Original Article J. Clin. Biochem.Nutr., 34, 43-47, 2003

Potent Antiulcerogenic Activity of Methanol Extract of quadrangularis by Antioxidative Mechanism

Mallika Jainu and C.S. Shyamala Devi*

Departmentof Biochemistryand MolecularBiology, University of Madras, Guindy Campus,Chennai 600 025,

Received8 May, 2003; Accepted14 October,2003

Summary Oxidative stress is considered to be one of the important etiological factors in various diseases including gastric ulcers. The mechanism of aspirin induced gastric lesion is mediated through lipid peroxidation. The effects of Cissus quadrangularis extract on aspirin- induced ulceration in rats with respect to its antioxidant status, lipid peroxidation, quantity of mucus and alkaline phosphatase and myeloperoxidase (MPO) activities was estimated. Cissus quadrangularis facilitated the healing of experimental gastric ulcer by increased mucus content, antioxidant defense enzyme activity and decreased MPO activity and lipid peroxides levels in the gastric mucosa. These results indicate that Cissus quadrangularis extract protects against aspirin-induced gastric mucosal lesions in rats possibly through its antioxidative action.

Key Words:aspirin, Cissus quadrangularis, gastric mucosal lesion, antioxidant status

ties. In Indian traditional medicine, the has Introduction also been used to cure dyspepsia, eye disease, piles, fracture swelling, wounds, burns, spleen disorder, Free radicals have aroused significant interest asthma, 'vatta' and 'kapha' conditions [6]. The anti- among scientists in the past decade [1]. Oxygen- ulcerogenic effect of Cissus quadrangularis against derived free radicals, which play an important role in gastric ulcer model in rats has been reported [7]. the pathogenesis of peptic ulcer apart from the inter- Although Cissusquadrangularis has been investigated active processes, like many other tissue degenerative for its fracture healing [8] and analgesic properties situations [2]. In the recent years there has been con- [9], detailed studies on its antioxidativepotential and siderable interest in natural products with antioxi- lipid peroxidation level in ulcerated rat are still lack- dants of plant origin, which have been identified and ing. Among the various factors that lead to initiation used as effective protective agents against oxidative of peptic ulcer, alcohol abuse, acute and chronic stress [3]. stress and prolonged use of non-steroidal inflamma- Most of the synthetic compounds given for gastric tory drug (NSAID) are the major causative ones, ulcer contribute for various undesirable side effects apart from the much controversialHelicobacter pylori or drug interaction [4]. There are many antiulcero- infection [10]. Reactive oxygen species (ROS) may genic , which might provide useful sources for play an important role in the pathophysiology of the development of drugs, in the treatment of ulcer acute ulceration induced by NSAIDs, the treatment [5]. Cissus quadrangularis Linn. () exhibits with potent scavengers of free radicals conferred wide range of beneficial and pharmacological proper- mucosal protection, and significantly reduced the severity of these lesions [11]. Further, we thought * To whom correspondence sho uld be addressed. that it would be worthwhile to evaluate the antiulcer Tel: +044-24412575 effect of Cissus quadrangularis against aspirin- E-mail:[email protected] induced ulcer, in terms of its antioxidative activity.

43 44 M. Jainu and C. S. Shyamala Devi

Materials and Methods Group III: Rats given Cissus quadrangularis extract (500mg/kg/day, orally) for 15 days Plant material Group IV: Pretreatment with Cissus quadrangu- Stem part of Cissus quadrangularis was collected laris (500mg/kg/day orally for 15 from Native Care and Cure Centre, India. The fresh days) and then oral administration of air-dried materials were powdered in a mechanical aspirin (400mg/kg) grinder. Crude powder was then soaked in methanol For estimation of biochemical parameters, gastric with intermittent shaking. The filtrate was collected mucosal tissue was taken from the antral portion of and used for extraction using Soxhlet apparatus. The the stomach.The surface gastric mucus was removed residual solvent was removed under vacuum drier by scraping the mucus with glass slide and it was and the solid brown mass obtained was stored at immediately homogenized in 4ml of distilled water. -4℃ for further use . The extract was dissolved in The mucus weight (mg) was obtained from the dif- distilled water and used in the present study. ference between the weight of homogenate and that of water [13]. Total proteins were determined by the Animals method of Lowry et al. [14]. Alkaline phosphatase Male albino rats of weight 175-200g were pur- (ALP) activity was measured using the method of chased from Tamil Nadu University of Veterinary Kind and King [15]. The changes in the activities of and Animal Sciences, Chennai. The animals were gastric mucosal myeloperoxidase(MPO) an index of housed in polypropylene cages maintained at tissue neutrophil infiltration were measured [16]. 27±2℃ in temperature, 55% in humidity, and a 12- Lipid peroxidation was estimated in terms of h light /12-h dark cycle.They were fed standard lab- thiobarbituric acid reactive substances (TBARS), oratory chow (Hindustan Lever Foods, Bangalore, using malondialdehyde (MDA) [11 as standard. India) and provided with water ad libitum.The study Superoxide dismutase (SOD) and catalase (CAT) was conducted after obtaining animal ethical com- activities were determined by the method of Misra mittee clearance (360/01/a/CPSEA dated 19-1- and Fridovich [18] and Luck [19], respectively. 2001). Reduced glutathione (GSH) content was determined after deproteinization by the method of Beautler and Dosagefixation Kelley [20]. Glutathione peroxidase (GPX) and glu- Normal rats were treated with 250, 500, and tathione-S-transferase (GST) were assayed by the 1,000mg/kg of Cissusquadrangularis extract respec- method of Paglia and Valentaina [21] and Habig et tively for 3, 7 and 15 days and 1h prior to ulcer al. [22]. induction. On 3rd, 7th and 15th day the rats were challenged with a single dose of 400mg/kg of aspi- Statistical analysis rin administered orally.The rats were killed after 4h The results were presented as the mean±SD. Stu- post-aspirin exposure for determination of gastric dent's t-test was used to analyze statistical signifi- lesions in gastric glandular portion under micro- cance in various groups of animals. scope. The area of mucosal damage of glandular stomach was calculated in square mm [12].This pre- Results liminary study suggested that Cissusquadrangularis at a dose of 500mg/kg given for 15 days appear to be The methanolic extract of Cissus quadrangularis more effective in preventing aspirin-induced gastric exhibited significant healing effect on NSAID- lesions. Therefore, this dose was chosen as optimum induced gastric ulcer, as evidenced from various bio- dosage for further studies. chemical parameters. Oral administration of the extract showed reduction in the ulcer index and Experimental methods healed at a faster rate with in 15 days. Rats were divided into 4 groups of 6 animals each Figure 1 shows the effect of Cissus quadrangularis as follows: Animals were fasted for 24h provided on mucus content, MPO and ALP activities in aspi- with water ad libitum. rin-induced ulcer. Pretreatment with Cissus quadran- Group I: Normal control gularis significantly (p<0.001) enhanced the mucus Group II: Rats given oral administration of aspi- content in ulcer induced rats and prevented the rin (400mg/kg) increase in gastric mucosal MPO and ALP activities.

J. Clin. Biochem.Nutr. Antioxidant Activity of Cissus quadrangularis on Ulcer Healing 45

Fig. 1. Effect of Cissus quadrangularis on gastric mucus content, alkaline phosphatase and myloperoxidase activity in exper- imental groups. Group I, normal control; Group II, aspirin induced; Group III, Cissus quadrangularis treated;

Group IV, Cissus guadrangularis+aspirin. Values are expressed as mean±SD for 6 animals in each group. *p<0.001 as compared with Group I; @p<0.001 as compared with Group II; NS, non-significant as compared with Group I.

Table 1. Effect of Cissus quadrangularis on the antioxidative status in the gastric mucosa of experimental groups.

Values are mean±SD of 6 animals in each group. *p<0.001 as compared with Group I; **p<0.001 as compared with Group II; NS, non-significant as compared with Group I.

Activities of antioxidative enzymes and concentra- with that of control rats (Group I). In Cissus qua- tion of TBARS are presented in Table 1. Levels of drangularis pretreated rats (Group IV), the level of GSH, SOD, CAT, GPX, and GST showed a signif- TBARS and antioxidative defense enzymes regis- icant decline (p<0.001) and increase in LPO con- tered a near normal values when compared with tent in aspirin administered rats, when compared ulcerated animals.

Vol.34, No.2, 2003 46 M. Jainu and C. S. Shyamala Devi

Discussion the activities of SOD, CAT, GPX, and GST and in GSH content in the extract-treated group. The efforts of scientists to understand how free Reactive oxygen species production may damage radicals cause destruction as well as how antioxidants cellular components; this damage, in part, is neutral- protect cells from damage could provide clues to ized by mucosal antioxidant (ascorbic acid and β- treat or prevent disease [23]. The oxygen-derived carotene), which are good scavengers of free radicals free radicals in the process of aspirin induced injury [33]. Phytochemical studies carried out on Cissus may arise from a blockade of the physiologicalfunc- guadrangularis revealed the presence of β-carotene, tion of prostaglandin E2 and thromboxane A2 and in ascorbic acid, ketosteroids, triterpenoids [34], calci- this way remove this cytoprotective effect; thus, the um, β-amyrin, and α-amyrin [35]. The observed induction of ulcers is promoted [24]. cytoprotective and antioxidative effect of Cissus qua- Pretreatment with Cissus quadrangularis extract drangularis is attributed to the presence of these anti- (500mg/kg) given for 15 days resulted in healing of oxidative phytoconstituents. aspirin-induced gastric lesions. Gastric mucus is In conclusion, it can be said that a possible healing believed to play an important role in the defensive action of Cissus quadrangularis could be due to anti- mechanism against gastric ulceration [25]. Oral pre- oxidative mechanism. Further studies are in progress treatment with the extract, although preventing to investigate the detailed mechanism of action of ulceration, originated a limited increase in the quan- this potent herbal extract before clinical trials. tity of gastric mucus. The release of ALP has been suggested to play a References role in tissue necrosis associatedwith various models of gastrointestinalulceration [26].The extract signif- [1] Aruoma, O.I. and Guppet, S.L.: Antioxidant Method- ology in vivo and in vitro Concepts, AOCS Press, icantly reduced the ALP activity when compared Champaign, IL, pp.41-172, 1997. with the aspirin control. MPO activity is used as an [2] Phian, G., Regillo,C., and Szabo, S.: Free radical and index of granulocyte content/infiltration in tissues lipid peroxidationin ethanol or aspirin induced gastric [27]. The previous studies indicate that aspirin can mucosalinjury. Dig. Dis. Sci.,32, 1395-1401, 1987. promote increase in neutrophil adhesion to venular [3] Shukla Mukherjee Sur, A. and Maid, B.R.: Hepato- endothelium in vivo [28]. In addition, it has been protective effect of Swertia chirata on rat. Indian J. reported that neutrophils mediate lipid peroxidation Exp. Biol.,35, 384-388, 1997. through the production of superoxide anions by the [4] Prakash, A. and Faulds, D.: Rebaprazole.Drugs, 2, 55-56, 1998. activated NADPH oxidoreductase in the cells [29]. [5] Akthar, H. H. and Ahmed, K.U.: Antiulcerogeniceval- TBARS and tissues associated MPO activity were uation of the methanolic extract of some indegenous measured in gastric mucosa as indices of lipid perox- medicinalplants of Pakistan in aspirinulcerated rats. J. idation and neutrophil infiltration [30]. The increase Ethnopharmacol.,46, 1-6, 1998. in TBARS and MPO activity after aspirin adminis- [6] Kritikar, K.R. and Basu, B.D.: Indian Medicinal tration was significantly inhibited by treatment with Plants (Third Revisedand Enlarged ed.), ed. by Basu, Cissusquadrangularis. This suggests that Cissusqua- L.M., Allahabad,Vol.3, pp.841-843, 2000. drangularis may promote ulcer healing through addi- [7] Anoop, A. and Jagdeesan,M.: Gastric and duodenal antiulcer and cytoprotectiveeffect of Cissusquadrangu- tional mechanisms. laris Linn. variant II in rat. Nig.J. Nat. Prod.Med., 6, In the current study, the TBARS concentration 1-7, 2002. was significantly reduced and antioxidative defence [8] Deka, D. K., Lahon, L. C., Saikia, J., and Mukit, A.: enzyme level increased in the Cissusquadrangularis- Effect of Cissus quadrangularis in acceleration healing treated groups compared with the aspirin induced process of experimentally fractured radius-ulna of dog; group. This indicates that Cissusquadrangularis con- A experimental study. Indian J. Pharmacol., 26, 44-45, fers an equivalent anti-lipid peroxidation effect on 1994. the gastric tissue. Body et al. suggested that GSH [9] Singh, S.P., Misra, N., Dixit, K. S., Singh, N., and levels were found to be decreasedin gastric ulcer tis- Kohli, R. P.: An experimental study of analgesic activity of Cissus quadrangularis. Indian J Pharmacol., 2, 162- sue [31]. Depletion of GSH will rendered enzymes 263, 1984. (GPX and GST) inactive or less active. SOD con- [10] Biswajit, M., Ray Chaudhuri, S.G., Arun, R., and tent in gastric tissue is a point of controversy [32]. Sandip Bandyopadhyay, K.: Effect of ethanol extract of The protective action may be, due to the increase in Piper betle Linn. leaf on healing of NSAID-induced

J. Clin. Biochem.Nutr. Antioxidant Activity of Cissus quadrangularis on Ulcer Healing 47

experimentalulcer-A novel role of free radical scav- [25] Martin, M. J., Alarcon de la Lastra, C., and Motilva, enging action.Indian J. Exp. Biol.,41, 311-315, 2003. V.: Bases fisiopatologicas farmacologicas de la ulcera [11] Sata, N., Kawano, S., Tsuji, S., and Kamada, T.: peptica, Secretariado de publications, Universidad de Microvascularbasis of gastric mucosal protection. Sevilla Press, Sevilla, pp.25-30, 1993. Clin. Gastroenterol.,10, S13-S18, 1988. [26] Obi, E., Meh, J. K., Orisakwe, O. E., Afonne, O. J., [12] Szabo, S., Trier, J.S., and Brown, A.: A quantitative Ilondu, N. A., and Agbasi, P.U.: Investigation of the method for assessingthe extent of experimentalgastric biochemical evidence for the antiulcerogenic activity of erosions and ulcer.J. Pharmacol.Methods, 13, 59-66, Synclisia scarbrida. Indian J. Pharmacol., 32, 381-383, 1985. 2000. [13] Martin, M.J., Marhuenda, E., and Alarcon, L.: Escu- [27] Krawisz, J. F., Sharan, P., and Stenson, W. F.: Quantita- line, ranitidine and carbenoxolonedifferent modes of tive assay for acute intestinal inflammation based on action on gastric mucosa. Gen.Pharmacol., 22, 1001 MPO activity. Assessment of inflammation in rat and 1004, 1991. hamster models. Gastroenterology, 87, 1344-1350, [14] Lowry, O.H., Rosebrough,N. J., and Fars, A. L.: Pro- 1984. tein measurement with folin phenol reagent. J. Biol. [28] Yoshida, N., Takemura, T., Granger, D. N., Anderson, Chem.,193, 265-278, 1951. D. C., Wolf, R.E., Mclntive, L. V., and Kvietys, P.R.: [15] Kind, P.R. N. and King, E.J.: Determination of serum Molecular determinations of aspirin-induced neutro- alkalinephosphatase. Clin. Pathol.,7, 322-326, 1954. phil adherence to endothelial cells. Gastroenterology, [16] Suzuki, K., Sasagawa,S., Sakatani, T., and Fujikura, 105, 715-724, 1993. T.: Assay method for myeloperoxidasein human poly- [29] Zimmerman, J.J., Ceisielski, W., and Lewandoski, J.: morphonuclear leukocytes.Anal. Biochem.,132, 345 Neutrophil-mediated phospholipid peroxidation 353, 1983. assessed chromatography-mass spectroscopy. Am. J. [17] Ohkawa, H., Ohishi, N., and Yagi, K.: Assay for lipid Physiol.,273, C653-661, 1997. peroxides in animal tissues by thiobarbituric acid reac- [30] Naito, T., Takagi, T., Matsuyama, K., Yoshida, N., and tion. Anal. Biochem.,95, 351-358, 1979. Yoshikawa, T.: Pioglitazone, a specific PPAR-γ ligand [18] Misra, H. P. and Fridovich, I.: The role of superoxide inhibits aspirin-inducedgastric mucosal injury in rats. anion in the autooxidation of epinephine and a simple Aliment.Pharmacol. Ther., 15, 865-873, 2001. assay for SOD. J. Biol. Chem., 247, 3170-3175, 1972. [31] Body, S.C., Samasa, H. A., and Body, M. R.: Gastric [19] Luck, H.: Catalase, in Methods of Enzymatic Analy- glutathionedepletion and acute ulcerogenesisby dieth- sis, ed. by Bergmeyer, H. U., Academic Press, New ylamaleate given subcutaneouslyin rats. Life Sci., 28, York, London, pp. 885-888, 1963. 2987-2992, 1981. [20] Beautler, E. and Kelley, B.M.: The effect of sodium [32] Mahendran, P. and Shyamala Devi, C. S.: The modu- nitrate on red cell glutathione. Experientia, 19, 96-97, lating effect of Garciniacombogia extracts on ethanol 1963. induced peroxidativedamage in rats. IndianJ. Pharma- [21] Paglia, D. E. and Valentaina, W. N.: Studies on the col.,33, 87-91, 2001. GSH and Glutathione characterization of erythrocyte [33] Daneses, S., Candelli,M., Cremonini,F., and Stevens, GPX. J. Lab. Clin. Med., 70, 158-169, 1967. T. R.: High oxygenfree radicals generation in gastric [22] Habig, W. H., Pabst, M. J., and Jakoby, W. B.: Glu- mucosa infected by H. pylori cytotoxic strains tathione-S-transferase. The first enzymatic step in (abstract).Gastroenterology, 118, A739-A743. mercapturic acid formation. J. Biol. Chem., 240, [34] Madan, M. G. and Ram, K.V.: Unsymmetric tetracy- 71130-71139, 1974. clic triterpenoid from Cissusquadrangularis. Phytochem- [23] Pillai, K. S. and Pillai, C.: Antioxidant in health. istry,29, 336-337, 1990. Indian J. Physiol.Pharmacol., 46, 1-5, 2002. [35] Udupa, K.N., Prasad, G., and Sen, S.P.: The effect of [24] Steinmeyer, J.: Pharmacological basis for the therapy phytogenicanabolic steroid in the accelerationof frac- of pain and inflammation with NSAIDs. Arthritis Res., ture repair.Life Sci.,4, 317-327, 1965. 2, 379-385, 2000.

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