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Protective effect of Cissus quadrangularis on neutrophil mediated tissue injury induced by aspirin in rats
Journal of Ethnopharmacology 104 (2006) 302–305
Protective effect of Cissus quadrangularis on neutrophil mediated tissue injury induced by aspirin in rats Mallika Jainu, K. Vijai Mohan, C.S. Shyamala Devi ∗ Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India Received 13 July 2004; received in revised form 4 August 2005; accepted 4 August 2005 Available online 9 December 2005
Abstract Cissus quadrangularis (family: Vitaceae) is well known for the treatment of gastric disorders in traditional medicine, owing to its rich source of carotenoids, triterpenoids and ascorbic acid, and has received considerable attention regarding its role in human nutrition. In the search of new potential antiulcer agents, the present study evaluated the ethanol extract of Cissus quadrangularis (CQE) against the gastric toxicity induced by aspirin in rats. The optimum protective dose of 500 mg/kg of extract was selected by the pretreatment of gastric ulcers with different doses of CQE (250, 500 and 750 mg/kg) for 7 days which showed ulcer protection by 40, 71.2 and 72.6%, respectively, as compared to ranitidine (RTD) (30 mg/kg) by 71.9% in the aspirin model. In addition, results have shown that administration of aspirin increases lipid peroxidation status, xanthine oxidase (XO), myeloperoxidase and decrease in selenium–glutathione peroxidase activities in the gastric mucosa, resulting in mucosal damage at both cellular and subcellular level. Pretreatment with CQE ameliorated the observed effect significantly in the gastric mucosa of ulcerated rats. These findings suggest that the gastroprotective activity of CQE could be mediated possibly through its antioxidant effect as well as by the attenuation of the oxidative mechanism and neutrophil infiltration. © 2005 Published by Elsevier Ireland Ltd. Keywords: Aspirin; Cissus quadrangularis; Ulcer lesions; Lipid peroxidation
1. Introduction In recent times, many medicinal plants continue to provide valuable therapeutic agents for the treatment of ulcers both in modern medicine and by the traditional system throughout the world. Since chemical compounds are known to have undesirable side-effects, the present study focused on natural products. 1.1. Plant Cissus quadrangularis Linn. Wall. Ex. Wight (family: Vitaceae) is an edible plant, commonly known as “bone setter” found in hotter parts of India, Ceylon, East Africa and Malaysia and Thailand. Cissus quadrangularis is used as a common food supplement in southern India. Stem parts of Cissus quadrangu∗ Corresponding author. Present address: 66, White House, II Main road, Gandhi Nagar, Adyar, Chennai 600020, Tamil Nadu, India. Tel.: +91 44 24412575; fax: +91 44 22352494. E-mail address: [email protected] (C.S.S. Devi).
0378-8741/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2005.08.076
laris were collected from Native Care and Cure Center, India and were duly authenticated by Dr. P. Brindha, Pharmacology Department, Captain Srinivasa Murthy Drug Research Institute, Arumbakkam, Chennai 600106. A voucher specimen PP. 502 has been deposited in the department. 1.2. Uses in traditional medicine The stout fleshy quadrangular stem is traditionally used for the treatment of gastritis, bone fractures, skin infections, constipations, eye diseases, piles, anemia, asthma, irregular menstruation, burns and wounds (Asolkar et al., 1992; Kritikar and Basu, 2000). Cissus quadrangularis has potent fracture healing property, antimicrobial, antiulcer, antioxidative, cholinergic activity and beneficial effect on cardiovascular diseases (Udupa and Prasad, 1964; Subbu, 1970; Anoop and Jagdeesan, 2002; Murthy et al., 2003; Jainu and Devi, 2003). Previously, we have demonstrated that methanolic extract of Cissus quadrangularis possesses antiulcer and cytoprotective property in indomethacin-induced gastric mucosal injury (Jainu and Devi,
M. Jainu et al. / Journal of Ethnopharmacology 104 (2006) 302–305
2004). Due to its widespread health use and pharmacologic actions, this study will highlight the health promoting and therapeutic effects of Cissus quadrangularis. 1.3. Previously isolated class of constituents The phytochemical analysis of the plant showed the presence of Vitamin C, -carotene, two asymmetric tetracyclic triterpenoids, -sitosterol, ␣-amyrin, ␣-amyrone and three stillbene derivatives, quadrangularins A, B, C, etc. (Chopra et al., 1956; Attawish et al., 2002). Some experimental studies have demonstrated that oxygengenerated free radicals derived from infiltrated neutrophils and enhanced lipid peroxidation play important roles in the pathogenesis of acute gastric lesions induced by aspirin (Konturek et al., 1994). Substances that are able to hinder their formation or capture the free oxygen radicals formed are thus potential antiulcerogenic agents. These facts form the basis for a study of whether the antioxidant mechanisms are involved in CQE mediated protection in aspirin-induced gastric damage. The objective of the study is to investigate the effect of CQE on neutrophil infiltration tissue injury induced by aspirin in order to reveal the mechanism underlying the antiulcer effect of the plant. 2. Materials and methods 2.1. Preparation of alcoholic extract Dried parts of Cissus quadrangularis were coarsely powdered and 1 kg of this powdered plant material was soaked in ethanol for 48 h and extracted by soxhlet extraction. The extract was vacuum dried and was stored at −4 ◦ C until further use. The yield of the extract was 5.2% (w/w) of powdered ethanolic extract. For administration, the extract was dissolved in distilled water and used for the present study. 2.2. Animals Male albino rats’ weighing 150–200 g were purchased from Tamil Nadu University of Veterinary and Animal Sciences, India. The animals were housed in polypropylene cages maintained in controlled temperature and light cycle. The animals were fed with food pellets and water was given ad libitum. The experiments were initiated only after the approval of the Institutes of Animal Ethics Committee (No: 360/01/a/CPSEA/ 2001). 2.3. Acute toxicity Adult albino rats of either sex were divided into four groups (n = 6) and orally fed with CQE at dose levels of 0.5, 1.5, 3.0 and 5.0 g/kg. Animals were watched carefully for 72 h after CQE administration and then for the next 7 days. At the end of this experimental period, the rats were observed for signs of toxicity, morphological behaviour and mortality. A separate group of controls received only the vehicle.
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2.4. Induction of aspirin-induced gastric lesions Adult male Wistar rats weighing 120–180 g were used for the experiment. All the animals were fasted for 24 h prior to the experiments. Aspirin at a dose of 400 mg/kg (Kamsiah et al., 2002) was administered to the animals and after 6 h, the animals were sacrificed by cervical dislocation and the stomach was incised along the greater curvature and the area of mucosal damage of glandular stomach was calculated in units of square millimeter (Szabo et al., 1985). 2.5. Dosage fixation A group of animals, which served as control, received only distilled water. The CQE at dose levels of 250, 500 and 750 mg/kg and ranitidine (RTD), the reference drug, in the dose of 30 mg/kg was administered orally for 7 days. A dosedependent antiulcer effect of CQE (250, 500 and 750 mg/kg) was seen on gastric ulcers. Therefore, for the test drug (CQE), an optimum dose of 500 mg/kg was selected for further studies and the percentage of ulcer protection was nearly equipotent to RTD, a standard antiulcer drug. 2.6. Treatment protocol Animals were divided into four groups of six animals in each group. Group 1 animals served as control. They received only distilled water equivalent to the volume of plant extract. Group 2 represented the ulcerated group. Ulceration was produced by the administration of aspirin (400 mg/kg) orally. Group 3 animals were pretreated with the test drug CQE (500 mg/kg) orally, once daily for 7 days and then ulceration was induced by aspirin. After 6 h, the animals were sacrificed, stomach was taken out and the scrapped gastric mucosal tissues were used for the estimation of biochemical enzymes. 2.6.1. Assay of Se-glutathione peroxidase (Se-GSHPx) and xanthine oxidase (XO) For the assays of Se-GSHPx and XO enzymes, gastric mucosal tissues were homogenized in nine volumes of icecold 0.05 M Tris–HCl buffer (pH 7.4). The homogenate was centrifuged at 4 ◦ C (10,000 × g, 20 min) and the resultant supernatent was dialyzed against 100 volumes of the same buffer at 4 ◦ C for 24 h. Gastric mucosal Se-GSHPx enzyme activity was assayed by the methods of Hochstein and Utley (1968). The enzyme activity was determined at 37 ◦ C by recording the decrease in absorbance at 340 nm following the oxidation of NADPH in the presence of H2 O2 , GSH and yeast glutathione reductase. One unit of this activity is defined as the amount of enzyme oxidizing 1 mol of NADPH per min. Gastric mucosal XO was assayed by the method of Hashimoto (1974). Three millilitres of incubation mixture contains 150 M phosphate buffer, 0.2 M xanthine, 0.3 M potassium oxonate and 0.2 ml of tissue homogenate. The tubes were incubated at 30 ◦ C for 30 min, 0.1 ml of 100% of TCA was added and mixed well. The content was centrifuged at 10,000 × g for
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15 min and the supernatant was analysed for XO activity by measuring the increase in absorbance at 292 nm following formation of uric acid at 30 ◦ C. One unit (U) of this enzyme is defined as the amount of enzyme forming 1 mol uric acid/min. The activity of XO was expressed as mU/g tissue. 2.6.2. Assay of lipid peroxidation The LPO product malondialdehyde (MDA) was estimated using 1,1,3,3-tetramethoxypropane as the standard, according to the method described by Ohkawa et al. (1979). To 0.5 ml of tissue homogenate, 1.5 ml of 20% acetic acid, 0.2 ml of SDS and 1.5 ml of TBA were added. The mixture was made up to 4.0 ml with distilled water and then heated for 60 min at 95 ◦ C using glass ball as condenser. After cooling, 4.0 ml of butanol–pyridine mixture was added and shaken well. After centrifugation at 4000 rpm for 10 min, the organic layer was taken and its absorbance was read at 532 nm and the results were expressed as nmol/mg protein. 2.6.3. Assay of myeloperoxidase Gastric mucosal myeloperoxidase (MPO), a marker enzyme of neutrophil infiltration, was assayed by the method of Suzuki et al. (1983). A sample of tissue homogenate (0.5 l) was added to a 0.5 ml of reaction volume containing PBS, pH 5.4, hexadecyl–trimethyl ammonium bromide and tetraethylbenzidine at 37 ◦ C. The reaction was started by the addition of H2 O2 and terminated by the sequential addition of catalase and sodium acetate. One unit of this enzyme activity was defined as the amount of enzyme causing a change in absorbance at 655 nm and the results were expressed as U/mg protein. Protein content was assessed according to the method of Lowry et al. (1951). 2.7. Statistical analysis The data were expressed as mean ± S.E.M. One-way analysis of variance followed by Dunnett’s multiple comparison tests were used to assess statistical significance of differences between groups.
Fig. 1. Effect of Cissus quadrangularis extract (CQE) on Se-glutathione peroxidase (Se-GSHPx) and xanthine oxidase (XO) activities in the aspirin-induced gastric ulcer model. Results are expressed as mean ± S.E.M. of six animals in each group. a P < 0.001 vs. control; b P < 0.001 vs. aspirin group.
750 mg/kg given orally for 7 days. Therefore, an optimal dose of CQE (500 mg/kg) given for 7 days significantly protected the animals against gastric ulcer. Hence, further studies were carried out with the same dose of CQE. As shown in Fig. 1 the increase in XO activity upon aspirin administration was attenuated by pretreatment with CQE at the dose of 500 mg/kg. Our data show that according to gastric mucosal damage, Se-GSHPx activity decreased significantly after aspirin administration when compared to control. CQE pretreatment replenish the Se-GSHPx activity to the levels seen in control rats. Fig. 2 depicts the activity of MPO, as an index of neutrophil infiltration and LPO level in the control and experimental animals. Aspirin induction strongly increased the activity of MPO and LPO levels in comparison with control. While pretreatment with CQE restored these alterations to near-normal levels in ulcerated rats, it was effective against aspirin-induced gastric ulcer.
3. Results Rats that received oral doses of 0.5, 1.5, 3.0 and 5.0 g/kg, did not manifest any clinical signs of toxicity. None of the doses tested could produce mortality in rats during the treatment period. In tests on rats, we found that doses of CQE up to 5 g/kg were non-toxic and we were unable to establish its oral LD50 value. Aspirin administered rats showed multiple gastric mucosal lesions, most often 1–2 mm in size or petechial, bleeding at the moment of the observation. The CQE showed significant antiulcer effect against ulcers induced in the aspirin model in a dose-dependent manner. In ulcerated rats, CQE at dose levels of 250, 500 and 750 mg/kg showed protection indexes of 40.0, 71.2 and 72.6%, respectively, whereas RTD showed 71.9% protection at a dose of 30 mg/kg. The percentage of ulcer protection of CQE at dose of 500 mg/kg was nearly the same as that of 750 mg/kg. There was no significant difference in the ulcer protection indexes of CQE at a dose of 500 mg/kg as compared with
Fig. 2. Effect of Cissus quadrangularis extract (CQE) on gastric mucosal lipid peroxide content (LPO) and myeloperoxidase (MPO) in aspirin administered rats. Results are expressed as mean ± S.E.M. of six animals in each group. a P < 0.001 vs. control; b P < 0.001 vs. aspirin group.
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4. Discussion The results of this study demonstrate that the CQE possesses antiulcer property as evidenced by its significant inhibition in the formation of gastric lesions induced by aspirin. The acute toxicity tests on rats showed that doses of CQE up to 5 g/kg were non-toxic and the oral LD50 value might be higher than this dose. Aspirin caused rise in the lipid peroxidation status possibly due to the activation of neutrophils, which play an important role in the damaging activity (Yoshikawa et al., 1992). The measurement of local enzymatic activity of MPO has been used to quantify neutrophil sequestration in tissues and the severity of inflammation (Nishizawa et al., 1996). The activity of Se-GSHPx, an index of tissue neutrophil infiltration increased, with lesion development in the aspirin administered rats (Krawisz et al., 1984). However, inhibition of MPO and increase in Se-GSHPx activity by CQE may be simple by a reflection of decreased inflammation independent of any direct effect on neutrophils. The gastric ulcer induced by aspirin was almost completely prevented, with attenuation of increased gastric mucosal XO and MPO activities and LPO content, by treatment with CQE, thereby decreasing the neutrophil sequestration at the wound site. Triterpenoids and -sitosterols present in Cissus quadrangularis possess antilipidperoxidative effect (Somova et al., 2003) and thus prevent gastric damage. The plant constituent such as -sitosterol has the ability to reduce the enzyme MPO, indicating a reduction of neutrophil influx in the inflamed tissues (DelaPureta et al., 2000). Murthy et al. (2003) have reported that Cissus quadrangularis possesses antioxidant activity in both the in vitro and in vivo models due to the presence of -carotene in this plant. Previous reports suggested that the healing action of Cissus quadrangularis is due to its antioxidative property (Jainu and Devi, 2003). Thus, the protective role of CQE against neutrophil mediated tissue injury may be due, in part, to a reduction in neutrophil infiltration into the gastric mucosa, via its antioxidant property. In conclusion, the antiulcer efficacy of CQE in gastric ulcer model could be due to inhibition of neutrophil infiltration and antioxidant properties. These properties of CQE merit detailed analysis relating to active principles and the mechanism of action, which might provide new alternatives for the clinical management of gastric ulcer diseases. Acknowledgement The authors would like to thank Dr. P. Brindha for his expert comments and suggestions in regard to this work. References Anoop, A., Jagdeesan, M., 2002. Gastric and duodenal antiulcer and cytoprotective effect of Cissus quadrangularis Linn. variant II in rats. Nigerian Journal of Natural Products and Medicine 6, 1–7.
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Asolkar, L.V., Kakkar, K.K., Chakre, O.J., 1992. Second Supplement to Glossary of Indian Medicinal Plants with Active Principles. CSIR Publication, New Delhi, p. 61. Attawish, A., Chavalttumrong, D., Chivapat, S., Chuthaputti, S., Rattarajarasroj, S., Punyamong, S., 2002. Subchronic toxicity of Cissus quadrangularis Linn. Songklanakarin Journal of Science and Technology 24, 39–51. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. Publication and Information Directorate, CSIR, New Delhi, p. 66. DelaPureta, R., Martinez-Dominguez, E., Ruiz-Gutierrez, V., 2000. Effect of minor components of virgin olive oil on topical anti-inflammatory assays. Zetschrift Fur Naturforschong 55, 814–819. Hashimoto, S., 1974. New spectrophotometric assay method of xanthine oxidase in crude tissue homogenate. Analytical Biochemistry 62, 426–433. Hochstein, P., Utley, H., 1968. Hydrogen peroxide detoxication by glutathione peroxidase and catalase in rat liver homogenate. Analytical Biochemistry 4, 574–579. Jainu, M., Devi, C.S.S., 2003. Potent antiulcerogenic activity of methanolic extract of Cissus quadrangularis by antioxidative mechanism. Journal of Clinical Biochemistry and Nutrition 34, 43–47. Jainu, M., Devi, C.S.S., 2004. Effect of Cissus quadrangularis on gastric mucosal defensive factors in experimentally induced gastric ulcer—a comparative study with sucralfate. Journal of Medicinal Food 7, 372–376. Kamsiah, J., Gapor, M.T., Nafeeza, M.I., Fauzee, A.M., 2002. Effect of various doses of palm Vitamin E and tocopherol on aspirin-induced gastric lesions in rats. International Journal of Experimental Pathology 83, 295–301. Konturek, S.J., Brozozowski, T., Stachura, J., Majka, J., 1994. Role of neutrophils and mucosal blood flow in gastric adaptation to aspirin. European Journal of Pharmacology 253, 107–114. Krawisz, J.E., Sharon, P., Stenson, W.F., 1984. Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models. Gastroenterology 87, 1344–1350. Kritikar, K.R., Basu, B.D., 2000. In: Basu, L.M., (Ed.), Indian Medicinal Plants, Lalit Mohan Basu Publisher, Allahabad, India, pp. 841–843. Lowry, O.H., Rosebrough, N.H., Farr, A.L., Randal, R.I., 1951. Protein measurement with Folin’s-phenol reagent. Journal of Biological Chemistry 193, 265–275. Murthy, K.N.C., Vanitha, A., Swamy, M.M., Ravishankar, G.A., 2003. Antioxidant and antimicrobial activity of Cissus quadrangularis L. Journal of Medicinal Food 6, 99–105. Nishizawa, H., Yamamda, H., Miyazaki, H., Ohara, M., Kaneko, K., Yamakawa, T., Wiener-kronish, J., Kodoh, I., 1996. Soluble complement receptor type I inhibited the systemic organic injury caused by acid instillation into a lung. Anesthiology 85, 1120–1128. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Somova, L.I., Shode, F.O., Ramananan, P., Nadar, A., 2003. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves. Journal of Ethnopharmacology 84, 299–305. Subbu, V.S.V., 1970. Mechanism of action of Vitis glucoside on myocardial tissue. Indian Journal of Medicinal Science 25, 400–403. Suzuki, K., Ota, H., Sasagawa, S., Sakatani, T., Fujikura, T., 1983. Assay method of myeloperoxidase in human polymorphonuclear leukocytes. Analytical Biochemistry 132, 345–353. Szabo, S., Trier, J.S., Brown, A., Schoor, J., 1985. Early vascular injury and increased permeability in gastric mucosal injury caused by ethanol in rat. Gastroenterology 88, 228–236. Udupa, K.N., Prasad, G., 1964. Biochemical Ca45 studies on effect of Cissus quadrangularis in fracture repair. Indian Journal of Medicinal Research 52, 480–487. Yoshikawa, T., Naito, Y., Ueda, S., 1992. Ischemia reperfusion injury and free radical involvement in gastric mucosal disorders. Advanced Experimental Medicinal Biology 316, 231–238.
Protective effect of Cissus quadrangularis on neutrophil mediated tissue injury induced by aspirin in rats Mallika Jainu, K. Vijai Mohan, C.S. Shyamala Devi ∗ Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India Received 13 July 2004; received in revised form 4 August 2005; accepted 4 August 2005 Available online 9 December 2005
Abstract Cissus quadrangularis (family: Vitaceae) is well known for the treatment of gastric disorders in traditional medicine, owing to its rich source of carotenoids, triterpenoids and ascorbic acid, and has received considerable attention regarding its role in human nutrition. In the search of new potential antiulcer agents, the present study evaluated the ethanol extract of Cissus quadrangularis (CQE) against the gastric toxicity induced by aspirin in rats. The optimum protective dose of 500 mg/kg of extract was selected by the pretreatment of gastric ulcers with different doses of CQE (250, 500 and 750 mg/kg) for 7 days which showed ulcer protection by 40, 71.2 and 72.6%, respectively, as compared to ranitidine (RTD) (30 mg/kg) by 71.9% in the aspirin model. In addition, results have shown that administration of aspirin increases lipid peroxidation status, xanthine oxidase (XO), myeloperoxidase and decrease in selenium–glutathione peroxidase activities in the gastric mucosa, resulting in mucosal damage at both cellular and subcellular level. Pretreatment with CQE ameliorated the observed effect significantly in the gastric mucosa of ulcerated rats. These findings suggest that the gastroprotective activity of CQE could be mediated possibly through its antioxidant effect as well as by the attenuation of the oxidative mechanism and neutrophil infiltration. © 2005 Published by Elsevier Ireland Ltd. Keywords: Aspirin; Cissus quadrangularis; Ulcer lesions; Lipid peroxidation
1. Introduction In recent times, many medicinal plants continue to provide valuable therapeutic agents for the treatment of ulcers both in modern medicine and by the traditional system throughout the world. Since chemical compounds are known to have undesirable side-effects, the present study focused on natural products. 1.1. Plant Cissus quadrangularis Linn. Wall. Ex. Wight (family: Vitaceae) is an edible plant, commonly known as “bone setter” found in hotter parts of India, Ceylon, East Africa and Malaysia and Thailand. Cissus quadrangularis is used as a common food supplement in southern India. Stem parts of Cissus quadrangu∗ Corresponding author. Present address: 66, White House, II Main road, Gandhi Nagar, Adyar, Chennai 600020, Tamil Nadu, India. Tel.: +91 44 24412575; fax: +91 44 22352494. E-mail address: [email protected] (C.S.S. Devi).
0378-8741/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2005.08.076
laris were collected from Native Care and Cure Center, India and were duly authenticated by Dr. P. Brindha, Pharmacology Department, Captain Srinivasa Murthy Drug Research Institute, Arumbakkam, Chennai 600106. A voucher specimen PP. 502 has been deposited in the department. 1.2. Uses in traditional medicine The stout fleshy quadrangular stem is traditionally used for the treatment of gastritis, bone fractures, skin infections, constipations, eye diseases, piles, anemia, asthma, irregular menstruation, burns and wounds (Asolkar et al., 1992; Kritikar and Basu, 2000). Cissus quadrangularis has potent fracture healing property, antimicrobial, antiulcer, antioxidative, cholinergic activity and beneficial effect on cardiovascular diseases (Udupa and Prasad, 1964; Subbu, 1970; Anoop and Jagdeesan, 2002; Murthy et al., 2003; Jainu and Devi, 2003). Previously, we have demonstrated that methanolic extract of Cissus quadrangularis possesses antiulcer and cytoprotective property in indomethacin-induced gastric mucosal injury (Jainu and Devi,
M. Jainu et al. / Journal of Ethnopharmacology 104 (2006) 302–305
2004). Due to its widespread health use and pharmacologic actions, this study will highlight the health promoting and therapeutic effects of Cissus quadrangularis. 1.3. Previously isolated class of constituents The phytochemical analysis of the plant showed the presence of Vitamin C, -carotene, two asymmetric tetracyclic triterpenoids, -sitosterol, ␣-amyrin, ␣-amyrone and three stillbene derivatives, quadrangularins A, B, C, etc. (Chopra et al., 1956; Attawish et al., 2002). Some experimental studies have demonstrated that oxygengenerated free radicals derived from infiltrated neutrophils and enhanced lipid peroxidation play important roles in the pathogenesis of acute gastric lesions induced by aspirin (Konturek et al., 1994). Substances that are able to hinder their formation or capture the free oxygen radicals formed are thus potential antiulcerogenic agents. These facts form the basis for a study of whether the antioxidant mechanisms are involved in CQE mediated protection in aspirin-induced gastric damage. The objective of the study is to investigate the effect of CQE on neutrophil infiltration tissue injury induced by aspirin in order to reveal the mechanism underlying the antiulcer effect of the plant. 2. Materials and methods 2.1. Preparation of alcoholic extract Dried parts of Cissus quadrangularis were coarsely powdered and 1 kg of this powdered plant material was soaked in ethanol for 48 h and extracted by soxhlet extraction. The extract was vacuum dried and was stored at −4 ◦ C until further use. The yield of the extract was 5.2% (w/w) of powdered ethanolic extract. For administration, the extract was dissolved in distilled water and used for the present study. 2.2. Animals Male albino rats’ weighing 150–200 g were purchased from Tamil Nadu University of Veterinary and Animal Sciences, India. The animals were housed in polypropylene cages maintained in controlled temperature and light cycle. The animals were fed with food pellets and water was given ad libitum. The experiments were initiated only after the approval of the Institutes of Animal Ethics Committee (No: 360/01/a/CPSEA/ 2001). 2.3. Acute toxicity Adult albino rats of either sex were divided into four groups (n = 6) and orally fed with CQE at dose levels of 0.5, 1.5, 3.0 and 5.0 g/kg. Animals were watched carefully for 72 h after CQE administration and then for the next 7 days. At the end of this experimental period, the rats were observed for signs of toxicity, morphological behaviour and mortality. A separate group of controls received only the vehicle.
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2.4. Induction of aspirin-induced gastric lesions Adult male Wistar rats weighing 120–180 g were used for the experiment. All the animals were fasted for 24 h prior to the experiments. Aspirin at a dose of 400 mg/kg (Kamsiah et al., 2002) was administered to the animals and after 6 h, the animals were sacrificed by cervical dislocation and the stomach was incised along the greater curvature and the area of mucosal damage of glandular stomach was calculated in units of square millimeter (Szabo et al., 1985). 2.5. Dosage fixation A group of animals, which served as control, received only distilled water. The CQE at dose levels of 250, 500 and 750 mg/kg and ranitidine (RTD), the reference drug, in the dose of 30 mg/kg was administered orally for 7 days. A dosedependent antiulcer effect of CQE (250, 500 and 750 mg/kg) was seen on gastric ulcers. Therefore, for the test drug (CQE), an optimum dose of 500 mg/kg was selected for further studies and the percentage of ulcer protection was nearly equipotent to RTD, a standard antiulcer drug. 2.6. Treatment protocol Animals were divided into four groups of six animals in each group. Group 1 animals served as control. They received only distilled water equivalent to the volume of plant extract. Group 2 represented the ulcerated group. Ulceration was produced by the administration of aspirin (400 mg/kg) orally. Group 3 animals were pretreated with the test drug CQE (500 mg/kg) orally, once daily for 7 days and then ulceration was induced by aspirin. After 6 h, the animals were sacrificed, stomach was taken out and the scrapped gastric mucosal tissues were used for the estimation of biochemical enzymes. 2.6.1. Assay of Se-glutathione peroxidase (Se-GSHPx) and xanthine oxidase (XO) For the assays of Se-GSHPx and XO enzymes, gastric mucosal tissues were homogenized in nine volumes of icecold 0.05 M Tris–HCl buffer (pH 7.4). The homogenate was centrifuged at 4 ◦ C (10,000 × g, 20 min) and the resultant supernatent was dialyzed against 100 volumes of the same buffer at 4 ◦ C for 24 h. Gastric mucosal Se-GSHPx enzyme activity was assayed by the methods of Hochstein and Utley (1968). The enzyme activity was determined at 37 ◦ C by recording the decrease in absorbance at 340 nm following the oxidation of NADPH in the presence of H2 O2 , GSH and yeast glutathione reductase. One unit of this activity is defined as the amount of enzyme oxidizing 1 mol of NADPH per min. Gastric mucosal XO was assayed by the method of Hashimoto (1974). Three millilitres of incubation mixture contains 150 M phosphate buffer, 0.2 M xanthine, 0.3 M potassium oxonate and 0.2 ml of tissue homogenate. The tubes were incubated at 30 ◦ C for 30 min, 0.1 ml of 100% of TCA was added and mixed well. The content was centrifuged at 10,000 × g for
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15 min and the supernatant was analysed for XO activity by measuring the increase in absorbance at 292 nm following formation of uric acid at 30 ◦ C. One unit (U) of this enzyme is defined as the amount of enzyme forming 1 mol uric acid/min. The activity of XO was expressed as mU/g tissue. 2.6.2. Assay of lipid peroxidation The LPO product malondialdehyde (MDA) was estimated using 1,1,3,3-tetramethoxypropane as the standard, according to the method described by Ohkawa et al. (1979). To 0.5 ml of tissue homogenate, 1.5 ml of 20% acetic acid, 0.2 ml of SDS and 1.5 ml of TBA were added. The mixture was made up to 4.0 ml with distilled water and then heated for 60 min at 95 ◦ C using glass ball as condenser. After cooling, 4.0 ml of butanol–pyridine mixture was added and shaken well. After centrifugation at 4000 rpm for 10 min, the organic layer was taken and its absorbance was read at 532 nm and the results were expressed as nmol/mg protein. 2.6.3. Assay of myeloperoxidase Gastric mucosal myeloperoxidase (MPO), a marker enzyme of neutrophil infiltration, was assayed by the method of Suzuki et al. (1983). A sample of tissue homogenate (0.5 l) was added to a 0.5 ml of reaction volume containing PBS, pH 5.4, hexadecyl–trimethyl ammonium bromide and tetraethylbenzidine at 37 ◦ C. The reaction was started by the addition of H2 O2 and terminated by the sequential addition of catalase and sodium acetate. One unit of this enzyme activity was defined as the amount of enzyme causing a change in absorbance at 655 nm and the results were expressed as U/mg protein. Protein content was assessed according to the method of Lowry et al. (1951). 2.7. Statistical analysis The data were expressed as mean ± S.E.M. One-way analysis of variance followed by Dunnett’s multiple comparison tests were used to assess statistical significance of differences between groups.
Fig. 1. Effect of Cissus quadrangularis extract (CQE) on Se-glutathione peroxidase (Se-GSHPx) and xanthine oxidase (XO) activities in the aspirin-induced gastric ulcer model. Results are expressed as mean ± S.E.M. of six animals in each group. a P < 0.001 vs. control; b P < 0.001 vs. aspirin group.
750 mg/kg given orally for 7 days. Therefore, an optimal dose of CQE (500 mg/kg) given for 7 days significantly protected the animals against gastric ulcer. Hence, further studies were carried out with the same dose of CQE. As shown in Fig. 1 the increase in XO activity upon aspirin administration was attenuated by pretreatment with CQE at the dose of 500 mg/kg. Our data show that according to gastric mucosal damage, Se-GSHPx activity decreased significantly after aspirin administration when compared to control. CQE pretreatment replenish the Se-GSHPx activity to the levels seen in control rats. Fig. 2 depicts the activity of MPO, as an index of neutrophil infiltration and LPO level in the control and experimental animals. Aspirin induction strongly increased the activity of MPO and LPO levels in comparison with control. While pretreatment with CQE restored these alterations to near-normal levels in ulcerated rats, it was effective against aspirin-induced gastric ulcer.
3. Results Rats that received oral doses of 0.5, 1.5, 3.0 and 5.0 g/kg, did not manifest any clinical signs of toxicity. None of the doses tested could produce mortality in rats during the treatment period. In tests on rats, we found that doses of CQE up to 5 g/kg were non-toxic and we were unable to establish its oral LD50 value. Aspirin administered rats showed multiple gastric mucosal lesions, most often 1–2 mm in size or petechial, bleeding at the moment of the observation. The CQE showed significant antiulcer effect against ulcers induced in the aspirin model in a dose-dependent manner. In ulcerated rats, CQE at dose levels of 250, 500 and 750 mg/kg showed protection indexes of 40.0, 71.2 and 72.6%, respectively, whereas RTD showed 71.9% protection at a dose of 30 mg/kg. The percentage of ulcer protection of CQE at dose of 500 mg/kg was nearly the same as that of 750 mg/kg. There was no significant difference in the ulcer protection indexes of CQE at a dose of 500 mg/kg as compared with
Fig. 2. Effect of Cissus quadrangularis extract (CQE) on gastric mucosal lipid peroxide content (LPO) and myeloperoxidase (MPO) in aspirin administered rats. Results are expressed as mean ± S.E.M. of six animals in each group. a P < 0.001 vs. control; b P < 0.001 vs. aspirin group.
M. Jainu et al. / Journal of Ethnopharmacology 104 (2006) 302–305
4. Discussion The results of this study demonstrate that the CQE possesses antiulcer property as evidenced by its significant inhibition in the formation of gastric lesions induced by aspirin. The acute toxicity tests on rats showed that doses of CQE up to 5 g/kg were non-toxic and the oral LD50 value might be higher than this dose. Aspirin caused rise in the lipid peroxidation status possibly due to the activation of neutrophils, which play an important role in the damaging activity (Yoshikawa et al., 1992). The measurement of local enzymatic activity of MPO has been used to quantify neutrophil sequestration in tissues and the severity of inflammation (Nishizawa et al., 1996). The activity of Se-GSHPx, an index of tissue neutrophil infiltration increased, with lesion development in the aspirin administered rats (Krawisz et al., 1984). However, inhibition of MPO and increase in Se-GSHPx activity by CQE may be simple by a reflection of decreased inflammation independent of any direct effect on neutrophils. The gastric ulcer induced by aspirin was almost completely prevented, with attenuation of increased gastric mucosal XO and MPO activities and LPO content, by treatment with CQE, thereby decreasing the neutrophil sequestration at the wound site. Triterpenoids and -sitosterols present in Cissus quadrangularis possess antilipidperoxidative effect (Somova et al., 2003) and thus prevent gastric damage. The plant constituent such as -sitosterol has the ability to reduce the enzyme MPO, indicating a reduction of neutrophil influx in the inflamed tissues (DelaPureta et al., 2000). Murthy et al. (2003) have reported that Cissus quadrangularis possesses antioxidant activity in both the in vitro and in vivo models due to the presence of -carotene in this plant. Previous reports suggested that the healing action of Cissus quadrangularis is due to its antioxidative property (Jainu and Devi, 2003). Thus, the protective role of CQE against neutrophil mediated tissue injury may be due, in part, to a reduction in neutrophil infiltration into the gastric mucosa, via its antioxidant property. In conclusion, the antiulcer efficacy of CQE in gastric ulcer model could be due to inhibition of neutrophil infiltration and antioxidant properties. These properties of CQE merit detailed analysis relating to active principles and the mechanism of action, which might provide new alternatives for the clinical management of gastric ulcer diseases. Acknowledgement The authors would like to thank Dr. P. Brindha for his expert comments and suggestions in regard to this work. References Anoop, A., Jagdeesan, M., 2002. Gastric and duodenal antiulcer and cytoprotective effect of Cissus quadrangularis Linn. variant II in rats. Nigerian Journal of Natural Products and Medicine 6, 1–7.
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