PROTECTIVE EFFECT OF TRITERPENES ON CALCIUM OXALATE CRYSTAL-INDUCED PEROXIDATIVE CHANGES IN EXPERIMENTAL UROLITHIASIS

Pharmacological Research, Vol. 41, No. 4, 2000 doi:10.1006rphrs.1999.0601, available online at http:rrwww.idealibrary.com on

PROTECTIVE EFFECT OF TRITERPENES ON CALCIUM OXALATE CRYSTAL-INDUCED PEROXIDATIVE CHANGES IN EXPERIMENTAL UROLITHIASIS M.M. MALINI, M. LENIN and P. VARALAKSHMIU Department of Biochemistry, Dr. Alm Post-graduate Institute of Basic Medical Sciences, Uni¨ ersity of Madras, Taramani Campus, Chennai-600 113, India Accepted 14 September 1999

Naturally occurring pentacyclic triterpenes of plant origin have been identified as possessing a wide range of pharmacological effects. Lupeol ŽLupa-21,20Ž29. dien, 3 ␤-ol. has been found to be efficient in reducing the risk of stone formation in animals by way of preventing crystal-induced tissue damage and dilution of urinary stone-forming constituents. In the present study, two structurally related triterpenes, lupeol and betulin ŽLupa-20Ž29.ene-3,28 diol. were assessed for their antilithiatic effect. Foreign body implantation method followed by supplementation of ammonium oxalate was adapted to induce stone formation in the bladder. This led to elevated lipid peroxidation and depleted antioxidant status in the renal tissues. Both the triterpenes were equally efficient in minimizing crystal-induced renal peroxidative changes measured in terms of malondialdehyde and subsequent tissue damage. The antioxidant status, comprising of the enzymatic and non-enzymatic components, was found to be significantly depleted in the kidney and bladder of stone-forming animals. Both lupeol and betulin were comparable in their ability to restore the thiol status and the antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase. The mechanism by which the two compounds render protection against oxalate-induced toxic manifestations and free radical production may involve the inhibition of calcium oxalate crystal aggregation and enhancement of the body defence systems. 䊚 2000 Academic Press KEY

WORDS:

triterpenes, lupeol, betulin, lipid peroxidation, antioxidants.

INTRODUCTION Toxic oxygen metabolites are generated normally by aerobic metabolism in cells and their generation can significantly increase in certain pathological conditions. Subsequently, when endogenous antioxidant defence capabilities are exceeded by this oxidant flux, tissue injury occurs. The biochemical mechanism by which various factors lead to initiation of calcium oxalate stone formation is still not known. However, free radical production has been known to be intricately involved in the process of crystal deposition in the renal tissues w1x. Our earlier studies have also indicated increased lipid peroxidation and decreased levels of antioxidant potential in the kidneys of rats supplemented with a calculi-producing diet w2, 3x. Oxalate, the major stone-forming constituent, has also been reported to induce lipid peroxidation and to cause tissue damage by reacting with polyunsaturated fatty 1043᎐6618r00r040413᎐06r$35.00r0

acids in cell membranes w4x. In recent times, various structurally-related triterpene compounds of plant origin have been reported to possess a variety of pharmacological activities, such as hepatoprotective w5x, hypolipidaemic w6x, antilithogenic w7x, anticancer w8x, and anti-inflammatory w9x effects. Different triterpenes like oleanolic acid, ursolic acid w10x and celastrol w11x have been reported to exhibit antioxidant properties in various pathological conditions. Cratae¨ a nur¨ ala. Buch Ham ŽCapparidaceae. is one of the medicinal plants in the Indian System of Medicine, well recognized for its antilithic properties w12x. Lupeol, isolated from the stem bark of this plant in our laboratory was identified as the active principle behind the antiurolithiatic effects w13, 14x. The pharmacological efficacy of triterpenes prompted us to investigate the activity of lupeol and another structurally related triterpene, betulin against free radical damage manifested during bladder stone formation. 䊚 2000 Academic Press

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MATERIALS AND METHODS

Chemicals Lupeol was isolated from the stem bark powder of Cratae¨ a nur¨ ala. Buch Ham. in our laboratory according to the method of Baskar w12x and betulin was obtained from Sigma Chemical Co. ŽSt. Louis, MO, USA.. Thiobarbituric acid and reduced glutathione were purchased from Sisco Research Laboratories Private Limited, Mumbai. Dithionitro benzoic acid was obtained from BDH Chemicals Ltd., Poole, England. All other chemical reagents and solvents were of analytical grade.

Induction of urolithiasis in rats The rats were anaesthetized under anaesthetic ether and a suprapubic incision was made to expose the bladder. It was then cut at the tip to insert a sterile piece of zinc Ž10᎐12 mg. and closed by a single stitch. The abdomen was finally closed by two or three stitches and the animal allowed to recover for 3 days.

Experimental set-up The animals were divided into six groups with six rats in each: Group Iᎏsham-operated control animals, wherein the bladder was cut open and sutured without zinc implantation to nullify the changes due to the trauma of surgery; Group IIᎏzinc implanted animals administered 2% ammonium oxalate ŽAOx. for 15 days Ž1 mlrrat per day, p.o..; Group IIIᎏsham-operated animals administered lupeol Ž35 mg kgy1 body wt. per day, p.o.. for 15 days; Group IVᎏsham-operated animals administered betulin Ž35 mg per kg body wt. per day, p.o.. for 15 days; Group Vᎏzinc implanted rats administered 2% ammonium oxalate q lupeol; Group VIᎏzinc implanted rats administered 2% ammonium oxalate q betulin.

Assay procedures Lipid peroxidation was assayed by the method of Devasagayam w15x in which malondialdehyde, a TBA reactive end product, served as the index of lipid peroxidation. Total GSH content was determined based on its reaction with 5,5⬘-dithiobis Ž2-nitrobenzoic acid. ŽDTNB. to give a compound that absorbs at 412 nm, according to the method of Moron et al. w16x. The thiol content of the tissues were estimated by the method of Sedlack Lindsay w17x. Ascorbic acid Žvitamin C. level was estimated by the method of Omaye et al. w18x and vitamin E was determined by the method of Baker and Frank w19x. Catalase activity was assayed by the method of Sinha w20x. In this method, potassium dichromate in acetic acid is reduced to chromic acetate when heated in the presence of H 2 O 2 , which was measured colorimetrically at 540 nm. Glutathione perox-

idase ŽGPx., was assayed by measuring the amount of GSH consumed in the reaction mixture according to the method of Rotruck et al. w21x. Superoxide dismutase ŽSOD. activity was assayed by the degree of inhibition of the autoxidation of pyrogallol at an alkaline pH by SOD according to the method of Marklund and Marklund w22x. Glutathione-S-transferase ŽGST. was assayed by the method of Habig et al. w23x. Glutathione reductase ŽGR. which utilizes NADPH to convert oxidized glutathione to the reduced form was assayed by the method of Staal et al. w24x. The method of Beutler w25x in which the amount of NADPH formed was measured spectrophotometrically was adopted to assay glucose-6-phosphate dehydrogenase ŽG6PD.. Protein content was estimated by the method of Lowry et al. w26x using bovine serum albumin as standard.

Statistical analysis

All data were expressed as mean " SD. Statistical significance between the data of different groups were evaluated using one-way analysis of variance followed by Student’s Neuman᎐Keul’s test. P values less than 0.05 were considered significantly altered.

RESULTS As seen in Table I, the lithogenic Group II animals showed increased basal levels of lipid peroxidation Ž P- 0.001., measured in terms of malondialdehyde, in the kidney and bladder tissues, when compared to the control animals. Oxalate is recognized to function as a ligand for numerous metal cations and can react with ferric ion to form a coordination complex. Treatment of the stone-forming animals with the two triterpenes ŽGroups V and VI. restricted the oxalate and crystal-induced peroxidative changes in the renal tissues to a great extent. Tables II and III delineate the changes in the levels of various non-enzymic antioxidants in kidney and bladder, respectively. A marked reduction in the level of GSH in the kidney Ž P- 0.001. and bladder Ž P- 0.001. of group II animals might be responsible for the abysmal state of the other antioxidants like ␣-tocopherol, ascorbic acid and the sulphydryl groups ŽTSH and NPSH. in these tissues Ž P- 0.001., which were remarkably depleted when compared to the control animals. Both lupeol and betulin treatment ŽGroups V and VI, respectively. were efficient in impeding the breakdown of antioxidant potential of the tissues. Tables IV and V show marked inhibition of the various enzymes constituting the antioxidative domain in kidney and bladder. In the kidney and bladder the activity of the glutathione-dependent

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Table I Effect of the triterpenes on the level of malondialdehyde in the tissues of control and experimental animals Parameters Group I (nmol of MDA Sham control formedr mg protein)

Group II Imp. AOx

Group III Shamq lupeol

Group IV Shamq betulin

Group V Imp.q AOxq lupeol

Kidney Basal 0.2 mM Ascorbate 10 mM FeSO4

2.77" 0.21 5.39" 0.41 8.47" 0.51

3.99" 0.23a ‡ 2.50" 0.21a b ‡ 2.79" 0.18b ‡ 2.99" 0.24b ‡c † 6.85" 0.63a ‡ 5.29" 0.63b ‡ 5.41" 0.28b ‡ 5.70" 0.28b ‡ U 9.78" 0.57a † 8.22" 0.72b ‡ 8.36" 0.56b † 8.60" 0.46b

Bladder Basal 0.2 mM Ascorbate 10 mM FeSO4

3.59" 0.15 4.15" 0.40 6.08" 0.31

4.84" 0.39a ‡ 3.33" 0.29b ‡ U 5.10" 0.44a 4.07" 0.25b † a 8.61" 0.53 ‡ 6.03" 0.35b ‡

Group VI Imp.q AOxq betulin

U

U

U

3.16" 0.23a b ‡c ‡d 5.75" 0.29b ‡ U 8.71" 0.57b U

U

3.41" 0.20b ‡ 3.92" 0.24b ‡c †d 4.06" 0.34a b ‡c ‡d † U 4.12" 0.26b 4.36" 0.20 4.45" 0.21 U U b 6.05" 0.21 ‡ 6.70" 0.53b ‡c d 6.79" 0.43a ‡b ‡c ‡d ‡

Notes. Values are expressed as mean " SD. Comparisons are made between: a with Group I; b with Group II; c with Group U III; d with Group IV; e with Group V. Symbols represent statistical significance: P - 0.05; †P- 0.01; ‡ P- 0.001. AOx, ammonium oxalate; Imp., implanted with zinc.

enzymesᎏGST, GR and G6PD Ž P- 0.001. were found to be decreased in Group II rats, when compared to control animals, with a concomitant fall in the activities of hydroperoxidases Ž P- 0.001.. Treatment with lupeol and betulin restored the levels of various antioxidant enzymes and the glutathione-related enzymes in both kidney and bladder.

DISCUSSION Several types of crystals are able to cause damage to biological membranes. Membrane lipids are particularly susceptible to oxidation not only because of their high polyunsaturated fatty acid content but also because of their association in the cell membrane with enzymic and non-enzymic systems capable of generating free radical species w27x. In the present study, supplementation of ammonium oxalate after implantation of a zinc piece in the urinary bladder led to deposition of calcium oxalate crystals, based on the fact that the zinc piece present in the urinary tract acts as a nidus around which a stone tends to form.

An increased level of oxalate in the tissues has been reported to cause lipid peroxidation. Similarly, the kidney and bladder of the lithogenic rats ŽGroup II. exhibited an increased level of lipid peroxides measured as TBA reactive substances ŽTable I.. Upon treatment with lupeol and betulin ŽGroups V and VI., the level of lipid peroxides were found to be restricted. This might be attributed to the ability of these triterpenes to reduce the level of oxalate supersaturation in the tissues, by way of their diuretic activity which has been already documented w28x. Apart from reducing oxalate supersaturation, they might also render cytoprotection against tissue injury, since various other triterpenes like oleanolic acid and ursolic acid have been reported to be cytoprotective, thereby rendering protection against free radical-induced derangements w29x. Ascorbic acid has been shown to promote non-enzymatic lipid peroxidation and it was also observed that oxalate along with FeSO4 , produced a highly significant increase in malondialdehyde, the end product of lipid peroxidation w30x. Treatment of the tissue homogenates of the calculogenic group ŽGroup II. with ascorbate and FeSO4 in ¨ itro, brought to light their increased susceptibility to peroxidative changes. At the same time, the groups treated with

Table II Effect of lupeol and betulin on non-enzymic antioxidant status in kidney of control and experimental animals Parameters Group I (␮ g mg Sham control proteiny 1) GSH TSH NPSH Vitamin C Vitamin E

4.68" 0.30 12.25" 0.90 3.26" 0.12 2.13" 0.05 0.48" 0.01

Group II Imp.q AOx

Group III Shamq lupeol

Group IV Shamq betulin

Group V Imp.q AOx q lupeol

Group VI Imp.q AOxq betulin

3.10" 0.13a ‡ 4.68" 0.36b ‡ 4.63" 0.43b ‡ 4.56" 0.35b ‡ 4.18" 0.40b ‡ U U U a b b b 7.02" 0.35 ‡ 12.31" 0.93 ‡ 12.36" 0.77 ‡ 11.02" 0.84 ‡ 10.79" 0.83a b ‡c d a b b a b c d a b c d 1.91" 0.09 ‡ 3.21" 0.10 ‡ 3.30" 0.08 ‡ 2.91" 0.08 ‡ ‡ ‡ ‡ 2.83" 0.12 ‡ ‡ ‡ ‡ U U 1.06" 0.08a ‡ 2.13" 0.09b ‡ 2.05" 0.06b ‡ 1.97" 0.12a b ‡c 1.93" 0.09a †b ‡c † U a b b cU b c 0.34" 0.02 ‡ 0.51" 0.01 ‡ 0.47" 0.01 ‡ 0.45" 0.02 ‡ ‡ 0.42" 0.03a ‡b ‡c ‡d ‡e

Notes. Values are expressed as mean " SD. Comparisons are made between: a with Group I; b with Group II; c with Group III; d with Group IV; e with Group V. Symbols represent statistical significance: U P - 0.05; †P- 0.01; ‡ P - 0.001. AOx, ammonium oxalate; Imp., implanted with zinc.

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Table III Effect of triterpene supplementation on non-enzymic antioxidant status in bladder of control and experimental animals Parameters (␮ g mg proteiny 1)

Group I Sham control

Group II Imp.q AOx

Group III Shamq lupeol

Group IV Shamq betulin

Group V Imp.q AOxq lupeol

Group VI Imp.q AOxq betulin

GSH TSH NPSH Vitamin C Vitamin E

3.46" 0.18 4.77" 0.16 2.04" 0.12 0.72" 0.02 1.06" 0.05

2.05" 0.19a ‡ 2.86" 0.12a ‡ 1.02" 0.08a ‡ 0.38" 0.03a ‡ 0.71" 0.04a ‡

3.37" 0.20b ‡ 4.63" 0.10b ‡ 2.12" 0.08b ‡ 0.78" 0.03b ‡ 1.04" 0.06b ‡

3.51" 0.31b ‡ 4.67" 0.19b ‡ 2.07" 0.13b ‡ 0.76" 0.02b ‡ 1.02" 0.04b ‡

3.25" 0.23b ‡ 4.11" 0.30a ‡b ‡c ‡d ‡ 1.62" 0.05a ‡b ‡c ‡d ‡ 0.70" 0.04b ‡c † 0.88" 0.05a ‡b ‡c ‡d †

3.08" 0.13a b ‡d 3.98" 0.19a ‡b ‡c ‡d ‡ 1.67" 0.08a ‡b ‡c ‡d ‡ 0.65" 0.01b ‡c ‡d † U U 0.94" 0.07a b ‡c

U

U

Notes. Values are expressed as mean " SD. Comparisons are made between: a with Group I; b with Group II; c with Group III; d with Group IV; e with Group V. Symbols represent statistical significance: U P- 0.05; †P- 0.01 and ‡ P- 0.001. AOx, ammonium oxalate; Imp., implanted with zinc.

the triterpenes, were found to be less susceptible to oxidative assault. Though there is induction of lipid peroxidation by the prooxidants under different pathological conditions, the level of LPO in cells is controlled by various cellular defence mechanisms consisting of enzymatic and non-enzymatic scavenger systems w31x. The physiological defence strategy appears to be a

complex process involving a large number of components. These cellular antioxidants are armoured with the capacity to deal with the reactive radical species produced by normal metabolic processes w32x. Their ability to render protection against the oxidative insults encountered during various pathological conditions is limited. In the present study, the level of glutathione

Table IV Alterations in the activities of tissue antioxidant enzymes in calculous conditions and the effect of lupeol and betulin Enzymes Group I (Units miny 1 Sham control mg proteiny 1)

Group II Imp.q AOx

Group III Shamq lupeol

Group IV Shamq betulin

Group V Imp.q AOxq lupeol

Kidney SOD GPx CAT

6.54" 0.51 3.24" 0.13a ‡ 6.37" 0.39b ‡ 6.44" 0.25b ‡ 6.34" 0.52b ‡ 10.30" 0.92 6.14" 0.52a ‡ 10.11" 0.64b ‡ 9.88" 0.74b ‡ 9.57" 0.55b ‡ b b 170.11" 14.08 103.44" 6.74 154.63" 13.19 ‡ 161.38" 10.55 ‡ 151.13" 12.68b ‡

Bladder SOD GPx CAT

7.07" 0.54 5.86" 0.50 139.11" 9.79

Group VI Imp.q AOxq betulin 6.00" 0.42b ‡ 9.18" 0.49b ‡ 150.43" 11.75b ‡

3.07" 0.12a ‡ 6.84" 0.57b ‡ 6.80" 0.40b ‡ 5.95" 0.45a †b ‡c †d † 5.62" 0.31a ‡b ‡c ‡d ‡ U a b b 2.87" 0.24 ‡ 5.98" 0.33 ‡ 5.51" 0.27 ‡ 4.88" 0.42a ‡b ‡c ‡d 4.96" 0.32a †b ‡c ‡ U a b b b 83.39" 4.53 ‡ 131.48" 10.02 ‡ 142.80" 5.91 ‡ 134.94" 4.92 ‡ 121.92" 8.55a †b ‡d ‡e

Notes. Enzyme units are expressed as: SOD, the amount of enzyme required to bring about 50% inhibition of autoxidation of pyrogallol; GPx, ␮ mol of GSH consumed; CAT, ␮ mol of H 2 O 2 consumed. Values are expressed as mean " SD. Comparisons are made between: a with Group I; b with Group II; c with Group III; d with Group IV; e with Group V. Symbols represent statistical significane. U P- 0.05; †P- 0.001; ‡ P- 0.001. AOx, ammonium oxalate; Imp., implanted with zinc. Table V Effect of lupeol and betulin on some glutathione-related enzymes in the tissues of control and experimental animals Enzymes Group I (Units miny 1 Sham control mg proteiny 1) Kidney GST GR G6PD Bladder GS GR G6PD

Group II Imp.q AOx

Group III Shamq lupeol

23.81" 1.13 17.93" 1.50a ‡ 25.69" 1.33b ‡ 3.48" 0.08 2.01" 0.08a ‡ 3.52" 0.10b ‡ 2.10" 0.09 1.04" 0.07a ‡ 1.99" 0.11b ‡ 7.82" 0.13 1.87" 0.04 1.66" 0.07

5.74" 0.42a ‡ 7.58" 0.36b ‡ 0.91" 0.05a ‡ 2.01" 0.09a †b ‡ 0.45" 0.02a ‡ 1.59" 0.06b ‡

Group IV Shamq betulin

Group V Imp.q AOxq lupeol

23.40" 1.43b ‡ 19.02" 1.20a ‡c ‡d ‡ 3.46" 0.07b ‡ 3.35" 0.12b ‡ U 2.05" 0.14b ‡ 1.87" 0.06a †b ‡d 7.78" 0.12b ‡ 1.92" 0.03b ‡ 1.63" 0.06b ‡

U

Group VI Imp.q AOxq betulin 21.85" 1.75b ‡c ‡e † 3.25" 0.15a †b ‡c †d † U 1.81" 0.08a ‡b ‡c d †

U

7.20" 0.29a b ‡d 6.98" 0.32a ‡b ‡c †d ‡ b cU 1.88" 0.08 ‡ 1.73" 0.04a †b ‡c ‡d ‡e † 1.42" 0.09a ‡b ‡c †d ‡ 1.32" 0.08a ‡b ‡c ‡d ‡

Notes. Enzyme units are expressed as: GST, nmol of CDNB᎐GSH conjugate formed; GR, nmol of NADPH oxidized; G6PD, nmol of NADPH formed. Values are expressed as mean " SD. Comparisons are made between: a with Group I; b with Group II; c with Group III; d with Group IV; e with Group V. Symbols represent statistical significance: U P- 0.05; †P- 0.01; ‡ P - 0.001. AOx, ammonium oxalate; Imp., implanted with zinc.

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ŽGSH., an important reducing power capable of reducing oxidized tissue components was found to be diminished in calculogenic rats ŽTables II and III.. This might be attributed to the increased utilization of GSH during oxalate-induced oxidative stress. Although a causal relationship is not yet established, there is an inverse correlation between the lower content of GSH and higher level of peroxides w33x. Accordingly, in this study, we have found that both lupeol and betulin treatment diminished lipid peroxidation in the tissues and increased GSH levels significantly. Vitamins C and E act synergistically through the interactions between water and lipid-soluble substances by both non-enzymatic and enzymatic mechanisms to confer protection in tissues and membranes against oxidative damage w34x. As seen from Table II the levels of these compounds were also found to be reduced drastically in the tissues of calculogenic rats ŽGroup II.. The loss of these vitamins virtually depict the extent of potential peroxidative assault caused by increased concentration of oxalate in the system. The tissues of animals treated with the triterpenes showed significantly higher levels of these vitamins along with GSH, which exhibit the protective effect of both lupeol and betulin against calcium oxalate-induced tissue damage. Physiologically, the superoxide dismutases ŽSOD. and the glutathione system are the primary contributors of the cellular antioxidant capacity w35x. SOD which is responsible for dismutation of reactive radicals was found to be significantly lowered in the tissues of rats given AOx after zinc implantation ŽTable IV.. Such a change may be attributed to the oxidative assault rendered by increased oxalate content in the tissues w36x. In the group of animals supplemented with the triterpenes, its activity was found to be markedly increased when compared with the kidney and bladder of lithogenic Žgroup II. animals. The activity of glutathione peroxidase ŽGPx., the first line of defence against membrane damaging peroxidative assault was found to be inhibited in both the kidneys and bladders of calculogenic rats. Kidneys, being the most vulnerable tissue to damage by lipid peroxides, has been shown to exhibit drastic alteration in the antioxidant capacity during peroxidative changes. Inhibition of GPx, which disposes of cellular H 2 O 2 by utilizing GSH as the co-factor, might be due to the depletion of GSH along with the high degree of peroxides being formed w37x. Catalase, which also detoxifies H 2 O 2 was found to be low in the tissues of stone-forming animals. As a major by-product of these enzymatic and non-enzymatic antioxidative reactions GSH is transformed to its oxidized form ŽGSSG., which is cytotoxic. It may be reduced back to GSH by glutathione reductase ŽGR. in the presence of NADPH w38x. In the present study, GR and G6PD, both of which are

417

responsible for the regeneration of GSH, were found to be inhibited ŽTable V. in the tissues of stone-forming rats ŽGroup II.. This might be attributed to the excessive production of GSSG, which fails to match the capacity of GR and thereby G-6-PD, to reduce it. Treatment with the triterpenes restored the activities of these enzymes towards normalcy, which is indicative of reduced oxidative stress. Since oxalate itself has been reported to incite tissue damage, the antioxidant protective mechanisms of cells and tissues are disturbed when oxalate-induced oxidative stress evade or overwhelm the cellular balance of pro- and antioxidants. Hence, under such conditions control over the urinary concentration of oxalate and further crystallization process seems to be the only way out. The two triterpenes lupeol and betulin, are capable of achieving this objective by way of increasing the urine volume, the mechanism of which is still to be explored. Apart from reducing urinary supersaturation of oxalate, they might also have a certain amount of cytoprotective action. It is evident from the present study that both triterpenes offer remarkable protection against lipid peroxidation, in ¨ i¨ o and also render the tissues less susceptible to lipid peroxidation when exposed to prooxidants like ascorbate and ferrous sulphate in ¨ itro. Various triterpenes like oleanolic acid, ursolic acid w10x and celastrol w39x which possess structural resemblance to lupeol and betulin, have been reported to exhibit protection against peroxidative changes by imparting cellular membrane stability. Mitigation of LPO in turn rectifies the stress on the antioxidant potency, which leads to normalization of non-enzymic antioxidant content and the activities of different antioxidant enzymes in the kidney and bladder of lithogenic rats. The mechanism of antilithic activity by these two compounds may involve the inhibition of oxalateinduced toxic manifestations and free radical production along with enhancement of the body defence systems. However, the exact role of these triterpenes in this line along with their influence on the various promotors and inhibitors of calculogenesis warrants further investigations.

ACKNOWLEDGEMENTS The work was supported by CSIR, India, Grant No. 27 Ž0066.r95rEMR-II, awarded to Prof. P. Varalakshmi, Department of Biochemistry. Dr ALM PGIBMS, Chennai, India.

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