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Gastroprotective activity of Trichosanthes cucumerina in rats
Journal of Ethnopharmacology 127 (2010) 750–754
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Gastroprotective activity of Trichosanthes cucumerina in rats L.D.A.M. Arawwawala a,∗ , M.I. Thabrew b , L.S.R. Arambewela a a b
Industrial Technology Institute, Bauddhaloka Mawatha, Colombo 07, Sri Lanka Department of Biochemistry & Clinical Chemistry, Faculty of Medicine, University of Kelaniya, Thallagolla Rd., Ragama, Sri Lanka
a r t i c l e
i n f o
Article history: Received 20 April 2009 Received in revised form 7 November 2009 Accepted 26 November 2009 Available online 4 December 2009 Keywords: Trichosanthes cucumerina Cucurbitaceae Gastroprotection Gastric acidity Mucus content Antihistamine activity
a b s t r a c t Aim of the study: The aim of the present study was to scientifically investigate whether Trichosanthes cucumerina Linn (Family: Cucurbitaceae) has gastroprotective activity. Materials and methods: All the experiments were conducted using Wistar strain rats (weight: 200–220 g). The food and water given to rats was withdrawn for 36 and 12 h respectively, before the commencement of the experiment. These rats were randomly divided into 6 groups (n = 8 rats/group; 4 males + 4 females) and groups 1–3 were orally administrated with hot water extract (HWE) at a dose of 375, 500 and 750 mg/kg, respectively. Group 4 was orally treated with equal volume of distilled water (1 mL; control), group 5 was orally treated with a reference drug, cimetidine (100 mg/kg) while the group 6 was orally treated with another reference drug, sucralfate (400 mg/kg). In the indomethacin experiment, only one dose of HWE (750 mg/kg) was tested, as this was found to have the maximum effect in the alcohol model also. Results: Results show that the HWE of Trichosanthes cucumerina possesses significant (P ≤ 0.05) and dose dependent gastroprotective effects in the alcohol model in terms of the length and number of gastric lesions mediated by alcohol, with a maximum effect at 750 mg/kg (inhibition of lesion length by 92%; number of gastric lesions by 88%). The same dose also mediated a significant (P ≤ 0.05) gastroprotective activity in the indomethacine model (inhibition of lesion length by 88%; number of gastric lesions by 84%). In both models, the protective effect demonstrated by the HWE was comparable with that produced by cimetidine. The HWE significantly (P ≤ 0.05) increased the amount of mucus produced by the rat gastro mucosa (by 39%) and reduced the gastric acidity (total acidity by 36%; free acidity by 40%). pH of the gastric juice increased from 4.1 to 6.0. However, no change in the volume of gastric juice was observed. Further, HWE showed potent antihistamine activity. Conclusion: It may be concluded that HWE of Trichosanthes cucumerina exerts a significant protection against ethanol or indomethacin induced gastric damage. Increasing the protective mucus layer, decreasing the acidity of the gastric juice and antihistamine activity are probable mechanisms by which the HWE of Trichosanthes cucumerina mediates its gastroprotective actions. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Trichosanthes cucumerina L. is an annual, dioecious climber belongs to the family Cucurbitaceae. It is widely distributed in Asian countries including Sri Lanka, India, Malay Penisula and Philippine (Jayaweera, 1980). Studies on the pharmacological activities have shown the presence of antiinflammatory activity in root tubers (Kolte et al., 1996–1997) and antidiabetic activity in seeds (Kar et al., 2003) of Trichosanthes cucumerina. In addition, to these pharmacological activities, a few studies have been conducted to investigate the properties of galactose specific seed lectin of Trichosanthes cucumerina. These include the thermodynamic and kinetic analysis of porphyrin binding studies (Kenoth et al., 2001), physicochemical
∗ Corresponding author. Tel.: +94 1112693807; fax: +94(11) 268 6567. E-mail address: [email protected] (L.D.A.M. Arawwawala). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.11.026
and saccharide binding properties (Kenoth et al., 2003) and steadystate and time-resolved fluorescence studies on the seed (Kenoth and Swamy, 2003). The whole plant including roots, leaves, fruits, seeds have medicinal properties. The root is used as a cure for bronchitis, headache and boils. Externally, the leaf juice is rubbed over the liver to relieve liver congestion. Both the root and fruit are considered to be cathartic. The fruit is used as an anthelmintic in French Guiana. The seeds are used for stomach disorders in Malabar Coast and is also considered antifebrile and anthelmintic. The aerial parts of Trichosanthes cucumerina are used along with other plant materials for indigestion, bilious fevers, boils, sores, skin eruptions such as urticaria, eczema, dermatitis, psoriasis diabetes and ulcers (Anonymous, 1976, 2002; Jayaweera, 1980). In Sri Lanka, a polyherbal preparation named as Patoladi decoction is given as a remedy for gastric ulcers. It contains Trichosanthes cucumerina aerial parts and another four plant ingredients: pericarp
L.D.A.M. Arawwawala et al. / Journal of Ethnopharmacology 127 (2010) 750–754
of dry fruit of Terminalia chebula Retz (Combretaceae), Terminalia belerica Rox (Combretaceae), Phyllanthus emblica Linn (Euphorbiaceae) and dry bark of Azadirachta indica A. Juss (Meliaceae). For the preparation of this decoction, 12 g of each ingredient in dried form is introduced into a clay pot and gently boiled in 1920 mL of water for about 3 h and the final volume reduced to 240 mL using a low flame. The dosage recommended for administration to an adult is a half a cup (120 mL) two times per day. According to Munasinghe et al. Patoladi decoction (2002a) and semi-solid preparation of this decoction (2002b) had strong gastroprotective activity against ethanol-induced gastric lesions in rats. However, it is not clear whether all five plant ingredients of Patoladi decoction contribute to its action. Bandyopadhyay et al. (2002) has reported that bark of Azadirachta indica has gastroprotective activity. Therefore, the aim of the present investigation was to determine whether Trichosanthes cucumerina also has gastroprotective properties. 2. Materials and methods 2.1. Plant material Trichosanthes cucumerina plants were collected from Western province of Sri Lanka. The plant was identified and authenticated by the curator of National Herbarium, Royal Botanical Gardens, Peradeniya, Sri Lanka. A voucher specimen (TS 01) was deposited in the Industrial Technology Institute, Colombo 7, Sri Lanka. 2.2. Animals Healthy adult male and female Wistar rats (weighing 200–225 g) were used throughout the experiment. They were housed individually in raised mesh bottom cages under standardized animal house conditions and fed with standard rat feed and water ad libitum. All animal experiments were conducted in accordance with the internationally accepted laboratory animal use and care and guide lines and rules of the ethical committee, University of Kelaniya, for animal experimentations. Prior to the experiments, the rats were deprived of food for 36 h, water for 12 h and kept in raised mesh bottomed cages to prevent coprophagy. 2.3. Preparation of hot water extract (HWE) Trichosanthes cucumerina aerial parts were cut into small pieces and air dried. Then, 60 g of the plant material was boiled in 1.9 L of distilled water (DW) and the final volume was reduced to 240 mL by boiling over 4 h. The hot water extract was freeze dried and stored at 4 ◦ C until use (yield 12.5% dry weight basis). 2.4. Administration of extract Doses of 375, 500, 750 and 1000 mg/kg of HWE were administered orally by gastric gavage (each dose in a volume of 1 mL DW) to separate groups of rats. The dose of 750 mg/kg corresponds to the normal therapeutic dose administered to adult humans as calculated on the basis of relative surface areas of humans and rats (Paget and Barnes, 1996). 2.5. Phytochemical screening of HWE Qualitative testing of the HWE for alkaloids, polyphenols, flavonoids, steroids, saponins and tannins was carried out according to the method described by Farnsworth (1996).
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2.6. Quantitative determination of total polyphenolic content in HWE The total polyphenolic content was estimated according to the Folin–Ciocalteu method described by Spanos and Worlstad (1990). Known concentrations of HWE (0.1 mL) was diluted with DW (0.9 mL) and mixed with 5 mL of 10-fold diluted solution of Folin–Ciocalteu reagent. Four milliliters of saturated sodium carbonate solution was added to the above mixture and shaken. The absorbance of the reaction mixture was measured at 765 nm after 2 h. Total phenolic content was expressed as gallic acid equivalents (mg gallic acid/g extract). 2.7. Quantitative determination of total flavonoid content in HWE The total flavonoid content was determined using the Dowd method as described by Meda et al. (2005). In this experiment, 5 mL of 2% AlCl3 in methanol was mixed with the same volume of the plant extract in known concentrations. After 10 min the absorbance of the reaction mixture was measured at 415 nm. Total flavonoid content was expressed as quercetin equivalents (mg quercetin/g extract). 2.8. Effects on the ulceration induced by absolute ethanol The food and water given to rats were withdrawn for 36 and 12 h, respectively before the commencement of the experiment. These rats were randomly divided into 6 equal groups (n = 8/group; 4 females and 4 males) and treated orally in the following manner: each rat in group 1 received 1 mL of DW (control group), rats in groups 2, 3, 4 received 375, 500 and 750 mg/kg of HWE in 1 mL of DW respectively while rats in group 5 received 100 mg/kg of cimetidine, a reference drug and rats in group 6 received 400 mg/kg of sucralfate, a reference drug. In previous studies, H2 -receptor antagonists such as famotidine (Umamaheswari et al., 2007) and ranitidine (Mesia-Vela et al., 2007) were used as the reference drugs in ethanol induced gastric ulceration model. In present study, we used cimetidine, another H2 -receptor antagonist as one of the reference drugs. However, according to some researchers (Malairajan et al., 2007), ethanol-induced ulcers are mainly inhibited by agents that enhance mucosal defense factors such as prostaglandins. Therefore, sucralfate, was used as the secondary reference drug in the investigation with ethanol induced gastric ulceration model. After 1 h of oral treatment, each rat was given absolute ethanol (5 mL/kg) orally and kept for another 1 h. Then the rats of all groups were sacrificed after exposure to ether, stomachs were removed and inflated with 1% formalin solution and immersed in the same solution to fix the outer layer of the stomach. Each stomach was opened along the greater curvature, rinsed with tap water to remove gastric contents and blood clots. The numbers of haemorrhagic lesions were counted using a magnifying lens and the lengths of the linear lesions were measured with a vernier caliper as described by Robert (1979). 2.9. Effects on the ulceration induced by indomethacin In this study, only 750 mg/kg of HWE was tested, as this was found to have the maximum effect in the ethanol-induced gastric ulceration model. Gastric ulcers were induced by indomethacin (25 mg/kg) as described by Bhargava et al. (1973). The food and water given to rats were withdrawn for 36 and 12 h, respectively before the commencement of the experiment. Twenty-four rats were orally treated with 25 mg/kg of indomethacin in 1 mL of 1% methylcellulose and randomly divided into 3 equal groups (n = 8/group; 4 females and 4 males). After 1 h, rats were treated orally in the following manner: each rat in group 1 (control group)
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received 1 mL of DW, rats in group 2 received 750 mg/kg of HWE in 1 mL of DW while those in group 3 received 100 mg/kg of cimetidine, the reference drug. One hour later, all the rats were sacrificed with over dose of ether. The numbers of haemorrhagic lesions and the lengths of the linear lesions were measured as described above. 2.10. Evaluation of the mode of gastroprotective activity The mode of action by which Trichosanthes cucumerina mediates its gastroprotective effects was assessed by determining its effects on
treated orally in the following manner: each rat in group 1 received 750 mg/kg of HWE in 1 mL of DW, rats in group 2 received chlorpheniramine, an antihistamine receptor antagonist at a dose of 0.40 mg/kg while rats in group 3 received 1 mL of DW (control group). After 1 h, 0.05 mL of 200 g/mL of histamine dihydrochloride was subcutaneously injected under mild ether anesthesia in the area of the skin where the fur was removed previously (Spector, 1956). The radius of the wheal formed was determined after 2.5 min and the areas were computed. 2.11. Statistical analysis
a. acidity and volume of gastric juice b. mucus content of stomach c. antihistamine activity.
Data are given as means ± S.E.M. Statistical comparisons were made using one-way ANOVA followed by Duncans multiple range test. A P value ≤0.05 was considered as significant.
2.10.1. Evaluation of the effects on acidity and volume of the gastric juice Twelve rats (6 male rats and 6 female rats) were starved for 36 h and water was withdrawn for 12 h as described previously. They were randomly divided into 2 equal groups (3 male rats and 3 female rats/group). Rats in the two groups were orally treated with either 750 mg/kg of HWE or 1 mL of DW per rat. One hour later, these rats were laparotomised under ether anesthesia and at the pyloric end of the stomach was ligated with a cotton thread. The stomachs were then carefully placed back in the abdominal cavities and the rats were sutured and allowed to regain consciousness. Four hours later stomachs were removed, gastric content collected and centrifuged at 3500 × g for 15 min for determination of gastric juice volume (mL) and pH. Acidity (total and free) in gastric secretion was determined by titration with 0.01 N NaOH according to the method described by Reitman (1970).
3. Results
2.10.2. Determination of mucus content of stomach Alcian blue binding to gastric wall mucus was determined by a modified method of Corne et al. (1974). In this experiment, 12 rats (6 male rats and 6 female rats) were starved for 36 h and water was withdrawn for 12 h as described previously. They were randomly divided into 2 equal groups (3 male rats and 3 female rats/group). Rats in the two groups were orally treated with either 750 mg/kg of HWE or 1 mL of DW per rat. One hour later, these rats were laparotomised under ether anesthesia and at the pyloric end of the stomach was ligated with a cotton thread. The stomachs were then carefully placed back in the abdominal cavities and the rats were sutured and allowed to regain consciousness. After 4 h, the rats were sacrificed with over dose of ether, each stomach was opened along the greater curvature, rinsed with 0.25 M sucrose solution. These stomachs were incubated in 10 mL aliquots of 0.1% alcian blue solution for 2 h at room temperature (30 ◦ C). After 2 h stomachs were removed, washed with 0.25 M sucrose solution and separately incubated in 10 mL aliquots of 0.5 M magnesium chloride solution for 2 h at room temperature while shaking at 30 min intervals to elute the alcian blue bound to the mucosa of the stomachs. Two hours later, the stomachs were removed and 5 mL of each aliquot of magnesium chloride solution containing the alcian blue eluted from each stomach was shaken with 5 mL of diethyl ether. The aqueous phase was separated out, centrifuged at 3200 × g for 5 min and the absorbance of the supernatant was measured at 605 nm. The amount of alcian blue bound per stomach in micrograms was determined using a standard calibration curve. 2.10.3. Antihistamine activity Eighteen rats (9 male rats and 9 female rats) were selected and their fur on posterior left lateral side was shaved under ether anesthesia. Twenty-four hours later, these rats were randomly divided into 3 groups (3 male rats and 3 female rats/group) and
3.1. Phytochemical screening of HWE Phytochemical screening revealed the presence of alkaloids, polyphenols, flavonoids, steroids, saponins and tannins in the extract. 3.2. Total polyphenol and flavonoid content in HWE The total polyphenolic content and flavonoid content in HWE were 65.8 ± 0.50 mg gallic acid equivalents/g extract and 45.4 ± 0.76 mg quercetin equivalents/g extract, respectively. 3.3. Effects on ethanol-induced gastric lesions Trichosanthes cucumerina HWE caused a significant (P < 0.05) inhibition of the length and the number of gastric lesions (Table 1) induced by absolute ethanol in a dose dependent (r = 0.94) manner. Among the tested doses, high dose (750 mg/kg) showed the maximum inhibition of the length (by 92%) and number of gastric lesions (by 88%) followed by mid (length: by 78%; number: by 77%) and low (length: by 45%; number: by 48%) doses. The gastroprotective activity of 750 mg/kg dose of HWE was comparable with those of cimetidine and sucralfate, the reference drugs (cimetidine: inhibition of length by 93% and number by 92%: sucralfate: inhibition of length by 96% and number by 95%). 3.4. Effects on indomethacin-induced gastric lesions Trichosanthes cucumerina HWE at a dose of 750 mg/kg significantly (P < 0.05) reduced the length (by 88%) and number of gastric lesions (by 84%) compared to the control group (Table 1). Similar to the absolute ethanol-induced gastric ulceration model, gastroprotective activity of the 750 mg/kg dose of HWE was comparable to the reference drug, cimetidine (inhibition of length by 90% and number by 90%). 3.5. Acid secretion studies Compared with controls, the rats treated with HWE showed significant (P < 0.05) reduction in free acidity (by 40%) and total acidity (by 36%) in the gastric juice (Table 2). Further, pH of the gastric juice increased from 4.1 to 6.0. However, no change in the volume of gastric juice was observed. 3.6. Gastric mucus studies Administration of Trichosanthes cucumerina HWE significantly (P < 0.05) increased the amount of mucus produced by the rat
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Table 1 Effect of Trichosanthes cucumerina hot water extract (HWE) on the length of gastric lesions (in mm) and number of lesions induced by absolute ethanol and indomethacin. The length of haemorrhagic lesions (mm) Ethanol-induced gastric ulceration model Control 375 mg/kg of HWE 500 mg/kg of HWE 750 mg/kg of HWE Cimetidine (100 mg/kg) Sucralfate (400 mg/kg)
71.5 39.2 15.9 5.7 4.8 3.1
Indomethacin-induced gastric ulceration model Control 750 mg/kg of HWE Cimetidine (100 mg/kg)
74.8 ± 3.7 8.9 ± 1.1* 7.3 ± 0.9*
± ± ± ± ± ±
The numbers of haemorrhagic lesions
4.8 3.6* 2.2* 1.3* 0.9* 0.7*
17.4 9.0 4.0 2.1 1.2 0.9
± ± ± ± ± ±
1.8 0.8* 0.7* 0.5* 0.3* 0.2*
17.8 ± 1.6 2.9 ± 0.4* 1.7 ± 0.2*
Values are expressed as mean ± S.E.M.; n = 8. * Significant when compared with respective controls P ≤ 0.05. Table 2 Effect of Trichosanthes cucumerina hot water extract (HWE) on gastric juice volume, pH, free acidity and total acidity. Treatment
pH
Gastric juice volume (mL)
Free acidity (mol L−1 )
Total acidity (mol L−1 )
Control Hot water extract (750 mg/kg)
4.1 ± 0.3 6.0 ± 0.2*
5.8 ± 0.2 5.6 ± 0.2
0.05 ± 3.0 × 10−3 0.03 ± 3.1 × 10−3 *
0.082 ± 4.7 × 10−3 0.052 ± 2.5 × 10−3 *
Values are expressed as mean ± S.E.M.; n = 6. * Significant when compared with respective controls P ≤ 0.05.
gastro mucosa (control vs treatment: 189.6 ± 8.8 g/stomach vs 263.1 ± 4.4 g/stomach). 3.7. Antihistamine activity Trichosanthes cucumerina HWE showed potent antihistamine activity as there was a significant (P < 0.05) reduction (by 27%) in the wheal area of the rats when compared with the control group (Table 3). Further, the effect of the HWE was comparable with that of chlorpheniramine, the reference drug. 4. Discussion Ethanol and indomethacin are commonly used for inducing ulcer in experimental rats (Umamaheswari et al., 2007; Nguelefack et al., 2008; Rao et al., 2008). Ethanol is one of the ulcerogenic agents that induces intense damage in gastric mucosa by promoting disturbances of mucosal microcirculation, ischemia and appearance of free radicals, endothelin release, degranulation of mast cells, inhibition of prostaglandins and decrease of gastric mucus production (Park et al., 2004). Indomethacin is a nonsteroidal antiinflammatory drug (NSAID) that inhibits prostaglandin production that results in increased acid production and decreased cytoprotective mucus formation, which can lead to induce gastrointestinal ulcer (Wallace and Devchand, 2005). Trichosanthes cucumerina possesses marked gastroprotective properties as evidenced by its significant inhibition of the formation of gastric lesions (in terms of length and number) induced by absolute ethanol and indomethacin. The gastroprotective activity of 750 mg/kg dose of HWE was comparable to that reference drugs (cimetidine and Table 3 Antihistamine activity of hot water extract (HWE) of Trichosanthes cucumerina as indicated by the area of the wheal. Treatment
Area of the wheal (mm2 )
Control Hot water extract (750 mg/kg) Chlorpheniramine (0.40 mg/kg)
73.2 ± 4.2 53.1 ± 6.6* 46.2 ± 5.5*
Values are expressed as mean ± S.E.M.; n = 6. * Significant when compared with respective controls P ≤ 0.05.
sucralfate) used in each experimental model. Further, in the ethanol model, the gastroprotective effect was dose-dependent and dose response curve was linear. In the present investigation, it has been demonstrated that HWE can significantly enhance gastric mucus secretion while reducing the acidity of the gastric juice in rats. Gastric mucus is an important protective factor for the gastric mucosa and it is capable of acting as an antioxidant agent and reducing mucosal damage mediated by oxygen free radicals (Berenguer et al., 2007). However, the protective properties of the mucus barrier depend not only on the gel structure but also on the amount or thickness of the layer covering the mucosal surface (Penisi and Piezzi, 1999). Administration of HWE significantly (P < 0.05) increased the amount of mucus produced by the rat gastro mucosa compared to their respective controls. Therefore, the enhanced mucus secretion after administration of HWE may help to protect against the absolute ethanol and indomethacin-induced damage by preventing the action of acid and pepsin on the stomach mucous epithelium (Laurence and Bennet, 1992). Similar mode of action has been reported with several other plants also (Moraes et al., 2008; Hiruma-Lima et al., 2009). It is also well known that prostaglandins synthesized in large quantities by the gastrointestinal mucosa can prevent experimentally induced ulcers by ulcerogens. Thus, when the ulcer lesions are induced by absolute ethanol or indomethacin, the cytoprotective effect of the antiulcer agent can mediated through endogeneous prostaglandins (Yamamoto et al., 1992). Therefore, it can be thought that HWE may stimulate the secretion of prostaglandin or possess prostaglandin like substances. Another important protective factor is the inhibition of acid secretion (Bandyopadhyay et al., 2002; Konturek et al., 2005), since when levels of acid overwhelm mucosal defense mechanisms this lead to ulcer formation (Schubert, 2004). In our study, HWE caused significant inhibition in the acidity (both total and free) with an elevation in gastric pH. Similar mode of action has also been reported in some other plant extracts (Umamaheswari et al., 2007; Nguelefack et al., 2008; Hiruma-Lima et al., 2009). The reduction of acidity of gastric juice mediated by HWE could be attributed to its antihistamine effect, because it is well established that antihistamine drugs like cimetidine which block H2 receptors in the stomach, reduce the acidity of gastric juice (Laurence and Bennet, 1992). Although, H2 receptor blockers are also expected to reduce
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the secretion of gastric juice (Laurence and Bennet, 1992) such an effect was not observed in the present study. H2 antagonists are recommended for the prevention of gastric lesions induced by NSAIDs (Eccles et al., 1998). Therefore, the antihistamine activity in HWE may also contribute to the protective activity against indomethacin-induced gastric lesions. Phytochemical screening of Trichosanthes cucumerina revealed the presence of alkaloids, saponins, tannins, flavonoids and other phenolic compounds. Polyphenols, especially tannins, are phytochemicals with antioxidant properties. These compounds have also shown antiulcerogenic properties due to their protein precipitating and vasoconstricting effects (Berenguer et al., 2006). Their astringent action can help precipitating microproteins on the ulcer site, thereby forming an impervious layer over the lining that hinders gut secretions and protects the underlying mucosa from toxic and other irritants (Nwafor et al., 2000). The antiulcerogenic effect of many plants is related to their flavonoid content, as these antioxidants inhibit lipid peroxidation and other free radical mediated processes that lead to gastric damage (Gurbuz et al., 2009). Moreover, saponins (Morikawa et al., 2006) and alkaloids (Li et al., 2005) have been shown to have potent gastroprotective activities. Therefore, secondary metabolites such as alkaloids, saponins, tannins, flavonoids and other phenolic compounds present in Trichosanthes cucumerina may also contribute to its gastroprotective effect. In conclusion, this study showed a potent gastroprotective activity of Trichosanthes cucumerina hot water extract in absolute ethanol and indomethacin-induced gastric lesions for the first time and indicate its therapeutic potential to be used as a cost effective herbal gastroprotective agent. Acknowledgement The authors express their gratitude to National Science Foundation for the Research Grant (NSF/SCH/2005/13). References Anonymous, 1976. The Wealth of India, vol. 10. The Dictionary of Indian Raw Materials and Industrial Products. Publication and Information directorate, CSIR, New Delhi, p. 291. Anonymous, 2002. Compendium of Medicinal Plants, vol. 2. A Publication of Department of Ayurveda, Sri Lanka, pp. 146–150. Bandyopadhyay, U., Biswas, K., Chatterjee, R., Bandyopadhyay, D., Chattopadhyay, I., Ganguly, C.K., Chakraborty, T., Bhattacharya, K., Banerjee, R.K., 2002. Gastroprotective effect of Neem (Azadirachta indica) bark extract: possible involvement of H(+)–K(+)-ATPase inhibition and scavenging of hydroxyl radical. Life Science 71, 2845–2865. Berenguer, B., Sanchez, L.M., Quilez, A., Lopez-Barreiro, M., de Haro, O., Galvez, J., Martin, M.J., 2006. Protective and antioxidant effects of Rhizophora mangle L. against NSAID-induced gastric ulcers. Journal of Ethnopharmacology 103, 194–200. Berenguer, B., Trabadela, C., Sanchez-Fidalgo, S., Quilez, A., Mino, P., Puerta, R.D.I., Martin-Calero, M.J., 2007. The aerial parts of Guazuma ulmifolia Lam. protect against NSAID-induced gastric lesions. Journal of Ethnopharmacology 114, 153–160. Bhargava, K.P., Gupta, M.B., Tangri, K.K., 1973. Mechanism of ulcerogenic activity of indomethacin and oxyphenylbutazone. European Journal of Pharmacology 22, 191–195. Corne, S.J., Morrisey, S.M., Woods, K.J., 1974. A method for the quantitative estimation of gastric barrier mucus. Journal of Physiology 2452, 116–117. Eccles, M., Freemantle, N., Mason, J., 1998. North of England evidence based-guideline development project: summary guideline for non-steroidal anti-inflammatory drugs versus basic analgesia in treating the pain of degenerative arthritis. British Medical Journal 317, 526–530. Farnsworth, N.R., 1996. Biological and phytochemical screening of plants. Journal of Pharmaceutical Science 55, 225–276. Gurbuz, I., Yesilada, E., Ito, S., 2009. An anti-ulcerogenic flavonol diglucoside from Equisetum palustre L. Journal of Ethnopharmacology 121, 360–365. Hiruma-Lima, C.A., Rodrigues, C.M., Kushima, H., Moraes, T.M., Lolis, S.D.F., Feitosa, S.B., Magri, L.P., Soares, F.R., Cola, M.M., Andrade, F.D.P., Vilegas, W., Brito, A.R.M.S., 2009. The anti-ulcerogenic effects of Curatella americana L. Journal of Ethnopharmacology 121, 425–432. Jayaweera, D.M.A., 1980. Medicinal Plants (Indigenous and Exotic) used in Ceylon (Part 2). A Publication of the National Science Council of Sri Lanka, pp. 162–163.
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Gastroprotective activity of Trichosanthes cucumerina in rats L.D.A.M. Arawwawala a,∗ , M.I. Thabrew b , L.S.R. Arambewela a a b
Industrial Technology Institute, Bauddhaloka Mawatha, Colombo 07, Sri Lanka Department of Biochemistry & Clinical Chemistry, Faculty of Medicine, University of Kelaniya, Thallagolla Rd., Ragama, Sri Lanka
a r t i c l e
i n f o
Article history: Received 20 April 2009 Received in revised form 7 November 2009 Accepted 26 November 2009 Available online 4 December 2009 Keywords: Trichosanthes cucumerina Cucurbitaceae Gastroprotection Gastric acidity Mucus content Antihistamine activity
a b s t r a c t Aim of the study: The aim of the present study was to scientifically investigate whether Trichosanthes cucumerina Linn (Family: Cucurbitaceae) has gastroprotective activity. Materials and methods: All the experiments were conducted using Wistar strain rats (weight: 200–220 g). The food and water given to rats was withdrawn for 36 and 12 h respectively, before the commencement of the experiment. These rats were randomly divided into 6 groups (n = 8 rats/group; 4 males + 4 females) and groups 1–3 were orally administrated with hot water extract (HWE) at a dose of 375, 500 and 750 mg/kg, respectively. Group 4 was orally treated with equal volume of distilled water (1 mL; control), group 5 was orally treated with a reference drug, cimetidine (100 mg/kg) while the group 6 was orally treated with another reference drug, sucralfate (400 mg/kg). In the indomethacin experiment, only one dose of HWE (750 mg/kg) was tested, as this was found to have the maximum effect in the alcohol model also. Results: Results show that the HWE of Trichosanthes cucumerina possesses significant (P ≤ 0.05) and dose dependent gastroprotective effects in the alcohol model in terms of the length and number of gastric lesions mediated by alcohol, with a maximum effect at 750 mg/kg (inhibition of lesion length by 92%; number of gastric lesions by 88%). The same dose also mediated a significant (P ≤ 0.05) gastroprotective activity in the indomethacine model (inhibition of lesion length by 88%; number of gastric lesions by 84%). In both models, the protective effect demonstrated by the HWE was comparable with that produced by cimetidine. The HWE significantly (P ≤ 0.05) increased the amount of mucus produced by the rat gastro mucosa (by 39%) and reduced the gastric acidity (total acidity by 36%; free acidity by 40%). pH of the gastric juice increased from 4.1 to 6.0. However, no change in the volume of gastric juice was observed. Further, HWE showed potent antihistamine activity. Conclusion: It may be concluded that HWE of Trichosanthes cucumerina exerts a significant protection against ethanol or indomethacin induced gastric damage. Increasing the protective mucus layer, decreasing the acidity of the gastric juice and antihistamine activity are probable mechanisms by which the HWE of Trichosanthes cucumerina mediates its gastroprotective actions. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Trichosanthes cucumerina L. is an annual, dioecious climber belongs to the family Cucurbitaceae. It is widely distributed in Asian countries including Sri Lanka, India, Malay Penisula and Philippine (Jayaweera, 1980). Studies on the pharmacological activities have shown the presence of antiinflammatory activity in root tubers (Kolte et al., 1996–1997) and antidiabetic activity in seeds (Kar et al., 2003) of Trichosanthes cucumerina. In addition, to these pharmacological activities, a few studies have been conducted to investigate the properties of galactose specific seed lectin of Trichosanthes cucumerina. These include the thermodynamic and kinetic analysis of porphyrin binding studies (Kenoth et al., 2001), physicochemical
∗ Corresponding author. Tel.: +94 1112693807; fax: +94(11) 268 6567. E-mail address: [email protected] (L.D.A.M. Arawwawala). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.11.026
and saccharide binding properties (Kenoth et al., 2003) and steadystate and time-resolved fluorescence studies on the seed (Kenoth and Swamy, 2003). The whole plant including roots, leaves, fruits, seeds have medicinal properties. The root is used as a cure for bronchitis, headache and boils. Externally, the leaf juice is rubbed over the liver to relieve liver congestion. Both the root and fruit are considered to be cathartic. The fruit is used as an anthelmintic in French Guiana. The seeds are used for stomach disorders in Malabar Coast and is also considered antifebrile and anthelmintic. The aerial parts of Trichosanthes cucumerina are used along with other plant materials for indigestion, bilious fevers, boils, sores, skin eruptions such as urticaria, eczema, dermatitis, psoriasis diabetes and ulcers (Anonymous, 1976, 2002; Jayaweera, 1980). In Sri Lanka, a polyherbal preparation named as Patoladi decoction is given as a remedy for gastric ulcers. It contains Trichosanthes cucumerina aerial parts and another four plant ingredients: pericarp
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of dry fruit of Terminalia chebula Retz (Combretaceae), Terminalia belerica Rox (Combretaceae), Phyllanthus emblica Linn (Euphorbiaceae) and dry bark of Azadirachta indica A. Juss (Meliaceae). For the preparation of this decoction, 12 g of each ingredient in dried form is introduced into a clay pot and gently boiled in 1920 mL of water for about 3 h and the final volume reduced to 240 mL using a low flame. The dosage recommended for administration to an adult is a half a cup (120 mL) two times per day. According to Munasinghe et al. Patoladi decoction (2002a) and semi-solid preparation of this decoction (2002b) had strong gastroprotective activity against ethanol-induced gastric lesions in rats. However, it is not clear whether all five plant ingredients of Patoladi decoction contribute to its action. Bandyopadhyay et al. (2002) has reported that bark of Azadirachta indica has gastroprotective activity. Therefore, the aim of the present investigation was to determine whether Trichosanthes cucumerina also has gastroprotective properties. 2. Materials and methods 2.1. Plant material Trichosanthes cucumerina plants were collected from Western province of Sri Lanka. The plant was identified and authenticated by the curator of National Herbarium, Royal Botanical Gardens, Peradeniya, Sri Lanka. A voucher specimen (TS 01) was deposited in the Industrial Technology Institute, Colombo 7, Sri Lanka. 2.2. Animals Healthy adult male and female Wistar rats (weighing 200–225 g) were used throughout the experiment. They were housed individually in raised mesh bottom cages under standardized animal house conditions and fed with standard rat feed and water ad libitum. All animal experiments were conducted in accordance with the internationally accepted laboratory animal use and care and guide lines and rules of the ethical committee, University of Kelaniya, for animal experimentations. Prior to the experiments, the rats were deprived of food for 36 h, water for 12 h and kept in raised mesh bottomed cages to prevent coprophagy. 2.3. Preparation of hot water extract (HWE) Trichosanthes cucumerina aerial parts were cut into small pieces and air dried. Then, 60 g of the plant material was boiled in 1.9 L of distilled water (DW) and the final volume was reduced to 240 mL by boiling over 4 h. The hot water extract was freeze dried and stored at 4 ◦ C until use (yield 12.5% dry weight basis). 2.4. Administration of extract Doses of 375, 500, 750 and 1000 mg/kg of HWE were administered orally by gastric gavage (each dose in a volume of 1 mL DW) to separate groups of rats. The dose of 750 mg/kg corresponds to the normal therapeutic dose administered to adult humans as calculated on the basis of relative surface areas of humans and rats (Paget and Barnes, 1996). 2.5. Phytochemical screening of HWE Qualitative testing of the HWE for alkaloids, polyphenols, flavonoids, steroids, saponins and tannins was carried out according to the method described by Farnsworth (1996).
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2.6. Quantitative determination of total polyphenolic content in HWE The total polyphenolic content was estimated according to the Folin–Ciocalteu method described by Spanos and Worlstad (1990). Known concentrations of HWE (0.1 mL) was diluted with DW (0.9 mL) and mixed with 5 mL of 10-fold diluted solution of Folin–Ciocalteu reagent. Four milliliters of saturated sodium carbonate solution was added to the above mixture and shaken. The absorbance of the reaction mixture was measured at 765 nm after 2 h. Total phenolic content was expressed as gallic acid equivalents (mg gallic acid/g extract). 2.7. Quantitative determination of total flavonoid content in HWE The total flavonoid content was determined using the Dowd method as described by Meda et al. (2005). In this experiment, 5 mL of 2% AlCl3 in methanol was mixed with the same volume of the plant extract in known concentrations. After 10 min the absorbance of the reaction mixture was measured at 415 nm. Total flavonoid content was expressed as quercetin equivalents (mg quercetin/g extract). 2.8. Effects on the ulceration induced by absolute ethanol The food and water given to rats were withdrawn for 36 and 12 h, respectively before the commencement of the experiment. These rats were randomly divided into 6 equal groups (n = 8/group; 4 females and 4 males) and treated orally in the following manner: each rat in group 1 received 1 mL of DW (control group), rats in groups 2, 3, 4 received 375, 500 and 750 mg/kg of HWE in 1 mL of DW respectively while rats in group 5 received 100 mg/kg of cimetidine, a reference drug and rats in group 6 received 400 mg/kg of sucralfate, a reference drug. In previous studies, H2 -receptor antagonists such as famotidine (Umamaheswari et al., 2007) and ranitidine (Mesia-Vela et al., 2007) were used as the reference drugs in ethanol induced gastric ulceration model. In present study, we used cimetidine, another H2 -receptor antagonist as one of the reference drugs. However, according to some researchers (Malairajan et al., 2007), ethanol-induced ulcers are mainly inhibited by agents that enhance mucosal defense factors such as prostaglandins. Therefore, sucralfate, was used as the secondary reference drug in the investigation with ethanol induced gastric ulceration model. After 1 h of oral treatment, each rat was given absolute ethanol (5 mL/kg) orally and kept for another 1 h. Then the rats of all groups were sacrificed after exposure to ether, stomachs were removed and inflated with 1% formalin solution and immersed in the same solution to fix the outer layer of the stomach. Each stomach was opened along the greater curvature, rinsed with tap water to remove gastric contents and blood clots. The numbers of haemorrhagic lesions were counted using a magnifying lens and the lengths of the linear lesions were measured with a vernier caliper as described by Robert (1979). 2.9. Effects on the ulceration induced by indomethacin In this study, only 750 mg/kg of HWE was tested, as this was found to have the maximum effect in the ethanol-induced gastric ulceration model. Gastric ulcers were induced by indomethacin (25 mg/kg) as described by Bhargava et al. (1973). The food and water given to rats were withdrawn for 36 and 12 h, respectively before the commencement of the experiment. Twenty-four rats were orally treated with 25 mg/kg of indomethacin in 1 mL of 1% methylcellulose and randomly divided into 3 equal groups (n = 8/group; 4 females and 4 males). After 1 h, rats were treated orally in the following manner: each rat in group 1 (control group)
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received 1 mL of DW, rats in group 2 received 750 mg/kg of HWE in 1 mL of DW while those in group 3 received 100 mg/kg of cimetidine, the reference drug. One hour later, all the rats were sacrificed with over dose of ether. The numbers of haemorrhagic lesions and the lengths of the linear lesions were measured as described above. 2.10. Evaluation of the mode of gastroprotective activity The mode of action by which Trichosanthes cucumerina mediates its gastroprotective effects was assessed by determining its effects on
treated orally in the following manner: each rat in group 1 received 750 mg/kg of HWE in 1 mL of DW, rats in group 2 received chlorpheniramine, an antihistamine receptor antagonist at a dose of 0.40 mg/kg while rats in group 3 received 1 mL of DW (control group). After 1 h, 0.05 mL of 200 g/mL of histamine dihydrochloride was subcutaneously injected under mild ether anesthesia in the area of the skin where the fur was removed previously (Spector, 1956). The radius of the wheal formed was determined after 2.5 min and the areas were computed. 2.11. Statistical analysis
a. acidity and volume of gastric juice b. mucus content of stomach c. antihistamine activity.
Data are given as means ± S.E.M. Statistical comparisons were made using one-way ANOVA followed by Duncans multiple range test. A P value ≤0.05 was considered as significant.
2.10.1. Evaluation of the effects on acidity and volume of the gastric juice Twelve rats (6 male rats and 6 female rats) were starved for 36 h and water was withdrawn for 12 h as described previously. They were randomly divided into 2 equal groups (3 male rats and 3 female rats/group). Rats in the two groups were orally treated with either 750 mg/kg of HWE or 1 mL of DW per rat. One hour later, these rats were laparotomised under ether anesthesia and at the pyloric end of the stomach was ligated with a cotton thread. The stomachs were then carefully placed back in the abdominal cavities and the rats were sutured and allowed to regain consciousness. Four hours later stomachs were removed, gastric content collected and centrifuged at 3500 × g for 15 min for determination of gastric juice volume (mL) and pH. Acidity (total and free) in gastric secretion was determined by titration with 0.01 N NaOH according to the method described by Reitman (1970).
3. Results
2.10.2. Determination of mucus content of stomach Alcian blue binding to gastric wall mucus was determined by a modified method of Corne et al. (1974). In this experiment, 12 rats (6 male rats and 6 female rats) were starved for 36 h and water was withdrawn for 12 h as described previously. They were randomly divided into 2 equal groups (3 male rats and 3 female rats/group). Rats in the two groups were orally treated with either 750 mg/kg of HWE or 1 mL of DW per rat. One hour later, these rats were laparotomised under ether anesthesia and at the pyloric end of the stomach was ligated with a cotton thread. The stomachs were then carefully placed back in the abdominal cavities and the rats were sutured and allowed to regain consciousness. After 4 h, the rats were sacrificed with over dose of ether, each stomach was opened along the greater curvature, rinsed with 0.25 M sucrose solution. These stomachs were incubated in 10 mL aliquots of 0.1% alcian blue solution for 2 h at room temperature (30 ◦ C). After 2 h stomachs were removed, washed with 0.25 M sucrose solution and separately incubated in 10 mL aliquots of 0.5 M magnesium chloride solution for 2 h at room temperature while shaking at 30 min intervals to elute the alcian blue bound to the mucosa of the stomachs. Two hours later, the stomachs were removed and 5 mL of each aliquot of magnesium chloride solution containing the alcian blue eluted from each stomach was shaken with 5 mL of diethyl ether. The aqueous phase was separated out, centrifuged at 3200 × g for 5 min and the absorbance of the supernatant was measured at 605 nm. The amount of alcian blue bound per stomach in micrograms was determined using a standard calibration curve. 2.10.3. Antihistamine activity Eighteen rats (9 male rats and 9 female rats) were selected and their fur on posterior left lateral side was shaved under ether anesthesia. Twenty-four hours later, these rats were randomly divided into 3 groups (3 male rats and 3 female rats/group) and
3.1. Phytochemical screening of HWE Phytochemical screening revealed the presence of alkaloids, polyphenols, flavonoids, steroids, saponins and tannins in the extract. 3.2. Total polyphenol and flavonoid content in HWE The total polyphenolic content and flavonoid content in HWE were 65.8 ± 0.50 mg gallic acid equivalents/g extract and 45.4 ± 0.76 mg quercetin equivalents/g extract, respectively. 3.3. Effects on ethanol-induced gastric lesions Trichosanthes cucumerina HWE caused a significant (P < 0.05) inhibition of the length and the number of gastric lesions (Table 1) induced by absolute ethanol in a dose dependent (r = 0.94) manner. Among the tested doses, high dose (750 mg/kg) showed the maximum inhibition of the length (by 92%) and number of gastric lesions (by 88%) followed by mid (length: by 78%; number: by 77%) and low (length: by 45%; number: by 48%) doses. The gastroprotective activity of 750 mg/kg dose of HWE was comparable with those of cimetidine and sucralfate, the reference drugs (cimetidine: inhibition of length by 93% and number by 92%: sucralfate: inhibition of length by 96% and number by 95%). 3.4. Effects on indomethacin-induced gastric lesions Trichosanthes cucumerina HWE at a dose of 750 mg/kg significantly (P < 0.05) reduced the length (by 88%) and number of gastric lesions (by 84%) compared to the control group (Table 1). Similar to the absolute ethanol-induced gastric ulceration model, gastroprotective activity of the 750 mg/kg dose of HWE was comparable to the reference drug, cimetidine (inhibition of length by 90% and number by 90%). 3.5. Acid secretion studies Compared with controls, the rats treated with HWE showed significant (P < 0.05) reduction in free acidity (by 40%) and total acidity (by 36%) in the gastric juice (Table 2). Further, pH of the gastric juice increased from 4.1 to 6.0. However, no change in the volume of gastric juice was observed. 3.6. Gastric mucus studies Administration of Trichosanthes cucumerina HWE significantly (P < 0.05) increased the amount of mucus produced by the rat
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Table 1 Effect of Trichosanthes cucumerina hot water extract (HWE) on the length of gastric lesions (in mm) and number of lesions induced by absolute ethanol and indomethacin. The length of haemorrhagic lesions (mm) Ethanol-induced gastric ulceration model Control 375 mg/kg of HWE 500 mg/kg of HWE 750 mg/kg of HWE Cimetidine (100 mg/kg) Sucralfate (400 mg/kg)
71.5 39.2 15.9 5.7 4.8 3.1
Indomethacin-induced gastric ulceration model Control 750 mg/kg of HWE Cimetidine (100 mg/kg)
74.8 ± 3.7 8.9 ± 1.1* 7.3 ± 0.9*
± ± ± ± ± ±
The numbers of haemorrhagic lesions
4.8 3.6* 2.2* 1.3* 0.9* 0.7*
17.4 9.0 4.0 2.1 1.2 0.9
± ± ± ± ± ±
1.8 0.8* 0.7* 0.5* 0.3* 0.2*
17.8 ± 1.6 2.9 ± 0.4* 1.7 ± 0.2*
Values are expressed as mean ± S.E.M.; n = 8. * Significant when compared with respective controls P ≤ 0.05. Table 2 Effect of Trichosanthes cucumerina hot water extract (HWE) on gastric juice volume, pH, free acidity and total acidity. Treatment
pH
Gastric juice volume (mL)
Free acidity (mol L−1 )
Total acidity (mol L−1 )
Control Hot water extract (750 mg/kg)
4.1 ± 0.3 6.0 ± 0.2*
5.8 ± 0.2 5.6 ± 0.2
0.05 ± 3.0 × 10−3 0.03 ± 3.1 × 10−3 *
0.082 ± 4.7 × 10−3 0.052 ± 2.5 × 10−3 *
Values are expressed as mean ± S.E.M.; n = 6. * Significant when compared with respective controls P ≤ 0.05.
gastro mucosa (control vs treatment: 189.6 ± 8.8 g/stomach vs 263.1 ± 4.4 g/stomach). 3.7. Antihistamine activity Trichosanthes cucumerina HWE showed potent antihistamine activity as there was a significant (P < 0.05) reduction (by 27%) in the wheal area of the rats when compared with the control group (Table 3). Further, the effect of the HWE was comparable with that of chlorpheniramine, the reference drug. 4. Discussion Ethanol and indomethacin are commonly used for inducing ulcer in experimental rats (Umamaheswari et al., 2007; Nguelefack et al., 2008; Rao et al., 2008). Ethanol is one of the ulcerogenic agents that induces intense damage in gastric mucosa by promoting disturbances of mucosal microcirculation, ischemia and appearance of free radicals, endothelin release, degranulation of mast cells, inhibition of prostaglandins and decrease of gastric mucus production (Park et al., 2004). Indomethacin is a nonsteroidal antiinflammatory drug (NSAID) that inhibits prostaglandin production that results in increased acid production and decreased cytoprotective mucus formation, which can lead to induce gastrointestinal ulcer (Wallace and Devchand, 2005). Trichosanthes cucumerina possesses marked gastroprotective properties as evidenced by its significant inhibition of the formation of gastric lesions (in terms of length and number) induced by absolute ethanol and indomethacin. The gastroprotective activity of 750 mg/kg dose of HWE was comparable to that reference drugs (cimetidine and Table 3 Antihistamine activity of hot water extract (HWE) of Trichosanthes cucumerina as indicated by the area of the wheal. Treatment
Area of the wheal (mm2 )
Control Hot water extract (750 mg/kg) Chlorpheniramine (0.40 mg/kg)
73.2 ± 4.2 53.1 ± 6.6* 46.2 ± 5.5*
Values are expressed as mean ± S.E.M.; n = 6. * Significant when compared with respective controls P ≤ 0.05.
sucralfate) used in each experimental model. Further, in the ethanol model, the gastroprotective effect was dose-dependent and dose response curve was linear. In the present investigation, it has been demonstrated that HWE can significantly enhance gastric mucus secretion while reducing the acidity of the gastric juice in rats. Gastric mucus is an important protective factor for the gastric mucosa and it is capable of acting as an antioxidant agent and reducing mucosal damage mediated by oxygen free radicals (Berenguer et al., 2007). However, the protective properties of the mucus barrier depend not only on the gel structure but also on the amount or thickness of the layer covering the mucosal surface (Penisi and Piezzi, 1999). Administration of HWE significantly (P < 0.05) increased the amount of mucus produced by the rat gastro mucosa compared to their respective controls. Therefore, the enhanced mucus secretion after administration of HWE may help to protect against the absolute ethanol and indomethacin-induced damage by preventing the action of acid and pepsin on the stomach mucous epithelium (Laurence and Bennet, 1992). Similar mode of action has been reported with several other plants also (Moraes et al., 2008; Hiruma-Lima et al., 2009). It is also well known that prostaglandins synthesized in large quantities by the gastrointestinal mucosa can prevent experimentally induced ulcers by ulcerogens. Thus, when the ulcer lesions are induced by absolute ethanol or indomethacin, the cytoprotective effect of the antiulcer agent can mediated through endogeneous prostaglandins (Yamamoto et al., 1992). Therefore, it can be thought that HWE may stimulate the secretion of prostaglandin or possess prostaglandin like substances. Another important protective factor is the inhibition of acid secretion (Bandyopadhyay et al., 2002; Konturek et al., 2005), since when levels of acid overwhelm mucosal defense mechanisms this lead to ulcer formation (Schubert, 2004). In our study, HWE caused significant inhibition in the acidity (both total and free) with an elevation in gastric pH. Similar mode of action has also been reported in some other plant extracts (Umamaheswari et al., 2007; Nguelefack et al., 2008; Hiruma-Lima et al., 2009). The reduction of acidity of gastric juice mediated by HWE could be attributed to its antihistamine effect, because it is well established that antihistamine drugs like cimetidine which block H2 receptors in the stomach, reduce the acidity of gastric juice (Laurence and Bennet, 1992). Although, H2 receptor blockers are also expected to reduce
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the secretion of gastric juice (Laurence and Bennet, 1992) such an effect was not observed in the present study. H2 antagonists are recommended for the prevention of gastric lesions induced by NSAIDs (Eccles et al., 1998). Therefore, the antihistamine activity in HWE may also contribute to the protective activity against indomethacin-induced gastric lesions. Phytochemical screening of Trichosanthes cucumerina revealed the presence of alkaloids, saponins, tannins, flavonoids and other phenolic compounds. Polyphenols, especially tannins, are phytochemicals with antioxidant properties. These compounds have also shown antiulcerogenic properties due to their protein precipitating and vasoconstricting effects (Berenguer et al., 2006). Their astringent action can help precipitating microproteins on the ulcer site, thereby forming an impervious layer over the lining that hinders gut secretions and protects the underlying mucosa from toxic and other irritants (Nwafor et al., 2000). The antiulcerogenic effect of many plants is related to their flavonoid content, as these antioxidants inhibit lipid peroxidation and other free radical mediated processes that lead to gastric damage (Gurbuz et al., 2009). Moreover, saponins (Morikawa et al., 2006) and alkaloids (Li et al., 2005) have been shown to have potent gastroprotective activities. Therefore, secondary metabolites such as alkaloids, saponins, tannins, flavonoids and other phenolic compounds present in Trichosanthes cucumerina may also contribute to its gastroprotective effect. In conclusion, this study showed a potent gastroprotective activity of Trichosanthes cucumerina hot water extract in absolute ethanol and indomethacin-induced gastric lesions for the first time and indicate its therapeutic potential to be used as a cost effective herbal gastroprotective agent. Acknowledgement The authors express their gratitude to National Science Foundation for the Research Grant (NSF/SCH/2005/13). References Anonymous, 1976. The Wealth of India, vol. 10. The Dictionary of Indian Raw Materials and Industrial Products. Publication and Information directorate, CSIR, New Delhi, p. 291. Anonymous, 2002. Compendium of Medicinal Plants, vol. 2. A Publication of Department of Ayurveda, Sri Lanka, pp. 146–150. Bandyopadhyay, U., Biswas, K., Chatterjee, R., Bandyopadhyay, D., Chattopadhyay, I., Ganguly, C.K., Chakraborty, T., Bhattacharya, K., Banerjee, R.K., 2002. Gastroprotective effect of Neem (Azadirachta indica) bark extract: possible involvement of H(+)–K(+)-ATPase inhibition and scavenging of hydroxyl radical. Life Science 71, 2845–2865. Berenguer, B., Sanchez, L.M., Quilez, A., Lopez-Barreiro, M., de Haro, O., Galvez, J., Martin, M.J., 2006. Protective and antioxidant effects of Rhizophora mangle L. against NSAID-induced gastric ulcers. Journal of Ethnopharmacology 103, 194–200. Berenguer, B., Trabadela, C., Sanchez-Fidalgo, S., Quilez, A., Mino, P., Puerta, R.D.I., Martin-Calero, M.J., 2007. The aerial parts of Guazuma ulmifolia Lam. protect against NSAID-induced gastric lesions. Journal of Ethnopharmacology 114, 153–160. Bhargava, K.P., Gupta, M.B., Tangri, K.K., 1973. Mechanism of ulcerogenic activity of indomethacin and oxyphenylbutazone. European Journal of Pharmacology 22, 191–195. Corne, S.J., Morrisey, S.M., Woods, K.J., 1974. A method for the quantitative estimation of gastric barrier mucus. Journal of Physiology 2452, 116–117. Eccles, M., Freemantle, N., Mason, J., 1998. North of England evidence based-guideline development project: summary guideline for non-steroidal anti-inflammatory drugs versus basic analgesia in treating the pain of degenerative arthritis. British Medical Journal 317, 526–530. Farnsworth, N.R., 1996. Biological and phytochemical screening of plants. Journal of Pharmaceutical Science 55, 225–276. Gurbuz, I., Yesilada, E., Ito, S., 2009. An anti-ulcerogenic flavonol diglucoside from Equisetum palustre L. Journal of Ethnopharmacology 121, 360–365. Hiruma-Lima, C.A., Rodrigues, C.M., Kushima, H., Moraes, T.M., Lolis, S.D.F., Feitosa, S.B., Magri, L.P., Soares, F.R., Cola, M.M., Andrade, F.D.P., Vilegas, W., Brito, A.R.M.S., 2009. The anti-ulcerogenic effects of Curatella americana L. Journal of Ethnopharmacology 121, 425–432. Jayaweera, D.M.A., 1980. Medicinal Plants (Indigenous and Exotic) used in Ceylon (Part 2). A Publication of the National Science Council of Sri Lanka, pp. 162–163.
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