil extrction of P. mrsupium rk powder ws done using petroleum ether, chloroform, nd methnol using Soxhlet pprtus. The powder ws extrcted for 16 h with ech solvent to remove the solule mtter. The extrct ws evported to dryness in vcuum desicctors. Finlly, the powder ws extrcted with distilled wter, centrifuged, dried, nd used s the soxhlet queous extrct In vitro ntioxidnt cpcity ssys Whole queous extrct (Aq), soxhlet methnolic extrct (Met) nd soxhlet queous extrct (Aqs) of P. mrsupium were used for these ssys. All the ssys were replicted t lest three times nd vlues expressed re n verge of these replictes. DPPH (1,1'-diphenyl-1-picrylhydrzyl) rdicl scvenging ility of the extrcts ws checked ccording to Aquino, et l. (12). The ility of the extrcts to reduce ferric complex ws ssyed ccording to the protocol developed y Alzorkey, et l. (13), wheres the potentil of the extrcts to inhiit 2,2'-zois-3-ethylenzthizoline-6-sulfonic cid rdicl ction (ABTS + ) formtion ws evluted y mesuring the lg time in formtion of the rdicl y the ABTS + /ferrylmyogloin ssy (14). In ll these ssys, ntioxidnt ctivity is expressed s equivlent of μg/ml of stndrd ntioxidnt L-scoric cid ABTS nd CO 3 rdicl nions scvenging ssy y pulse rdiolysis The pulse rdiolysis experiments were crried out t Ntionl Centre for Free Rdicl Reserch, University of Pune. The ility of the extrcts to scvenge ABTS nd CO 3 rdicls ws determined y pulse rdiolysis. ABTS rdicl ws produced y the rection of rdiolyticlly generted zide rdicls with ABTS. CO 3 rdicls were generted using rection mixture contining 0.05 M NHCO 3 nd 0.05 M N 2 CO 3 sturted with N 2 O. In the presence of the extrct, the decy of ABTS nd CO 3 were correlted with the concentrtion of scoric cid equivlents (15) Isoltion of rt liver mitochondri nd exposure to oxidtive stress Three month old femle Wistr rts were used for the isoltion of mitochondri from liver (16). Oxidtive stress to mitochondri ws generted y 2.2'-zois (2-midinopropne) dihydrochloride (AAPH), which genertes peroxyl rdicl nd scorte-fe 2+ which genertes OH like rdicls (16) TBARs ssy The ility of the extrcts to protect lipids from oxidtive dmge ws ssessed y mesuring inhiition of lipid peroxide nd hydroperoxide formtion. Briefly, mitochondri were treted with AAPH nd or scorte-fe 2+ in the sence or presence of extrct nd were incuted t 37ºC in shker wter-th. These were then rected with TBA regent, oiled for 30 min nd the pink color developed due to thiorituric cid rective sustnce (TBARs) formed ws estimted t 532 nm spectrophotometriclly nd expressed s mlonldehyde (MDA) equivlents fter ccounting for pproprite lnks. Mlonldehyde ws prepred y cid hydrolysis of tetrmethoxypropne (16) Lipid hydroperoxide ssy Oxidtive dmge to mitochondri y AAPH nd scoric cid-fe 2+ ws mesured in terms of lipid hydroperoxides formed using FOXII regent (17). The working regent ws routinely clirted ginst solutions of cumene hydroperoxide of known concentrtion. Smples were incuted t 37ºC for 30 min, nd then rected with FOXII regent, spun nd

3 Drug Discov Ther. 2009; 3(4): sornce ws red t 560 nm Estimtion of protein cronyl Protein cronyls were mesured using 2,4-dinitrophenyl hydrzine (DNPH) (18) nd re expressed s nmoles of protein formed per mg protein Mesurement of protein sulphydryl Protein sulphydryls were quntitted using Ellmn's regent (5,5-dithiois-2-nitroenzoic cid) nd re expressed s nmoles protein sulphydryls per mg protein (19) Liver slice culture Liver slice cultures were s descried erlier (20,21). Liver slices were incuted with or without whole queous extrct nd dmge ws induced y ethnol. At the end of incution, the culture medium ws collected nd used for estimtion of lctte dehydrogense (LDH), glutmte pyruvte trnsminse (GPT), nd glutmte oxlocette trnsminse (GOT), which were used s cytotoxicity mrkers. The slices were homogenized nd the homogentes were centrifuged t 10,000 rpm for 10 min t 4ºC. The superntnt ws ssyed for LDH, GPT, GOT nd ntioxidnt enzymes ctlse, superoxide dismutse (SOD), nd glutthione peroxidse (GPx), nd ntioxidnt molecules glutthione nd uric cid. Oxidtive dmge induced y ethnol ws estimted y mesuring lipid peroxidtion. Cholesterol, triglyceride, nd iliruin were ssyed s lipid metolic mrkers Mesurement of cytotoxicity mrkers Smples were ssyed for cytotoxic mrker enzymes nmely LDH, GPT, nd GOT. LDH ctivity ws ssyed ccording to the protocol of Whlefeld (22). GPT nd GOT were estimted using Accurex kits (Accurex Biomedicl Pvt. Ltd., Mumi, Indi) (23). Enzyme units in oth the superntnt nd tissue homogente were mesured. Relese of enzymes from liver slices ws clculted s the rtio of enzyme ctivity found in the superntnt to the totl enzyme (superntnt + homogente) ctivity Mesurement of ntioxidnt enzymes Superoxide dismutse (SOD) ctivity ws mesured spectrophotometriclly using the method of Beuchmp nd Fridovich (24). The ctivity of ctlse ws mesured spectrophotometriclly t 240 nm s descried y Aei (25). Glutthione peroxidse ctivity ws determined ccording to the protocol descried y Lwrence nd Burk (26). Glutthione reductse (GR) ws mesured y the method of Golderg (27). Assys were repeted three times nd the vlues given re n verge of these replictes. Vlues re expressed s μmoles/100 mg tissue Mesurement of ntioxidnt molecules Levels of uric cid were determined using n Accurex kit (Accurex Biomedicl Pvt. Ltd.) (28). Uricse converts uric cid to llntoin nd hydrogen peroxide. The generted hydrogen peroxidse degrdes phenolic chromgen in the presence of peroxidse to form red colored compound, which is mesured t 510 nm. The vlues re expressed s mg/100 mg tissue nd the vlues given re replictes of three experiments. Totl glutthione levels were estimted in liver slice homogentes following the protocol of Teixeri nd Meineghini (29). For the determintion of GSSG, the cell extrct ws treted with 4-vinyl pyridine to finl concentrtion of 0.1% (v/v) nd then incuted for 1 h. The glutthione content ws determined s ove. The mount of GSH could e determined y sutrcting the GSSG content from totl glutthione. Vlues re expressed s micromoles/100 mg of tissue nd the vlues re n verge of three different experiments Mesurement of lipid peroxidtion Lipid peroxidtion in liver slices ws estimted in terms of thiorituric rective sustnces formed (30). Tissue homogente ws prepred in 5% TCA. To 1 ml of homogente, 4 ml of 0.5% TBA in 20% TCA ws dded nd this ws incuted t 95 C for 30 min. This ws immeditely cooled on ice, during which the color chnged from ornge to pink. After centrifugtion t 4,000 rpm for 10 min, the difference in the sornce of the superntnt t 532 nm (specific) nd 600 nm (non-specific) ws mesured to estimte pmol of mlonldehydes (MDA) Mesurement of lipid metolic mrkers Cholesterol ws estimted using n Accurex kit (Accurex Biomedicl Pvt. Ltd.) using threestep rection (31). Cholesterol esterse hydrolyses cholesterol esters into free cholesterol nd ftty cids. The free cholesterol is cted upon y cholesterol oxidse to form cholest-4-en-3one nd hydrogen peroxide. In the presence of peroxidse, hydrogen peroxide oxidtively couples with 4-mino-ntipyrine nd phenol to produce red quinoneimine dye which hs n sornce mximum t 510 nm. Triglyceride levels were estimted y n Accurex kit (32). Hydrolysis of triglycerides y lipoprotein lipse releses glycerol, which is converted to glycerol-3-phosphte y glycerol kinse. This glycerol-3-phosphte is oxidized to dihydroxycetone phosphte nd hydrogen peroxide. In the presence of peroxidse, hydrogen peroxide oxidizes

4 154 Drug Discov Ther. 2009; 3(4): the phenolic chromogen to red colored compound, which is mesured t 510 nm. Biliruin levels were mesured y n Accurex kit (33). In the presence of dimethyl sulphoxide, iliruin rects with dizotized sulphnilic cid to produce violet colored compound zoiliruin, which is mesured t 546 nm. The vlues given re n verge of three replictes nd re expressed s mg/100 mg tissue. 3. Results 3.1. P. mrsupium extrcts exhiit high in vitro ntioxidnt ctivity Antioxidnt ctivities of whole queous, soxhlet queous nd soxhlet methnolic extrcts ws evluted y their rdicl scvenging ility, ferric reducing ility, nd rdicl formtion inhiition ctivity. In DPPH nd ferrylmyogloin/abts + ssys, the whole queous extrct exhiited the highest rdicl scvenging s well s inhiition of ABTS + rdicl formtion, with vlue of 1.22 ± 0.01 nd 3.74 ± 0.05 μg/ml, respectively, of scoric cid equivlent ntioxidnt cpcity (AEAC) (Figures 1 nd 2). In Ferric reducing ntioxidnt power (FRAP) ssy, the mximum ferric reducing ility of ± 0.03 μg/ml scoric cid equivlent ws shown with the 0.05% soxhlet methnolic extrct (Figure 3). Additionlly, pulse rdiolysis ws used to quntitte rdicl scvenging ctivity of these extrcts. Decy of ABTS (Figure 4) nd CO 3 (Figure 5) rdicls ws monitored in the presence of scoric cid nd the liner plot of pseudo-first order rte constnt (K s ) versus scoric cid concentrtion ws used to clirte the stndrd curve for estimtion of scorte equivlents in the extrcts. The pseudo-first order rte constnts for the decy of ABTS nd CO 3 were determined for the known concentrtions of extrcts nd from the clirtion curve, the scorte equivlents, present in different extrcts were determined. Figures 4-d nd Figures 5-d show ABTS nd CO 3 rdicl scvenging ctivities y whole queous, queous soxhlet, nd methnolic soxhlet extrcts, respectively. Mximum ABTS rdicl scvenging ctivity ws exhiited y the 1% soxhlet methnolic extrct with vlue of 2.3 μg/ml of AEAC nd mximum CO 3 rdicl scvenging ctivity ws exhiited y 0.1% methnolic soxhlet extrct with vlue of 3.9 μg/ml of AEAC. cronyl were quntitted s mesure of dmge to protein. The whole queous extrct showed the highest ility to inhiit formtion of lipid peroxides followed y soxhlet queous ginst scorte-fe 2+ nd AAPH induced oxidtive dmge respectively (Tle 1). Similr results were otined for lipid hydroperoxide formed in response to dmge y scorte-fe 2+ nd AAPH. The nmoles of lipid hydroperoxide formed ws significntly (p < 0.05) reduced ginst scorte- Fe 2+ nd AAPH s compred to oxidtively dmged mitochondri (Tle 2). Protein cronyl nd protein sulphydryl were quntitted s mesure of oxidtive dmge to proteins induced y AAPH. Protein cronyl formtion ws reduced sustntilly (p < 0.05) y 1% whole Figure 1. DPPH rdicl scvenging ctivity of vrious P. mrsupium extrcts. Aq, whole queous extrct; Aqs, soxhlet queous extrct; Met, soxhlet methnolic extrct. Rdicl scvenging ctivity is expressed s scoric cid equivlent ntioxidnt ctivity (AEAC). Vlues re men ± SD of three different experiments. Figure 2. Inhiition of ABTS rdicl formtion y vrious P. mrsupium extrcts. Aq, whole queous extrct; Aqs, soxhlet queous extrct; Met, soxhlet methnolic extrct. Inhiition is mesured in terms of scoric cid equivlent ntioxidnt ctivity (AEAC). Vlues re men ± SD of three different experiments P. mrsupium extrcts protect iomolecules from oxidtive dmge These extrcts were checked for their ility to protect rt liver mitochondri ginst oxidtive dmge induced y AAPH nd scorte-fe 2+. Lipid peroxides nd hydroperoxides were used s mesure of dmge to lipids, while protein sulphydryl nd protein Figure 3. Ferric reducing ility of vrious P. mrsupium extrcts. Aq, whole queous extrct; Aqs, soxhlet queous extrct; Met, soxhlet methnolic extrct. Rdicl scvenging ctivity is expressed s scoric cid equivlent ntioxidnt ctivity (AEAC). Vlues re men ± SD of three different experiments.

5 Drug Discov Ther. 2009; 3(4): c d Figure 4. Kinetic decy of ABTS rdicl generted y pulse rdiolysis. ABTS rdicl scvenging ws compred in the presence of () different concentrtions of scoric cid, () 1% nd 0.1% of whole queous extrct, (c) 1% nd 0.1% of soxhlet queous extrct, nd (d) 1% nd 0.1% of soxhlet methnolic extrct of P. mrsupium. c d Figure 5. Kinetic decy of CO 3 rdicl generted y pulse rdiolysis. CO 3 rdicl scvenging ws compred in the presence of () different concentrtions of scoric cid, () 1% nd 0.1% of whole queous extrct, (c) 1% nd 0.1% of soxhlet queous extrct, nd (d) 0.1% nd 0.01% of soxhlet methnolic extrct of P. mrsupium. queous extrct s compred to dmged mitochondri. Similrly, the 1% whole queous extrct ws le to protect mitochondri significntly (p < 0.05) y inhiiting depletion of the protein sulphydryls ginst dmged mitochondri (Tle 3) Protection of liver slices from ethnol induced oxidtive stress The ility of whole queous extrct of P. mrsupium to protect liver cells ginst ethnol induced oxidtive

6 156 Drug Discov Ther. 2009; 3(4): Tle 1. Inhiition of lipid peroxidtion y P. mrsupium mesured in terms of nmoles of TBARs formed/mg protein in rt liver mitochondri dmged y scorte-fe 2+ nd AAPH. Lipid peroxidtion in controls nd oxidtively dmged mitochondri y scorte-fe 2+ nd AAPH ws 0.5 ± 0.1 nd 1.93 ± 0.37 nmoles of TBARs/mg protein, respectively. Lipid peroxidtion in controls nd oxidtively dmged mitochondri y APPH were 0.44 ± 0.04 nd 1.01 ± 0.08 nmoles of TBARs, respectively. Vlues re represented s men ± SD of three different experiments. Nmoles of TBARs formed/mg Extrct protein y Ascorte-Fe 2+ AAPH Aqueous extrct 1% Aqueous extrct 0.1% Aqueous soxhlet extrct 1% Aqueous soxhlet extrct 0.1% Methnol soxhlet extrct 1% Methnol soxhlet extrct 0.1% 0.32 ± ± ± ± ± ± ± ± ± ± ± ± 0.07 These vlues differ significntly (p < 0.05) from the AAPH nd scorte-fe 2+ induced dmge to rt liver mitochondri (Student's t-test). stress ws checked using liver slice culture nd ssying the relese of intrcellulr enzymes nmely LDH, GOT, nd GPT. As shown in Figure 6, the % relese for LDH, GOT, nd GPT ws significntly decresed to 12.6 ± 1.06, 15.3 ± 1.1, nd 16.4 ± 0.79, respectively, in slices treted with ethnol nd 1% whole queous extrct s compred to ethnol treted slices which hd percent relese of 53 ± 1.83, 41 ± 1.4, nd 41 ± 0.7 for LDH, GOT, nd GPT, respectively. The time course Tle 2. Inhiition of lipid peroxidtion mesured in terms of nmoles of lipid hydroperoxides formed/mg protein in rt liver mitochondri dmged y scorte-fe 2+ nd AAPH y different extrcts of P. mrsupium. Lipid hydroperoxides formed in controls nd oxidtively dmged mitochondri y scorte-fe 2+ were ± 3.8 nd ± 5.71 nmoles of lipid hydroperoxides/mg protein. Lipid hydroperoxides formed in controls nd oxidtively dmged mitochondri y APPH were ± 3.99 nd ± nmoles of lipid hydroperoxides/mg protein. Vlues re represented s men ± SD of three different experiments. Extrct Nmoles of lipid hydroperoxides formed/mg protein y Ascorte-Fe 2+ AAPH Aqueous extrct 1% Aqueous extrct 0.1% Aqueous soxhlet extrct 1% Aqueous soxhlet extrct 0.1% Methnol soxhlet extrct 1% Methnol soxhlet extrct 0.1% ± ± ± ± ± ± ± ± ± ± ± ± These vlues differ significntly (p < 0.05) from the AAPH nd scorte-fe 2+ induced dmge to rt liver mitochondri (Tukey's t-test). c Tle 3. Protein sulphydryl formed in rt liver mitochondri dmged y AAPH y different extrcts of P. mrsupium. Protein suplhydryl in controls nd oxidtively dmged mitochondri were ± 1.99 nd ± 0.18 nmoles protein suplhydryl/mg protein, respectively. Protein oxidtion mesured in terms of nmoles of protein cronyl formed/mg protein in oxidtively dmged rt liver mitochondri y AAPH y different extrcts of P. mrsupium. Protein cronyl in controls nd oxidtively dmged mitochondri were 7.59 ± 0.54 nd ± 1.09 nmoles protein cronyl/mg protein, respectively. Vlues re represented s men ± SD of three different experiments Extrct Aqueous extrct 1% Aqueous extrct 0.1% Aqueous soxhlet extrct 1% Aqueous soxhlet extrct 0.1% Methnol soxhlet extrct 1% Methnol soxhlet extrct 0.01% nmoles Protein suplhydryl/mg protein 31.6 ± ± ± ± ± ± 0.58 nmoles Protein cronyl/mg protein ± ± ± ± ± ± 0.31 These vlues differ significntly (p < 0.01) from the AAPH nd scorte-fe 2+ induced dmge to rt liver mitochondri (Student's t-test). d Figure 6. Effect of whole queous extrct of P. mrsupium on relese of vrious cytotoxicity mrkers from liver slice culture. () Mesurement of cytotoxicity mrkers in the presence or sence of ethnol (Eth.) lone or with different concentrtions of whole queous extrct (EX). (), (c) nd (d) re time course for LDH, GOT nd GPT, respectively. Vlues re men ± SD of three different experiments.

7 Drug Discov Ther. 2009; 3(4): of relese of these enzymes showed tht these enzymes were relesed over the period of 2 h nd this relese ws inhiited within 30 min in the presence of 1% P. mrsupium extrct (Figures 6-d) P. mrsupium extrct protects liver slices y lowering lipid peroxide formtion due to ethnol It ws oserved tht in ethnol-treted liver cells the mount of MDA formed ws sustntilly higher (1.29 pmol/100 mg tissue) compred to the control (0.27 pmol/100 mg tissue) (Figure 7). Following the time course for lipid peroxidtion, grdul increse in lipid peroxidtion over period of 2 h ws oserved, which ws inhiited within 30 min in the presence of 1% P. mrsupium extrct (Figure 7) P. mrsupium extrct modultes ntioxidnt sttus in liver cells in response to ethnol induced oxidtive stress Modultion of ntioxidnt enzymes Intrcellulr ntioxidnt enzymes superoxide dismutse (SOD), ctlse (CAT) nd glutthione peroxidse (GPx) were quntitted. In the presence of ethnol, the ctivities of SOD nd ctlse were found to e significntly incresed s compred to controls wheres smll decrese ws seen in GPx s compred to control (Tle 4). Activities of ll these enzymes were normlized in the presence of P. mrsupium extrct in concentrtion-dependent mnner. Following the time course for ll three enzymes, SOD nd ctlse showed grdul increse, while GPx ctivity grdully dropped in ethnol treted slices compred to control. The ctivity of ll the enzymes ws restored in the presence of extrct (Figure 8). GR is involved in synthesis of reduced glutthione, which is mjor ntioxidnt in the cell. Liver slices treted with ethnol showed decrese in the ctivity of this enzyme, which ws restored y whole queous extrct in concentrtion dependent mnner (Figure 9). Moreover time course reveled tht within 30 min, ctivity of GR ws restored (Figure 9) Modultion of ntioxidnt molecules Glutthione nd uric cid re smll ntioxidnt molecules, which directly scvenge free rdicls. Activity of oth these molecules ws decresed in liver slices treted with ethnol s compred to untreted slices (Figures 10 nd 11). The whole queous extrct of P. mrsupium normlized the levels of oth these molecules within 30 min in concentrtion dependent mnner (Figures 10 nd 11) P. mrsupium regultes lipid metolic mrkers Figure 7. Effect of whole queous extrct of P. mrsupium on lipid peroxidtion of liver slices. () Protection conferred y different concentrtions of whole queous extrct in the presence of ethnol, s mesured s picomoles of mlonldehyde formed per mg of tissue. () Time course for lipid peroxidtion mesured t n intervl of 30 min. Vlues re men ± SD of three different experiments. Cholesterol, triglyceride, nd iliruin levels were checked s mjor lipid metolic mrkers. In liver slices treted with ethnol oth cholesterol (0.9 mg/100 mg tissue) nd triglycerides (3.09 mg/100 mg tissue) were found to e mrkedly decresed s compred to untreted slices (12.18 nd mg/100 mg tissue, respectively). 1% P. mrsupium extrct ws le to restore these levels. Biliruin levels in liver slices treted with ethnol showed smll increse (0.39 ± 0.01 mg/100 mg tissue), s compred to control (0.2 ± 0.02 mg/100 mg tissue). Slices treted with 1% P. mrsupium extrct did not show ny chnge compred to control untreted slices (Tle 5). Cholesterol nd triglyceride levels decresed grdully over period of 2 h during ethnol tretment nd were normlized Tle 4. Specific ctivity of ctlse, SOD nd GPx (U/mg protein) under different conditions Control Extrct 1% EX 1% + OH EX 0.5% + OH EX 0.1% + OH OH CAT SOD GPx 1,014.1 ± ± ± ± ± ± ,198.2 ± ± ± ,366.4 ± ± ± ,724.5 ± c ± 5.28 c 1.32 ± c 2,040.7 ± ± ± These vlues differ significntly (p 0.001) from the control group (student's t-test); These vlues differ significntly (p 0.001) from ethnol treted group (student's t-test); c These vlues differ significntly (p 0.1) from ethnol treted group (student's t-test).

8 158 Drug Discov Ther. 2009; 3(4): c Figure 8. Time course for vrious ntioxidnt enzymes in the presence of ethnol nd ethnol with whole queous extrct. () Ctlse, () superoxide dismutse (SOD), nd (c) glutthione peroxidse (GPx). Vlues re men ± SD of three different experiments. Figure 9. Effect of P. mrsupium extrct on glutthione reductse in liver slice culture. () Level of glutthione reductse in the presence of ethnol nd ethnol long with different concentrtions of whole queous extrct. () Time course for glutthione reductse in the presence of ethnol nd ethnol with 1% whole queous extrct. Vlues re men ± SD of three different experiments. Figure 10. Effect of P. mrsupium extrct on glutthione mount in liver slice culture. () Amount of glutthione under different conditions. () Time course for glutthione mesured t n intervl of 30 min under vrious conditions. Vlues re men ± SD of three different experiments. Figure 11. Effect of P. mrsupium extrct on uric cid mount in liver slice culture. () Amount of uric cid under vrious conditions. () Time course for uric cid mesured t n intervl of 30 min under vrious conditions. Vlues re men ± SD of three different experiments.

9 Drug Discov Ther. 2009; 3(4): Tle 5. Amount of cholesterol, triglyceride, nd iliruin (mg/100 mg tissue) in liver slice culture under different conditions Control Extrct 1% EX 1% + OH EX 0.5% + OH EX 0.1% + OH OH Cholesterol Triglyceride Biliruin ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 These vlues differ significntly (p 0.001) from the control group (Student's t-test); These vlues differ significntly (p 0.001) from ethnol treted group (Student's t-test). within 30 min in the presence of 1% P. mrsupium extrct long with ethnol. Biliruin levels incresed grdully over period of 2 h during ethnol tretment nd ws restored within 30 min in the presence of 1% P. mrsupium extrct (Figure 12). 4. Discussion Rective oxygen species hve een implicted in the etiology of mny humn diseses such s crdiovsculr liments, cncer, dietes, Alzheimer's disese, rthritis, nd neurodegenertive, nd in the process of ging. Antioxidnts confer protection either y inhiiting formtion of rdicls or y scvenging free rdicls (34). Recently, lrge numer of plnt extrcts hve een explored for their ility s strong ntioxidnts. Pterocrpus mrsupium hs een used in yurvedic medicine for the tretment of dietes over long period of time. Two mjor phenolic constituents of P. mrsupium nmely pterostilene nd mrsupium were le to significntly decrese plsm glucose levels y 42 nd 33%, respectively, in streptozotocin induced dietic rts. Though the exct mechnism of the ntidietic effect of this extrct is yet to e understood, some dietics use it regulrly s n ntidietic drug. Demonstrtion of the strong ntioxidnt ctivity of this extrct would certinly increse its potentil s n ntidietic drug. Therefore, the im of the present study ws to evlute ntioxidnt ctivity of P. mrsupium using in vitro ssys nd liver slice cultures s model system. The totl ntioxidnt ctivity of P. mrsupium extrcts ws evluted using different in vitro ntioxidnt ctivity ssys. These ssys correspond to n ction of ntioxidnts t different levels. The DPPH rdicl scvenging ssy corresponds to the primry rdicl scvenging ctivity of n ntioxidnt, wheres ferrylmyogloin/abts + corresponds to the ility of n ntioxidnt to inhiit rdicl formtion. The FRAP ssy, on the other hnd, evlutes the reducing ility of the ntioxidnt. The whole queous extrct of P. mrsupium exhiited high ntioxidnt potentil in ll of the ove mentioned ssys except the FRAP ssy, wherein the soxhlet methnolic extrct showed high reducing power (Figures 1-3). Pulse rdiolysis revels the ility of ntioxidnt to scvenge secondry rdicls such s ABTS nd CO 3. The soxhlet methnolic extrct showed high ABTS s well s CO 3 rdicl c Figure 12. Effect of P. mrsupium extrct on vrious lipid metolic mrkers in liver slice culture. Time course for cholesterol (), triglyceride (), nd iliruin (c) were mesured t n intervl of 30 min under vrious conditions. Vlues re men ± SD of three different experiments. scvenging ctivity. The whole queous extrct on the other hnd ws est in inhiiting formtion of lipid peroxides nd lipid hydroperoxide induced y AAPH nd scorte-fe 2+ in rt liver mitochondri (Tles 1 nd 2). Similrly, the whole queous extrct inhiited formtion of protein cronyls s well s inhiited depletion of protein sulphydryls induced y AAPH in rt liver mitochondri (Tle 3). This dt clerly shows tht the whole queous extrct nd soxhlet methnolic extrct ehve differentilly in different ssys. This differentil ctivity of the extrcts could e ttriuted to different components present in the extrct. The ntioxidnt ctivity of the extrct ws

10 160 Drug Discov Ther. 2009; 3(4): dditionlly evluted using liver slice cultures s model system. Oxidtive stress ws induced y ddition of ethnol to the liver slice culture. These cultures offer very good test system ecuse they provide model of the considerle complexity of structurlly nd functionlly intct cells. These cultures retin intct tissue rchitecture nd more closely mimic the in vivo sitution s compred to isolted heptocytes. Ethnol used to induce oxidtive stress ws cytotoxic to the cells s mesured y the increse in LDH, GOT, nd GPT relese in treted cells. This cytotoxic effect ws sustntilly lowered in concentrtion dependent mnner on ddition of whole queous extrct (Figure 6) proly through reduction of oxidtive stress. Incresed lipid peroxidtion hs lredy een ssocited with liver injury in nimls fed ethnol (35) or in heptocytes treted with ethnol in vitro (36). In our studies when cultures were treted with P. mrsupium extrct long with ethnol, lipid peroxidtion ws sustntilly reduced s compred with control cells (Figure 7). The fct tht lipid peroxidtion ws significntly reduced in P. mrsupium-treted cultures indictes tht this extrct hs strong ntioxidnt ctivity. Activity of four ntioxidnt enzymes nmely ctlse, SOD, GR, nd GPx ws mesured. In the presence of ethnol the ctivities of SOD nd ctlse sustntilly incresed, proly in response to oxidtive stress induced y ethnol. Addition of whole queous extrcts long with ethnol led to decrese in these enzyme levels, which ws comprle to those seen in untreted cultures (Tle 4). The levels of GPx (Tle 4) nd GR (Figure 9), on the other hnd, decresed in the presence of ethnol nd were normlized on ddition of whole queous extrcts in concentrtion dependent mnner. This result correlted to the levels of glutthione, which lso decresed in the presence of ethnol nd ws restored in the presence of the extrct (Figure 10). The levels of uric cid, nother ntioxidnt molecule, were lso decresed with ethnol tretment nd were normlized within thirty minutes in the presence of P. mrsupium extrct (Figure 11). Cholesterol, iliruin, nd triglyceride levels were estimted s mrkers of liver specific lipid metolism. Both cholesterol nd triglycerides decresed sustntilly in the presence of ethnol nd in the presence of extrct this decrese ws prevented (Figures 12 nd ). We lso checked for iliruin, the product of cholesterol degrdtion nd it ws found to e incresed with ethnol tretment nd ws restored in the presence of the extrct (Tle 5). However, this increse ws not s prominent s the decrese in cholesterol. This ws it surprising ecuse the mjority of reports hve shown ssocition of ft ccumultion with lcohol intke. However, ll those oservtions were mde on chronic tretment of tissues wheres in our system cells re given cute exposure to ethnol (37). Our dt demonstrtes tht during cute exposure to lcohol P. mrsupium extrct helps to mintin cholesterol s well s triglyceride levels. Both the reduction in lipid peroxidtion nd restortion of the ctivity of AOEs clerly demonstrte tht in the presence of P. mrsupium extrcts free rdicl genertion is significntly reduced. The ntioxidnt ctivity exhiited y P. mrsupium extrct in this system seems to e due to its interction with free rdicls leding to their inctivtion rther thn ltering the ctivity of ntioxidnt enzymes, which scvenge them. Antioxidnt ctivity of the plnt extrcts is often ttriuted to the presence of flvonoids. P. mrsupium is known to contin pterostilene, n nlogue of resvertrol, which is shown to hve peroxyl rdicl scvenging ctivity (38). Other purified flvonoids, mrsupin, pterostilene, nd epictechin hve lso een shown to possess ntidietic ctivity (39). 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