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Antiulcer activity of methanolic extract of Momordica charantia L. in rats
Journal of Ethnopharmacology 123 (2009) 464–469
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Antiulcer activity of methanolic extract of Momordica charantia L. in rats Samsul Alam a , Mohammed Asad a,∗ , Syed Mohammed Basheeruddin Asdaq a , V. Satya Prasad b a b
Department of Pharmacology, Krupanidhi College of Pharmacy, Bangalore 560 034, India Department of Anatomy, MNR Medical College, Sangareddy, Andhra Pradesh, India
a r t i c l e
i n f o
Article history: Received 31 July 2008 Received in revised form 5 March 2009 Accepted 13 March 2009 Available online 26 March 2009 Keywords: Momordica charantia L. Cucurbitaceae Gastric ulcer Duodenal ulcer Gastric secretion Gastric cytoprotection
a b s t r a c t Aim of the study: Momordica charantia L. (Cucurbitaceae) commonly known as ‘bitter gourd’ is a multi purpose herb cultivated in different parts of the world for its edible fruits. The objective of the present study was to evaluate the effect of standardized methanolic extract of Momordica charantia L. fruits on gastric and duodenal ulcers. Materials and methods: The effect was evaluated in acetic acid induced chronic gastric ulcer, pylorus ligation induced gastric ulcer, ethanol induced gastric ulcer, stress induced gastric ulcer, indomethacin induced gastric ulcer and cysteamine induced duodenal ulcer model. The extract was administered orally at two different doses of 100 mg/kg and 500 mg/kg. Results: The healing of acetic acid induced gastric ulcer was increased by both doses of the extract. In pylorus-ligated rats, the extract showed significant decrease in ulcer index, total acidity, free acidity and pepsin content and an increase in gastric mucosal content. The extract also reduced the ulcer index in stress induced, ethanol induced and indomethacin induced gastric ulcers and cysteamine induced duodenal ulcer. Conclusion: The methanolic extract of Momordica charantia L. fruit increases healing of gastric ulcer and also prevents development of gastric ulcers and duodenal ulcers in rats. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The fruits of Momordica charantia L. (Cucurbitaceae) are used as vegetable in different parts of the world. Apart from their use for consumption, the fruits are reported to possess wide range of pharmacological activities such as hypoglycaemic (Zheng et al., 2005), antidiabetic (Sathishsekar and Subramanian, 2005a), antifungal (Schmourlo et al., 2005), inhibition of p-glycoproteins (Limtrakul et al., 2004), antihyperlipidemic (Chen and Li, 2005) and antioxidant effects (Sathishsekar and Subramanian, 2005b). The fruits are used traditionally as anthelmintic, antiemetic, carminative, purgative and for the treatment of anemia, jaundice, malaria, cholerea, etc. (Ross, 1999). In Turkey, the olive oil extract of fresh fruits or dry fruit mixed with honey is used for the treatment of peptic ulcer (Gurbuz et al., 2000; Dengiz and Gursan, 2005). Earlier study carried out with olive oil extract of fresh fruits, dry fruit in honey and ethanol extract of dry fruits reported that they all possess gastric cytoprotective effect in rats (Gurbuz et al., 2000). The fruits are also reported to prevent the growth of Helicobacter pylori, the organism responsible for development of gastric and duodenal ulcers
∗ Corresponding author at: Krupanidhi College of Pharmacy, # 5, Sarjapur Road, Koramangala, Bangalore 560 034, India. Tel.: +91 80 25535751; fax: +91 80 51309161. E-mail address: [email protected] (M. Asad). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.03.024
(Yesilada et al., 1999). However, there is no scientific report on the effect of fruits on gastric ulcer healing, on gastric offensive factors such as gastric acid and gastric pepsin secretion or on gastric defensive factors such as gastric mucus secretion. The present study evaluates the effect of standardized methanolic extract of Momordica charantia L. fruits on experimentally induced gastric and duodenal ulcers in rats to determine its effect on gastric secretion, gastric cytoprotection and development of duodenal ulcers in rats. 2. Materials and Methods 2.1. Extraction of fruits The fruits of Momordica charantia L. were purchased from local supplier, who collected them in the month of May 2006. The fruits were dried and extracted using 70% methanol in a Soxhlet apparatus. The yield was around 7.8% (w/w). 2.2. Estimation of bitter substances as lignan (Nalkami, 1989) The extract (3 g) was refluxed with 50 ml alcohol in a water bath for 30 min and filtered. The residue was further refluxed until bitterness. The alcohol from the combined filtrate was removed under reduced pressure and the residue was treated repeatedly with 25
S. Alam et al. / Journal of Ethnopharmacology 123 (2009) 464–469
and 15 ml hot water. The hot water washings were combined and shaken repeatedly with 25, 20 and 15 ml of ethyl acetate. The ethyl acetate fraction was collected and evaporated to dryness to get a constant weight. 2.3. Estimation of charantin (Pitiphanpong et al., 2005) This was carried out using high performance liquid chromatography (HPLC) procedure using a C-18 inertsil ODS-3 column (5 m particle, 4.6 mm × 250 mm ID). Methanol:water (100:2) was used as a mobile phase with a flow rate of 1 ml/min at 204 nm UV detection. A sample volume of 20 l was taken. A standard calibration curve was made by plotting peak area versus concentration. The retention time was around 12 min. Apart from estimation of bitter principles and charantin, the loss of weight on drying, ash content and heavy metals content were determined to ascertain whether these are within the limits described by the British Pharmacopoeia (2005). 2.4. Experimental animals Albino Wistar rats of either sex weighing between 200 and 250 g were used. Institutional Animal Ethics Committee approved the experimental protocol. Animals were maintained under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision on Experiment on Animals (CPCSEA). 2.5. Drugs and dosage The dried extract was suspended in water using 0.5% carboxy methyl cellulose. The extract was administered orally at two different doses of 100 and 500 mg/kg (Shibib et al., 1993; Sarkar et al., 1996). The effect observed with the extract was compared with standard antisecretory agent (ranitidine: 50 mg/kg) (Asad et al., 2001) or gastric cytoprotective agent (misoprostol: 100 g/kg) (Brzozowski et al., 1998).
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(a) Regenerated lining epithelial width: Regenerated lining epithelial width is defined as the average distance from the origin of regenerated lining epithelium to the surface of the ulcer. (b) Capillary density within the granulation tissues of the ulcers: The capillary density was determined using eyepiece reticule (×100 magnification) on the H and E stained sections in the ulcer centre, each field was 140 m × 140 m. At least four fields were examined on each section. Capillary density within the granulation tissues of the ulcers was expressed as the average capillary numbers in the field. (c) Collagen content within the scar tissues of ulcers: Collagen in scar tissues displayed blue colour in sections stained with Masson’s stain and was determined by point count using 1 square eye piece reticule (×100 magnification). Collagen content was expressed as: Volume =
Total number of points falling on the tissue Total number of points in the reticule
(d) Regenerated glandular epithelium: It is the total length of the epithelium found in the secretory gland. 2.6.2. Pylorus ligation induced ulcers (Shay et al., 1945; Kulkarni, 1999) Albino Wistar rats were fasted for 36 h with water ad libitum. Normal saline (1 ml/rat p.o.) was administered twice daily to all the animals during the fasting period. Under light ether anesthesia, pyloric ligation was performed. The drugs were administered intraduodenally immediately after pylorus ligation. The intraduodenal injection was made using a syringe with 26 G needle directly into the duodenum. The animals were deprived of food and water during the postoperative period and were sacrificed 6 h after pylorus ligation by over dose of ether anesthesia. The stomach was isolated and the content of the stomach was used for estimation of free and total acidity (Hawk et al., 1947), pepsin content (Debnath et al., 1974), mucin content (Corne et al., 1974) and total proteins (Lowry et al., 1951). The stomach was cut opened along the greater curvature and ulcer index was determined as mentioned above.
2.6. Experimental models 2.6.1. Acetic acid induced ulcer (Asad et al., 2001) Under light ether anaesthesia, laporatomy was performed on 24 h fasted rats. Glacial acetic acid (0.05 ml) was applied onto the serosal surface of the stomach using a cylindrical mould of 6 mm diameter for 1 min. The acid solution was then removed by rinsing the mould with normal saline to avoid damage to the surrounding tissues. The stomach was placed back carefully and the abdominal wall was closed by interrupted sutures. The animals were treated with ranitidine (50 mg/kg p.o.) or extract of Momordica charantia L. either at both doses once daily for 10 days. Animals in the control group received only vehicle. Rats were sacrificed on the 10th day, stomachs were removed and cut opened along the greater curvature and the ulcer index was determined using the formula (Ganguly, 1969). Ulcer index =
10 X
where X = total mucosal area/total ulcerated area. The ulcers were given scores based on their intensity as follows: 0: no ulcer, 1: superficial mucosal erosion, 2: deep ulcer or transmural necrosis, 3: perforated or penetrated ulcer. The tissues were subsequently processed for histological examination. Four indices namely regenerated glandular epithelial width; capillary density, collagen content and surface epithelium were selected to reflect the rate and quality of ulcer healing (Wang et al., 1999).
2.6.3. Ethanol induced ulcers (Brzozowski et al., 1998) The animals were fasted for 36 h before administration of ethanol. The drugs were administered 1 h before ethanol administration and 1 h after ethanol administration, the animals were sacrificed and ulcer index was determined. 2.6.4. Cold restraint stress induced ulcers (Vincent et al., 1977) The drugs were administered 30 min before subjecting the animals to cold restraint stress. Stress was induced by placing the animals in a restraint cage at a temperature of 2–3 ◦ C for 2.5 h. The animals were then sacrificed by over dose of ether anesthesia and ulcer index was determined as mentioned above. 2.6.5. Healing of indomethacin induced gastric ulcers (Majumdar et al., 2003) Gastric ulcers were induced by administering indomethacin (5 mg/kg p.o.) for 5 days, during this period the animals were fed normally. The last day of indomethacin treatment was considered as 0th day. The animals were then treated either with misoprostol (100 g/kg p.o.) or with extract of Momordica charantia L. at both doses once daily for 5 days after induction of ulcer while the control group received only vehicle. Rats were sacrificed on the 0th day and 5th day, 6 h after the last treatment. The stomachs were removed and they were cut opened along the greater curvature and ulcer score and ulcer index were determined. The glandular portion of the stomach was used for estimation of mucin content, total proteins and activities of antioxidant factors such as catalase
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Table 1 Effect of 70% methanolic extract of Momordica charantia L. fruits on healing of acetic acid induced chronic gastric ulcers. Treatment
Regenerated glandular epithelium width (m)
Vehicle (1 ml/kg p.o.) Ranitidine (50 mg/kg p.o.) Momordica charantia L. extract (100 mg/kg p.o.) Momordica charantia L. extract (500 mg/kg p.o.)
279.73 356.38 395.79 329.79
± ± ± ±
17.214 10.075** 09.738** 14.282*
Capillary density (no.) in 19,600 m2 5.63 5.24 7.00 6.37
± ± ± ±
0.427 0.433 0.223 0.657
Volume of collagen content 0.117 0.124 0.114 0.186
± ± ± ±
Surface epithelium (m)
0.003 0.004 0.007 0.002**
162.75 144.78 156.98 146.15
± ± ± ±
04.25 10.90 06.68 12.50
Ulcer score 1.75 0.50 1.00 0.66
± ± ± ±
Ulcer index
0.250 0.223** 0.250 0.210*
0.16 0.06 0.02 0.02
± ± ± ±
0.04 0.01** 0.01** 0.01**
All values are mean ± SEM, n = 5–6. * p < 0.05 when compared to vehicle treated group. ** p < 0.01 when compared to vehicle treated group.
(Link, 1988) and super oxide dismutase (SOD) (Elstnev and Heupel, 1976).
3.2. Effect of Momordica charantia L. on ulcer healing in acetic acid induced chronic gastric ulcers
2.6.6. Cysteamine induced duodenal ulcers (Szabo, 1978) Duodenal ulcers were induced by administering cysteamine hydrochloride (400 mg/kg p.o.) twice at an interval of 4 h. Extract of Momordica charantia L. at both doses or ranitidine (50 mg/kg p.o.) was administered 30 min prior to each dose of cysteamine hydrochloride. The animals were sacrificed 24 h after first dose of cysteamine and the duodena were excised carefully. The duodenum was cut opened along the antimesentric side and the duodenal ulcer area, ulcer score and ulcer index were determined. The ulcers were given scores based on their intensity as follows: 0: No ulcer, 1: superficial mucosal erosion, 2: deep ulcer or transmural necrosis and 3: perforated or penetrated ulcer. The ulcer index was calculated using the following equation (Desai et al., 1999):
The higher dose of extract and ranitidine showed a significant reduction in ulcer index and ulcer score while the lower dose significantly reduced ulcer index when compared to the control (p < 0.01). Histopathological studies of the ulcerated tissue supported the macroscopic findings (Table 1).
Ulcer index = Arithmetic mean of intensity in a group +
Number of ulcer positive animals ×2 Total number of animals
3.3. Effect of Momordica charantia L. extract on pylorus ligation induced gastric ulcers The higher dose of Momordica charantia L. extract (500 mg/kg p.o.) and ranitidine showed antiulcer effect as indicated by a significant reduction in ulcer index, free and total acidity and pepsin activity (p < 0.01). However, the lower dose of extract did not show any significant reduction in ulcer index but increased the protein content of gastric juice. The mucin content was significantly increased by all treatments (Table 2).
3.4. Effect of Momordica charantia L. extract on ulcer index in ethanol induced gastric ulcers
2.6.7. Statistical analysis The statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Dunnet comparison test. For comparing nonparametric ulcer scores, ANOVA followed by nonparametric Dunn post-test was used. The values are expressed as mean ± SEM and p < 0.05 was considered significant.
The methanolic extract of both doses of Momordica charantia L. and misoprostol showed a significant reduction in ulcer index when compared to the control. The ulcer index was 0.83 ± 0.07 in the vehicle treated group, while the ulcer index in lower dose treated group was 0.32 ± 0.019 and in higher dose treated group, it was 0.19 ± 0.011. Misoprostol was most effective; it reduced the ulcer index to 0.091 ± 0.011.
3. Results 3.1. Analysis of extract
3.5. Effect of Momordica charantia L. extract on ulcer index in stress induced gastric ulcers
The extract was found to contain 8.8% (w/w) of bitter principles and 0.51% w/w of charantin. The loss of weight on drying (1%), ash content (32%) and level of heavy metals were within the British Pharmacopoeia limits.
The methanolic extract of Momordica charantia L. and ranitidine showed a significant reduction in ulcer index when compared to the control (Table 3).
Table 2 Effect of 70% methanolic extract of Momordica charantia L. fruits on pylorus-ligated rats. Treatment
Free acidity (mEq/l)
Vehicle (1 ml/kg p.o.) Ranitidine (50 mg/kg p.o.) Momordica charantia L. extract (100 mg/kg p.o.) Momordica charantia L. extract (500 mg/kg p.o.)
7.035 2.548 4.235 3.714
All values are mean ± SEM, n = 5–6. * p < 0.05 when compared to vehicle treated group. ** p < 0.01 when compared to vehicle treated group.
± ± ± ±
0.804 0.179** 0.384** 0.515**
Total acidity (mEq/l) 13.28 7.34 8.72 9.55
± ± ± ±
0.639 0.685** 0.508** 1.001**
Ulcer index 0.0515 0.0219 0.0461 0.0257
± ± ± ±
0.00217 0.00507** 0.00146 0.00761**
Mucin content (g/g) 0.055 0.108 0.092 0.127
± ± ± ±
0.007 0.009** 0.009* 0.011**
Pepsin content (g/6 h) 0.355 0.207 0.240 0.183
± ± ± ±
0.0206 0.0105** 0.0186** 0.0091**
Total proteins (mg/ml) 11.86 0.81 4.67 9.00
± ± ± ±
0.800 1.298** 0.957* 2.150
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Table 3 Effect of 70% methanolic extract of Momordica charantia L. fruits on stress induced gastric ulcers and cysteamine induced duodenal ulcers. Treatment
Vehicle (1 ml/kg p.o.) Ranitidine (50 mg/kg) Momordica charantia L. extract (100 mg/kg) Momordica charantia L. extract (500 mg/kg)
Stress induced
Cysteamine induced duodenal ulcer
Ulcer index
Ulcer area
0.177 0.021 0.075 0.020
± ± ± ±
0.041 0.007** 0.012* 0.006**
7.166 1.833 2.166 1.660
± ± ± ±
Ulcer score
1.956 1.167* 1.406* 0.760*
2.00 0.33 0.66 0.50
± ± ± ±
Ulcer index
0.564 0.210** 0.330* 0.223*
3.66 0.99 1.66 1.50
All values are mean ± SEM, n = 5–6. * p < 0.05 when compared to vehicle treated group. ** p < 0.01 when compared to vehicle treated group.
Table 4 Effect of 70% methanolic extract of Momordica charantia L. fruits on healing of indomethacin induced gastric ulcers. Treatment
Mucin content (g/gm)
Control 1 (5-day indomethacin + 5-day vehicle) Control 2 (5-day indomethacin) Misoprostol (100 g/kg) Momordica charantia L. extract (100 mg/kg) Momordica charantia L. extract (500 mg/kg)
0.585 0.733 1.597 1.040 0.768
± ± ± ± ±
0.045 0.795 0.180** 0.045 0.036
Ulcer index (mg/ml) 0.606 0.246 0.048 0.182 0.062
± ± ± ± ±
0.186 0.046 0.010** 0.049 0.031**
Total proteins of proteins) 25.46 30.37 89.99 55.19 29.20
± ± ± ± ±
2.277 2.213 2.983** 7.069** 4.044
SOD (units/mg of proteins) 58.41 40.33 13.69 27.52 47.74
± ± ± ± ±
5.28 2.21 0.51** 3.93** 12.41
Catalase (units/mg) 784.85 412.22 91.68 130.62 99.44
± ± ± ± ±
99.46 25.29** 10.08** 29.66** 7.29**
All values are mean ± SEM, n = 5–6. ** p < 0.01 when compared to control 1 group.
3.6. Effect of Momordica charantia L. extract on healing of indomethacin induced gastric ulcers The ulcer index was significantly reduced by higher dose of extract of (500 mg/kg p.o.) and misoprostol. The protein content was increased by lower dose of the extract (100 mg/kg p.o.) and misoprostol. The activities of antioxidant enzymes were reduced, but the higher dose of extract showed nonsignificant reduction in SOD activity (Table 4). 3.7. Effect of Momordica charantia L. extract on ulcer area, ulcer score and ulcer index in cysteamine induced duodenal ulcers The methanolic extract of Momordica charantia L. showed significant reduction in ulcer area and ulcer score when compared to the control (Table 3). 4. Discussion The standardized methanolic extract of Momordica charantia L. fruits showed good antiulcer activity as indicated by its effect on the healing of gastric ulcers induced by acetic acid model and prevention of gastric and duodenal ulcer formation in other models. The ulcer formation in each of these models occurs by different mechanisms. The healing of chronic gastric ulcer induced by acetic acid closely resembles to the healing of human peptic ulcers (Okabe et al., 1971). Pylorus ligation induces gastric ulcers due to accumulation of gastric secretion in the stomach (Shay et al., 1945). The methanolic extract of Momordica charantia L. significantly decreased the total acidity and free acidity and increased the mucus content indicating that it has both gastric antisecretory and gastric cytoprotective effects. Ethanol is a corrosive agent that damages the gastric mucosa (Soll, 1990; Surendra, 1999). The methanolic extract of Momordica charantia L. was effective in this model and this effect may be due to increased mucus secretion that protects gastric mucosa from corrosive effects of ethanol. Stress induced gastric ulcer is a method used to evaluate the effect of drugs on gastric secretion, stress and gastrointestinal (G.I.) motility (Brodie and Hanson, 1960; Michael and Charles, 1983). The effect of the extract against stress induced ulcer may be due to its gastric antisecretory action and it is also speculated that some of the constituents present in the Momordica charantia L. extract may
have antistress effect. Indomethacin inhibits prostaglandin synthesis and this deficiency in prostaglandins is responsible for ulceration (Vedavyasa, 1999). Like in the ethanol induced gastric ulcers, the methanolic extract of Momordica charantia L. was effective in reducing the ulcer index significantly and it also increased the mucus content. This model confirmed the cytoprotective effect of the extract. The extract of Momordica charantia L. significantly reduced the activities of antioxidant factors such as SOD and catalase. The reason for this effect cannot be explained. Cysteamine induced duodenal ulcer resembles duodenal ulcer in humans (Szabo, 1978). The methanolic extract of Momordica charantia L. was effective in reducing the ulcer area. The doses of the extract were selected based on earlier reports (Sarkar et al., 1996; Shibib et al., 1993). In traditional medicine, the olive oil extract of fresh fruits prepared by macerating the fruits with olive oil for 15 days or dried fruits mixed with honey are used. Gurbuz et al. (2000) reported that olive oil extract of fresh fruits, dry fruit mixed with honey and ethanolic extract of dried fruits possess gastric cytoprotective effect against ethanol induced and diethyldithiocarbamate induced ulcerogenesis. These authors also reported that ethanolic extract of Momordica charantia L. fruits was more potent than the olive oil extract in reducing the development of ethanol plus hydrochloric acid induced gastric ulcers. The present study was carried out using methanolic extract. Methanol and ethanol possess very close polarity and the compositions of both these extracts may be more or less alike. In the present study, the methanolic extract reduced the development of ethanol induced gastric ulcers indicating gastric cytoprotective effect and this result complements the earlier findings reported by Gurbuz et al. (2000). Previous studies by Yıldırım (1994) and Dengiz and Gursan (2005) reported that olive oil extract of Momordica charantia L. possesses weak activity against indomethacin-induced gastric ulcers. Furthermore, both these reports showed that olive oil itself possesses significant antiulcer effect against indomethacin induced ulcers. However, in the present study, methanolic extract of Momordica charantia L. showed good cytoprotective effect against indomethacin induced gastric ulcers. The findings of the present study and those of earlier study by Gurbuz et al. (2000) suggests that methanolic or ethanolic extract of Momordica charantia L. fruits may have better gastric cytoprotective effect than the olive oil extract. Apart from the gastric cytoprotective effect, the present study also evaluates the effect of Momordica charantia fruits on gastric
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ulcer healing, gastric secretion and duodenal ulcers. It was assumed earlier that Momordica charantia fruits may not have gastric antisecretory effect and the ulcer healing effect observed in humans may be due to their gastric cytoprotective action alone (Gurbuz et al., 2000). But it is well known that gastric cytoprotective property alone in rats does not produce significant ulcer healing in humans (Vogel, 2002). Gastric cytoprotective agents such as misoprostol increases ulcer healing only in doses that reduce gastric acid secretion in addition to their cytoprotective effect (Hoogerwerf and Pasricha, 2001). The results of the present study suggests that ulcer healing effect of Momordica charantia fruits observed in humans may be due to both gastric antisecretory and gastric cytoprotective effects. Momordica charantia L. was reported to contain a number of flavonoids, alkaloids and many other chemical constituents (Ross, 1999). The constituent(s) responsible for antiulcer effect of the fruits are not known. A number of flavonoids, present in different plants are known to reduce gastric ulcer formation (Suzuki et al., 1998). Hence, it is assumed that flavanoids may contribute at least in part to the antiulcer effect of the fruits. Apart from flavonoids, the fruits also contain steroids such as beta sitosterol that is known to reduce the development of gastric ulcers (Xiao et al., 1992) and carotenoids that possess gastric cytoprotective action (Kamath et al., 2008). Earlier study on gastroprotective effect of Momordica charantia L. fruits by Gurbuz et al. (2000) reported that hexane soluble and hexane insoluble fractions of ethanolic extract were equally effective in reducing the development of ethanol induced gastric ulcers. Hence, the gastroprotective effect observed in the present study may be due to both polar constituents such as flavonoids and non-polar constituents such as carotenoids and sterols. Furthermore, the active constituent responsible for many of the effects of Momordica charantia L. fruits is charantin. Hence, studies should also be carried out with charantin to determine its effect on the gastric and duodenal ulcer formation. 5. Conclusion The methanolic extract of Momordica charantia L. fruits was effective in increasing the healing of gastric ulcers induced by acetic acid. The methanolic extract of the fruits also decreased the development of gastric ulcers in Shay rat model, ethanol induced gastric ulcers, indomethacin induced gastric ulcers, stress induced gastric ulcers and duodenal ulcers induced by cysteamine. The antiulcer effect of methanol extract of Momordica charantia L. is due to its gastric antisecretory and gastric cytoprotective effect. Acknowledgements The authors are thankful to Prof. Suresh Nagpal, Chairman, Krupanidhi Educational Trust (Bangalore, India), Prof. Sunil Dhamanigi, Secretary, Krupanidhi Educational Trust and Dr. Amit Kumar Das, Professor and Principal, Krupanidhi College of Pharmacy for providing facilities to carry out this work. References Asad, M., Shewade, D.G., Koumaravelou, K., Abraham, B.K., Vasu, S., Ramaswamy, S., 2001. Gastric anti-secretary, anti-ulcer activity of oxytocin in rats and guinea pigs. Life Sciences 70, 17–24. British Pharmacopoeia, 2005. Her Majesty’s Stationary Office, London, Vol. 2, pp. 1103–1129. Brodie, D.A.Q., Hanson, H.M., 1960. A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38, 353–360. Brzozowski, T., Konturek, S.J., Kwiecien, S., Pajdo, R., Brzozowski, I., Hahn, E.G., 1998. Involvement of endogenous cholecystokinin and somatostatin in gastro protection induced by intra duodenal fat. Journal of Clinical Gastroenterology 27, 125–137.
Chen, Q., Li, E.T., 2005. Reduced adiposity in bitter melon (Momordica charantia) fed rats is associated with lower tissue triglyceride and higher plasma catecholamines. British Journal of Nutrition 93, 747–754. Corne, S.J., Morrissey, S.M., Woods, R.J., 1974. A method for the quantitative estimation of gastric barrier mucus. In: Proceeding of the Physiological Society, pp. 116–117. Debnath, P.K., Gode, K.O., Govinda, D.D., Sanyal, A.K., 1974. Effect of propranolol on gastric secretion in rats. British Journal of Pharmacology 51, 213– 216. Dengiz, G.O., Gursan, N., 2005. Effects of Momordica charantia L. (Cucurbitaceae) on indomethacin-induced ulcer model in rats. Turkish Journal of Gastroenterology 16, 85–88. Desai, J.K., Goyal, R.K., Parmar, N.S., 1999. Characterization of dopamine receptor subtypes involved in experimentally induced gastric and duodenal ulcers in rats. Journal of Pharmacy and Pharmacology 51, 187–192. Elstnev, E.F., Heupel, E., 1976. Inhibition of nitrite formation from hyroxylammoniumchloride. A simple assay for super oxide dismutase. Analytical Chemistry 70, 616–620. Ganguly, A.K., 1969. A method for quantitative assessment of experimentally produced ulcers in stomach of rats. Experientia 25, 11–24. Gurbuz, I., Akyuz, C., Yesilada, E., Sener, B., 2000. Anti-ulcerogenic effect of Momordica charantia L. fruits on various ulcer models in rats. Journal of Ethnopharmacology 71, 77–82. Hawk, P.B., Oser, B.L., Summerson, H.W., 1947. Practical physiological chemistry, 12th ed. Churchill, London, p. 347. Hoogerwerf, W.A., Pasricha, P.J., 2001. Agents used for the treatment of gastric acidity and treatment of peptic ulcers and gastroesophageal reflux disease. In: Hardman, J.E., Limbard, L.E. (Eds.), Goodman’s and Gilman’s The Pharmacological Basis of Therapeutics. McGraw-Hill, New Delhi, pp. 1011–1012. Kamath, B.S., Srikanta, B.M., Dharmesh, S.M., Sarada, R., Ravishankar, G.A., 2008. Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis. European Journal of Pharmacology 590, 387–395. Kulkarni, S.K., 1999. Hand Book of Experimental Pharmacology, 3rd ed. Vallabh Prakashan, New Delhi, pp. 148–150. Limtrakul, P., Khantamat, O., Pintha, K., 2004. Inhibition of P-glycoprotein activity and reversal of cancer multidrug resistance by Momordica charantia extract. Cancer Chemotherapy Pharmacology 54, 525–530. Link, E.M., 1988. Mechanism of pH dependent hydrogen per oxide cytotoxicity invitro. Archives of Biochemistry and Biophysics 365, 362–372. Lowry, C.H., Roseborough, N.I., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. Journal of Biological Chemistry 193, 265– 275. Majumdar, B., Chaudhri, S.G.R., Ray, A., Bandyopadhyay, S.K., 2003. Effect of ethanol extract of Piper betel Linn. leaf on healing of NSAID-induced experimental ulcer. A novel role of free radical scavenging action. Indian Journal of Experimental Biology 41, 311–315. Michael, N.P., Charles, T.R., 1983. Stressful life events, acid hyper secretion and ulcer disease. Gastroenterology 84, 114–119. Nalkami, 1989. Indian Materia Medica 2, 947. Okabe, S., Roth, J.L.A., Pfeiffer, C.J., 1971. Differential healing periods of the acetic acid ulcer model in rats and cats. Experientia 27, 146–148. Pitiphanpong, J., Goto, M., Sasaki, M., Shotipruk, A., 2005. Extraction of charantin from Momordica charantia. In: Symposium of Separation Science and Technology, Osaka, Japan June 3–4, p. 2005. Ross, I.A., 1999. Medicinal Plants of the World. Humana Press, New Jersey, USA, pp. 213–219. Sarkar, S., Pranava, M., Marita, R.A., 1996. Demonstration of the hypoglycemic action of Momordica charantia in a validated animal model of diabetes. Pharmacological Research 33, 1–4. Sathishsekar, D., Subramanian, S., 2005a. Beneficial effects of Momordica charantia seeds in the treatment of STZ-induced diabetes in experimental rats. Biological and Pharmaceutical Bulletin 28, 978–983. Sathishsekar, D., Subramanian, S., 2005b. Antioxidant properties of Momordica Charantia (bitter gourd) seeds on streptozotocin induced diabetic rats. Asia Pacific Journal of Clinical Nutrition 14, 153–158. Schmourlo, G., Mendonca-Filho, R.R., Alviano, C.S., Costa, S.S., 2005. Screening of antifungal agents using ethanol precipitation and bioautography of medicinal and food plants. Journal of Ethnopharmacology 96, 563–568. Shay, H., Komarov, S.A., Fele, S.S., Meranze, D., Gruenstein, H., Siplet, H., 1945. A simple method for uniform production of gastric ulceration in rat. Gastroenterology 5, 43–46. Shibib, B.A., Khan, L.A., Rahman, R., 1993. Hypoglycaemic activity of Coccinia indica and Momordica charantia in diabetic rats: depression of the hepatic gluconeogenic enzymes glucose-6-phosphatase and fructose-1.6-bisphosphatase and elevation of both liver and red-cell shunt enzyme glucose-6-phosphate dehydrogenase. Biochemical Journal 292, 267–270. Soll, A.H., 1990. Pathogenesis of peptic ulcers and implication for therapy. New England Journal of Medicine 322, 909–916. Surendra, S., 1999. Evaluation of gastric anti-ulcer activity of fixed oil of tulsi and possible mechanism. Indian Journal of Experimental Biology 36, 253– 257. Suzuki, Y., Ishihara, M., Segami, T., Ito, M., 1998. Anti-ulcer effects of antioxidants, quercetin, alpha-tocopherol, nifedipine and tetracycline in rats. Japan Journal of Pharmacology 78, 435–441. Szabo, S., 1978. Animal model for human disease: duodenal ulcer disease. American Journal of Pathology 73, 273–276.
S. Alam et al. / Journal of Ethnopharmacology 123 (2009) 464–469 Vedavyasa, S., 1999. Gastric mucosal cellular changes induced by indomethacin male albino rats. Indian Journal of Experimental Biology 37, 365–369. Vincent, G.P., Galvin, G.B., Rutkowski, J.L., Pare, W.P., 1977. Body orientation, food deprivation and potentiation of restraint induced gastric lesions. Gastroenterology and Clinical Biology 1, 539–543. Vogel, H.G., 2002. Drug Discovery and Evaluation, 2nd ed. Springer-Verlag, Berlin, Germany, p. 871. Wang, J.Z., Wuy, J., Rao, C.M., Gao, M.T., Li, W.G., 1999. Effect of recombinant human basic fibroblast growth factor on stomach ulcers in rats and mice. Acta Pharmacologica Sinica 209, 763–768. Xiao, M., Yang, Z., Jiu, M., You, J., Xiao, R., 1992. The anti-gastroulcerative activity of beta-sitosterol-beta-d-glucoside and its aglycone in rats. Hua Xi Yi Ke Da Xue Xue Bao 23, 98–101.
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Yesilada, E., Gurbuz, I., Shibata, H., 1999. Screening of Turkish anti-ulcerogenic folk remedies for anti-Helicobacter pylori activity. Journal of Ethnopharmacology 66, 289–293. Yıldırım, O.F., 1994. Momordica charantia L. (Kudret Narı) Bitkisinin Cesitli Ekstrelerinin Sicanlarda Deneysel Olarak Olusturulan Peptik Ulserde Etkisinin Incelenmesi (Studies on the Effect of Momordica charantia L. on the Experimentally- Induced Peptic Ulcer in Rats). Yuksek Lisans tezi (Master thesis), Gazi Univ. Saglık Bilimleri Enstitusu (Gazi University, Institute of Medical Sciences). Zheng, Z.X., Teng, J.Y., Liu, J.Y., Qiu, J.H., Ouyang, H., Xue, C., 2005. The hypoglycemic effects of crude polysaccharides extract from Momordica charantia in mice. Wei Sheng Yan Jiu 34, 361–363.
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Antiulcer activity of methanolic extract of Momordica charantia L. in rats Samsul Alam a , Mohammed Asad a,∗ , Syed Mohammed Basheeruddin Asdaq a , V. Satya Prasad b a b
Department of Pharmacology, Krupanidhi College of Pharmacy, Bangalore 560 034, India Department of Anatomy, MNR Medical College, Sangareddy, Andhra Pradesh, India
a r t i c l e
i n f o
Article history: Received 31 July 2008 Received in revised form 5 March 2009 Accepted 13 March 2009 Available online 26 March 2009 Keywords: Momordica charantia L. Cucurbitaceae Gastric ulcer Duodenal ulcer Gastric secretion Gastric cytoprotection
a b s t r a c t Aim of the study: Momordica charantia L. (Cucurbitaceae) commonly known as ‘bitter gourd’ is a multi purpose herb cultivated in different parts of the world for its edible fruits. The objective of the present study was to evaluate the effect of standardized methanolic extract of Momordica charantia L. fruits on gastric and duodenal ulcers. Materials and methods: The effect was evaluated in acetic acid induced chronic gastric ulcer, pylorus ligation induced gastric ulcer, ethanol induced gastric ulcer, stress induced gastric ulcer, indomethacin induced gastric ulcer and cysteamine induced duodenal ulcer model. The extract was administered orally at two different doses of 100 mg/kg and 500 mg/kg. Results: The healing of acetic acid induced gastric ulcer was increased by both doses of the extract. In pylorus-ligated rats, the extract showed significant decrease in ulcer index, total acidity, free acidity and pepsin content and an increase in gastric mucosal content. The extract also reduced the ulcer index in stress induced, ethanol induced and indomethacin induced gastric ulcers and cysteamine induced duodenal ulcer. Conclusion: The methanolic extract of Momordica charantia L. fruit increases healing of gastric ulcer and also prevents development of gastric ulcers and duodenal ulcers in rats. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The fruits of Momordica charantia L. (Cucurbitaceae) are used as vegetable in different parts of the world. Apart from their use for consumption, the fruits are reported to possess wide range of pharmacological activities such as hypoglycaemic (Zheng et al., 2005), antidiabetic (Sathishsekar and Subramanian, 2005a), antifungal (Schmourlo et al., 2005), inhibition of p-glycoproteins (Limtrakul et al., 2004), antihyperlipidemic (Chen and Li, 2005) and antioxidant effects (Sathishsekar and Subramanian, 2005b). The fruits are used traditionally as anthelmintic, antiemetic, carminative, purgative and for the treatment of anemia, jaundice, malaria, cholerea, etc. (Ross, 1999). In Turkey, the olive oil extract of fresh fruits or dry fruit mixed with honey is used for the treatment of peptic ulcer (Gurbuz et al., 2000; Dengiz and Gursan, 2005). Earlier study carried out with olive oil extract of fresh fruits, dry fruit in honey and ethanol extract of dry fruits reported that they all possess gastric cytoprotective effect in rats (Gurbuz et al., 2000). The fruits are also reported to prevent the growth of Helicobacter pylori, the organism responsible for development of gastric and duodenal ulcers
∗ Corresponding author at: Krupanidhi College of Pharmacy, # 5, Sarjapur Road, Koramangala, Bangalore 560 034, India. Tel.: +91 80 25535751; fax: +91 80 51309161. E-mail address: [email protected] (M. Asad). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.03.024
(Yesilada et al., 1999). However, there is no scientific report on the effect of fruits on gastric ulcer healing, on gastric offensive factors such as gastric acid and gastric pepsin secretion or on gastric defensive factors such as gastric mucus secretion. The present study evaluates the effect of standardized methanolic extract of Momordica charantia L. fruits on experimentally induced gastric and duodenal ulcers in rats to determine its effect on gastric secretion, gastric cytoprotection and development of duodenal ulcers in rats. 2. Materials and Methods 2.1. Extraction of fruits The fruits of Momordica charantia L. were purchased from local supplier, who collected them in the month of May 2006. The fruits were dried and extracted using 70% methanol in a Soxhlet apparatus. The yield was around 7.8% (w/w). 2.2. Estimation of bitter substances as lignan (Nalkami, 1989) The extract (3 g) was refluxed with 50 ml alcohol in a water bath for 30 min and filtered. The residue was further refluxed until bitterness. The alcohol from the combined filtrate was removed under reduced pressure and the residue was treated repeatedly with 25
S. Alam et al. / Journal of Ethnopharmacology 123 (2009) 464–469
and 15 ml hot water. The hot water washings were combined and shaken repeatedly with 25, 20 and 15 ml of ethyl acetate. The ethyl acetate fraction was collected and evaporated to dryness to get a constant weight. 2.3. Estimation of charantin (Pitiphanpong et al., 2005) This was carried out using high performance liquid chromatography (HPLC) procedure using a C-18 inertsil ODS-3 column (5 m particle, 4.6 mm × 250 mm ID). Methanol:water (100:2) was used as a mobile phase with a flow rate of 1 ml/min at 204 nm UV detection. A sample volume of 20 l was taken. A standard calibration curve was made by plotting peak area versus concentration. The retention time was around 12 min. Apart from estimation of bitter principles and charantin, the loss of weight on drying, ash content and heavy metals content were determined to ascertain whether these are within the limits described by the British Pharmacopoeia (2005). 2.4. Experimental animals Albino Wistar rats of either sex weighing between 200 and 250 g were used. Institutional Animal Ethics Committee approved the experimental protocol. Animals were maintained under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision on Experiment on Animals (CPCSEA). 2.5. Drugs and dosage The dried extract was suspended in water using 0.5% carboxy methyl cellulose. The extract was administered orally at two different doses of 100 and 500 mg/kg (Shibib et al., 1993; Sarkar et al., 1996). The effect observed with the extract was compared with standard antisecretory agent (ranitidine: 50 mg/kg) (Asad et al., 2001) or gastric cytoprotective agent (misoprostol: 100 g/kg) (Brzozowski et al., 1998).
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(a) Regenerated lining epithelial width: Regenerated lining epithelial width is defined as the average distance from the origin of regenerated lining epithelium to the surface of the ulcer. (b) Capillary density within the granulation tissues of the ulcers: The capillary density was determined using eyepiece reticule (×100 magnification) on the H and E stained sections in the ulcer centre, each field was 140 m × 140 m. At least four fields were examined on each section. Capillary density within the granulation tissues of the ulcers was expressed as the average capillary numbers in the field. (c) Collagen content within the scar tissues of ulcers: Collagen in scar tissues displayed blue colour in sections stained with Masson’s stain and was determined by point count using 1 square eye piece reticule (×100 magnification). Collagen content was expressed as: Volume =
Total number of points falling on the tissue Total number of points in the reticule
(d) Regenerated glandular epithelium: It is the total length of the epithelium found in the secretory gland. 2.6.2. Pylorus ligation induced ulcers (Shay et al., 1945; Kulkarni, 1999) Albino Wistar rats were fasted for 36 h with water ad libitum. Normal saline (1 ml/rat p.o.) was administered twice daily to all the animals during the fasting period. Under light ether anesthesia, pyloric ligation was performed. The drugs were administered intraduodenally immediately after pylorus ligation. The intraduodenal injection was made using a syringe with 26 G needle directly into the duodenum. The animals were deprived of food and water during the postoperative period and were sacrificed 6 h after pylorus ligation by over dose of ether anesthesia. The stomach was isolated and the content of the stomach was used for estimation of free and total acidity (Hawk et al., 1947), pepsin content (Debnath et al., 1974), mucin content (Corne et al., 1974) and total proteins (Lowry et al., 1951). The stomach was cut opened along the greater curvature and ulcer index was determined as mentioned above.
2.6. Experimental models 2.6.1. Acetic acid induced ulcer (Asad et al., 2001) Under light ether anaesthesia, laporatomy was performed on 24 h fasted rats. Glacial acetic acid (0.05 ml) was applied onto the serosal surface of the stomach using a cylindrical mould of 6 mm diameter for 1 min. The acid solution was then removed by rinsing the mould with normal saline to avoid damage to the surrounding tissues. The stomach was placed back carefully and the abdominal wall was closed by interrupted sutures. The animals were treated with ranitidine (50 mg/kg p.o.) or extract of Momordica charantia L. either at both doses once daily for 10 days. Animals in the control group received only vehicle. Rats were sacrificed on the 10th day, stomachs were removed and cut opened along the greater curvature and the ulcer index was determined using the formula (Ganguly, 1969). Ulcer index =
10 X
where X = total mucosal area/total ulcerated area. The ulcers were given scores based on their intensity as follows: 0: no ulcer, 1: superficial mucosal erosion, 2: deep ulcer or transmural necrosis, 3: perforated or penetrated ulcer. The tissues were subsequently processed for histological examination. Four indices namely regenerated glandular epithelial width; capillary density, collagen content and surface epithelium were selected to reflect the rate and quality of ulcer healing (Wang et al., 1999).
2.6.3. Ethanol induced ulcers (Brzozowski et al., 1998) The animals were fasted for 36 h before administration of ethanol. The drugs were administered 1 h before ethanol administration and 1 h after ethanol administration, the animals were sacrificed and ulcer index was determined. 2.6.4. Cold restraint stress induced ulcers (Vincent et al., 1977) The drugs were administered 30 min before subjecting the animals to cold restraint stress. Stress was induced by placing the animals in a restraint cage at a temperature of 2–3 ◦ C for 2.5 h. The animals were then sacrificed by over dose of ether anesthesia and ulcer index was determined as mentioned above. 2.6.5. Healing of indomethacin induced gastric ulcers (Majumdar et al., 2003) Gastric ulcers were induced by administering indomethacin (5 mg/kg p.o.) for 5 days, during this period the animals were fed normally. The last day of indomethacin treatment was considered as 0th day. The animals were then treated either with misoprostol (100 g/kg p.o.) or with extract of Momordica charantia L. at both doses once daily for 5 days after induction of ulcer while the control group received only vehicle. Rats were sacrificed on the 0th day and 5th day, 6 h after the last treatment. The stomachs were removed and they were cut opened along the greater curvature and ulcer score and ulcer index were determined. The glandular portion of the stomach was used for estimation of mucin content, total proteins and activities of antioxidant factors such as catalase
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Table 1 Effect of 70% methanolic extract of Momordica charantia L. fruits on healing of acetic acid induced chronic gastric ulcers. Treatment
Regenerated glandular epithelium width (m)
Vehicle (1 ml/kg p.o.) Ranitidine (50 mg/kg p.o.) Momordica charantia L. extract (100 mg/kg p.o.) Momordica charantia L. extract (500 mg/kg p.o.)
279.73 356.38 395.79 329.79
± ± ± ±
17.214 10.075** 09.738** 14.282*
Capillary density (no.) in 19,600 m2 5.63 5.24 7.00 6.37
± ± ± ±
0.427 0.433 0.223 0.657
Volume of collagen content 0.117 0.124 0.114 0.186
± ± ± ±
Surface epithelium (m)
0.003 0.004 0.007 0.002**
162.75 144.78 156.98 146.15
± ± ± ±
04.25 10.90 06.68 12.50
Ulcer score 1.75 0.50 1.00 0.66
± ± ± ±
Ulcer index
0.250 0.223** 0.250 0.210*
0.16 0.06 0.02 0.02
± ± ± ±
0.04 0.01** 0.01** 0.01**
All values are mean ± SEM, n = 5–6. * p < 0.05 when compared to vehicle treated group. ** p < 0.01 when compared to vehicle treated group.
(Link, 1988) and super oxide dismutase (SOD) (Elstnev and Heupel, 1976).
3.2. Effect of Momordica charantia L. on ulcer healing in acetic acid induced chronic gastric ulcers
2.6.6. Cysteamine induced duodenal ulcers (Szabo, 1978) Duodenal ulcers were induced by administering cysteamine hydrochloride (400 mg/kg p.o.) twice at an interval of 4 h. Extract of Momordica charantia L. at both doses or ranitidine (50 mg/kg p.o.) was administered 30 min prior to each dose of cysteamine hydrochloride. The animals were sacrificed 24 h after first dose of cysteamine and the duodena were excised carefully. The duodenum was cut opened along the antimesentric side and the duodenal ulcer area, ulcer score and ulcer index were determined. The ulcers were given scores based on their intensity as follows: 0: No ulcer, 1: superficial mucosal erosion, 2: deep ulcer or transmural necrosis and 3: perforated or penetrated ulcer. The ulcer index was calculated using the following equation (Desai et al., 1999):
The higher dose of extract and ranitidine showed a significant reduction in ulcer index and ulcer score while the lower dose significantly reduced ulcer index when compared to the control (p < 0.01). Histopathological studies of the ulcerated tissue supported the macroscopic findings (Table 1).
Ulcer index = Arithmetic mean of intensity in a group +
Number of ulcer positive animals ×2 Total number of animals
3.3. Effect of Momordica charantia L. extract on pylorus ligation induced gastric ulcers The higher dose of Momordica charantia L. extract (500 mg/kg p.o.) and ranitidine showed antiulcer effect as indicated by a significant reduction in ulcer index, free and total acidity and pepsin activity (p < 0.01). However, the lower dose of extract did not show any significant reduction in ulcer index but increased the protein content of gastric juice. The mucin content was significantly increased by all treatments (Table 2).
3.4. Effect of Momordica charantia L. extract on ulcer index in ethanol induced gastric ulcers
2.6.7. Statistical analysis The statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Dunnet comparison test. For comparing nonparametric ulcer scores, ANOVA followed by nonparametric Dunn post-test was used. The values are expressed as mean ± SEM and p < 0.05 was considered significant.
The methanolic extract of both doses of Momordica charantia L. and misoprostol showed a significant reduction in ulcer index when compared to the control. The ulcer index was 0.83 ± 0.07 in the vehicle treated group, while the ulcer index in lower dose treated group was 0.32 ± 0.019 and in higher dose treated group, it was 0.19 ± 0.011. Misoprostol was most effective; it reduced the ulcer index to 0.091 ± 0.011.
3. Results 3.1. Analysis of extract
3.5. Effect of Momordica charantia L. extract on ulcer index in stress induced gastric ulcers
The extract was found to contain 8.8% (w/w) of bitter principles and 0.51% w/w of charantin. The loss of weight on drying (1%), ash content (32%) and level of heavy metals were within the British Pharmacopoeia limits.
The methanolic extract of Momordica charantia L. and ranitidine showed a significant reduction in ulcer index when compared to the control (Table 3).
Table 2 Effect of 70% methanolic extract of Momordica charantia L. fruits on pylorus-ligated rats. Treatment
Free acidity (mEq/l)
Vehicle (1 ml/kg p.o.) Ranitidine (50 mg/kg p.o.) Momordica charantia L. extract (100 mg/kg p.o.) Momordica charantia L. extract (500 mg/kg p.o.)
7.035 2.548 4.235 3.714
All values are mean ± SEM, n = 5–6. * p < 0.05 when compared to vehicle treated group. ** p < 0.01 when compared to vehicle treated group.
± ± ± ±
0.804 0.179** 0.384** 0.515**
Total acidity (mEq/l) 13.28 7.34 8.72 9.55
± ± ± ±
0.639 0.685** 0.508** 1.001**
Ulcer index 0.0515 0.0219 0.0461 0.0257
± ± ± ±
0.00217 0.00507** 0.00146 0.00761**
Mucin content (g/g) 0.055 0.108 0.092 0.127
± ± ± ±
0.007 0.009** 0.009* 0.011**
Pepsin content (g/6 h) 0.355 0.207 0.240 0.183
± ± ± ±
0.0206 0.0105** 0.0186** 0.0091**
Total proteins (mg/ml) 11.86 0.81 4.67 9.00
± ± ± ±
0.800 1.298** 0.957* 2.150
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Table 3 Effect of 70% methanolic extract of Momordica charantia L. fruits on stress induced gastric ulcers and cysteamine induced duodenal ulcers. Treatment
Vehicle (1 ml/kg p.o.) Ranitidine (50 mg/kg) Momordica charantia L. extract (100 mg/kg) Momordica charantia L. extract (500 mg/kg)
Stress induced
Cysteamine induced duodenal ulcer
Ulcer index
Ulcer area
0.177 0.021 0.075 0.020
± ± ± ±
0.041 0.007** 0.012* 0.006**
7.166 1.833 2.166 1.660
± ± ± ±
Ulcer score
1.956 1.167* 1.406* 0.760*
2.00 0.33 0.66 0.50
± ± ± ±
Ulcer index
0.564 0.210** 0.330* 0.223*
3.66 0.99 1.66 1.50
All values are mean ± SEM, n = 5–6. * p < 0.05 when compared to vehicle treated group. ** p < 0.01 when compared to vehicle treated group.
Table 4 Effect of 70% methanolic extract of Momordica charantia L. fruits on healing of indomethacin induced gastric ulcers. Treatment
Mucin content (g/gm)
Control 1 (5-day indomethacin + 5-day vehicle) Control 2 (5-day indomethacin) Misoprostol (100 g/kg) Momordica charantia L. extract (100 mg/kg) Momordica charantia L. extract (500 mg/kg)
0.585 0.733 1.597 1.040 0.768
± ± ± ± ±
0.045 0.795 0.180** 0.045 0.036
Ulcer index (mg/ml) 0.606 0.246 0.048 0.182 0.062
± ± ± ± ±
0.186 0.046 0.010** 0.049 0.031**
Total proteins of proteins) 25.46 30.37 89.99 55.19 29.20
± ± ± ± ±
2.277 2.213 2.983** 7.069** 4.044
SOD (units/mg of proteins) 58.41 40.33 13.69 27.52 47.74
± ± ± ± ±
5.28 2.21 0.51** 3.93** 12.41
Catalase (units/mg) 784.85 412.22 91.68 130.62 99.44
± ± ± ± ±
99.46 25.29** 10.08** 29.66** 7.29**
All values are mean ± SEM, n = 5–6. ** p < 0.01 when compared to control 1 group.
3.6. Effect of Momordica charantia L. extract on healing of indomethacin induced gastric ulcers The ulcer index was significantly reduced by higher dose of extract of (500 mg/kg p.o.) and misoprostol. The protein content was increased by lower dose of the extract (100 mg/kg p.o.) and misoprostol. The activities of antioxidant enzymes were reduced, but the higher dose of extract showed nonsignificant reduction in SOD activity (Table 4). 3.7. Effect of Momordica charantia L. extract on ulcer area, ulcer score and ulcer index in cysteamine induced duodenal ulcers The methanolic extract of Momordica charantia L. showed significant reduction in ulcer area and ulcer score when compared to the control (Table 3). 4. Discussion The standardized methanolic extract of Momordica charantia L. fruits showed good antiulcer activity as indicated by its effect on the healing of gastric ulcers induced by acetic acid model and prevention of gastric and duodenal ulcer formation in other models. The ulcer formation in each of these models occurs by different mechanisms. The healing of chronic gastric ulcer induced by acetic acid closely resembles to the healing of human peptic ulcers (Okabe et al., 1971). Pylorus ligation induces gastric ulcers due to accumulation of gastric secretion in the stomach (Shay et al., 1945). The methanolic extract of Momordica charantia L. significantly decreased the total acidity and free acidity and increased the mucus content indicating that it has both gastric antisecretory and gastric cytoprotective effects. Ethanol is a corrosive agent that damages the gastric mucosa (Soll, 1990; Surendra, 1999). The methanolic extract of Momordica charantia L. was effective in this model and this effect may be due to increased mucus secretion that protects gastric mucosa from corrosive effects of ethanol. Stress induced gastric ulcer is a method used to evaluate the effect of drugs on gastric secretion, stress and gastrointestinal (G.I.) motility (Brodie and Hanson, 1960; Michael and Charles, 1983). The effect of the extract against stress induced ulcer may be due to its gastric antisecretory action and it is also speculated that some of the constituents present in the Momordica charantia L. extract may
have antistress effect. Indomethacin inhibits prostaglandin synthesis and this deficiency in prostaglandins is responsible for ulceration (Vedavyasa, 1999). Like in the ethanol induced gastric ulcers, the methanolic extract of Momordica charantia L. was effective in reducing the ulcer index significantly and it also increased the mucus content. This model confirmed the cytoprotective effect of the extract. The extract of Momordica charantia L. significantly reduced the activities of antioxidant factors such as SOD and catalase. The reason for this effect cannot be explained. Cysteamine induced duodenal ulcer resembles duodenal ulcer in humans (Szabo, 1978). The methanolic extract of Momordica charantia L. was effective in reducing the ulcer area. The doses of the extract were selected based on earlier reports (Sarkar et al., 1996; Shibib et al., 1993). In traditional medicine, the olive oil extract of fresh fruits prepared by macerating the fruits with olive oil for 15 days or dried fruits mixed with honey are used. Gurbuz et al. (2000) reported that olive oil extract of fresh fruits, dry fruit mixed with honey and ethanolic extract of dried fruits possess gastric cytoprotective effect against ethanol induced and diethyldithiocarbamate induced ulcerogenesis. These authors also reported that ethanolic extract of Momordica charantia L. fruits was more potent than the olive oil extract in reducing the development of ethanol plus hydrochloric acid induced gastric ulcers. The present study was carried out using methanolic extract. Methanol and ethanol possess very close polarity and the compositions of both these extracts may be more or less alike. In the present study, the methanolic extract reduced the development of ethanol induced gastric ulcers indicating gastric cytoprotective effect and this result complements the earlier findings reported by Gurbuz et al. (2000). Previous studies by Yıldırım (1994) and Dengiz and Gursan (2005) reported that olive oil extract of Momordica charantia L. possesses weak activity against indomethacin-induced gastric ulcers. Furthermore, both these reports showed that olive oil itself possesses significant antiulcer effect against indomethacin induced ulcers. However, in the present study, methanolic extract of Momordica charantia L. showed good cytoprotective effect against indomethacin induced gastric ulcers. The findings of the present study and those of earlier study by Gurbuz et al. (2000) suggests that methanolic or ethanolic extract of Momordica charantia L. fruits may have better gastric cytoprotective effect than the olive oil extract. Apart from the gastric cytoprotective effect, the present study also evaluates the effect of Momordica charantia fruits on gastric
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ulcer healing, gastric secretion and duodenal ulcers. It was assumed earlier that Momordica charantia fruits may not have gastric antisecretory effect and the ulcer healing effect observed in humans may be due to their gastric cytoprotective action alone (Gurbuz et al., 2000). But it is well known that gastric cytoprotective property alone in rats does not produce significant ulcer healing in humans (Vogel, 2002). Gastric cytoprotective agents such as misoprostol increases ulcer healing only in doses that reduce gastric acid secretion in addition to their cytoprotective effect (Hoogerwerf and Pasricha, 2001). The results of the present study suggests that ulcer healing effect of Momordica charantia fruits observed in humans may be due to both gastric antisecretory and gastric cytoprotective effects. Momordica charantia L. was reported to contain a number of flavonoids, alkaloids and many other chemical constituents (Ross, 1999). The constituent(s) responsible for antiulcer effect of the fruits are not known. A number of flavonoids, present in different plants are known to reduce gastric ulcer formation (Suzuki et al., 1998). Hence, it is assumed that flavanoids may contribute at least in part to the antiulcer effect of the fruits. Apart from flavonoids, the fruits also contain steroids such as beta sitosterol that is known to reduce the development of gastric ulcers (Xiao et al., 1992) and carotenoids that possess gastric cytoprotective action (Kamath et al., 2008). Earlier study on gastroprotective effect of Momordica charantia L. fruits by Gurbuz et al. (2000) reported that hexane soluble and hexane insoluble fractions of ethanolic extract were equally effective in reducing the development of ethanol induced gastric ulcers. Hence, the gastroprotective effect observed in the present study may be due to both polar constituents such as flavonoids and non-polar constituents such as carotenoids and sterols. Furthermore, the active constituent responsible for many of the effects of Momordica charantia L. fruits is charantin. Hence, studies should also be carried out with charantin to determine its effect on the gastric and duodenal ulcer formation. 5. Conclusion The methanolic extract of Momordica charantia L. fruits was effective in increasing the healing of gastric ulcers induced by acetic acid. The methanolic extract of the fruits also decreased the development of gastric ulcers in Shay rat model, ethanol induced gastric ulcers, indomethacin induced gastric ulcers, stress induced gastric ulcers and duodenal ulcers induced by cysteamine. The antiulcer effect of methanol extract of Momordica charantia L. is due to its gastric antisecretory and gastric cytoprotective effect. Acknowledgements The authors are thankful to Prof. Suresh Nagpal, Chairman, Krupanidhi Educational Trust (Bangalore, India), Prof. Sunil Dhamanigi, Secretary, Krupanidhi Educational Trust and Dr. Amit Kumar Das, Professor and Principal, Krupanidhi College of Pharmacy for providing facilities to carry out this work. References Asad, M., Shewade, D.G., Koumaravelou, K., Abraham, B.K., Vasu, S., Ramaswamy, S., 2001. Gastric anti-secretary, anti-ulcer activity of oxytocin in rats and guinea pigs. Life Sciences 70, 17–24. British Pharmacopoeia, 2005. Her Majesty’s Stationary Office, London, Vol. 2, pp. 1103–1129. Brodie, D.A.Q., Hanson, H.M., 1960. A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38, 353–360. Brzozowski, T., Konturek, S.J., Kwiecien, S., Pajdo, R., Brzozowski, I., Hahn, E.G., 1998. Involvement of endogenous cholecystokinin and somatostatin in gastro protection induced by intra duodenal fat. Journal of Clinical Gastroenterology 27, 125–137.
Chen, Q., Li, E.T., 2005. Reduced adiposity in bitter melon (Momordica charantia) fed rats is associated with lower tissue triglyceride and higher plasma catecholamines. British Journal of Nutrition 93, 747–754. Corne, S.J., Morrissey, S.M., Woods, R.J., 1974. A method for the quantitative estimation of gastric barrier mucus. In: Proceeding of the Physiological Society, pp. 116–117. Debnath, P.K., Gode, K.O., Govinda, D.D., Sanyal, A.K., 1974. Effect of propranolol on gastric secretion in rats. British Journal of Pharmacology 51, 213– 216. Dengiz, G.O., Gursan, N., 2005. Effects of Momordica charantia L. (Cucurbitaceae) on indomethacin-induced ulcer model in rats. Turkish Journal of Gastroenterology 16, 85–88. Desai, J.K., Goyal, R.K., Parmar, N.S., 1999. Characterization of dopamine receptor subtypes involved in experimentally induced gastric and duodenal ulcers in rats. Journal of Pharmacy and Pharmacology 51, 187–192. Elstnev, E.F., Heupel, E., 1976. Inhibition of nitrite formation from hyroxylammoniumchloride. A simple assay for super oxide dismutase. Analytical Chemistry 70, 616–620. Ganguly, A.K., 1969. A method for quantitative assessment of experimentally produced ulcers in stomach of rats. Experientia 25, 11–24. Gurbuz, I., Akyuz, C., Yesilada, E., Sener, B., 2000. Anti-ulcerogenic effect of Momordica charantia L. fruits on various ulcer models in rats. Journal of Ethnopharmacology 71, 77–82. Hawk, P.B., Oser, B.L., Summerson, H.W., 1947. Practical physiological chemistry, 12th ed. Churchill, London, p. 347. Hoogerwerf, W.A., Pasricha, P.J., 2001. Agents used for the treatment of gastric acidity and treatment of peptic ulcers and gastroesophageal reflux disease. In: Hardman, J.E., Limbard, L.E. (Eds.), Goodman’s and Gilman’s The Pharmacological Basis of Therapeutics. McGraw-Hill, New Delhi, pp. 1011–1012. Kamath, B.S., Srikanta, B.M., Dharmesh, S.M., Sarada, R., Ravishankar, G.A., 2008. Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis. European Journal of Pharmacology 590, 387–395. Kulkarni, S.K., 1999. Hand Book of Experimental Pharmacology, 3rd ed. Vallabh Prakashan, New Delhi, pp. 148–150. Limtrakul, P., Khantamat, O., Pintha, K., 2004. Inhibition of P-glycoprotein activity and reversal of cancer multidrug resistance by Momordica charantia extract. Cancer Chemotherapy Pharmacology 54, 525–530. Link, E.M., 1988. Mechanism of pH dependent hydrogen per oxide cytotoxicity invitro. Archives of Biochemistry and Biophysics 365, 362–372. Lowry, C.H., Roseborough, N.I., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. Journal of Biological Chemistry 193, 265– 275. Majumdar, B., Chaudhri, S.G.R., Ray, A., Bandyopadhyay, S.K., 2003. Effect of ethanol extract of Piper betel Linn. leaf on healing of NSAID-induced experimental ulcer. A novel role of free radical scavenging action. Indian Journal of Experimental Biology 41, 311–315. Michael, N.P., Charles, T.R., 1983. Stressful life events, acid hyper secretion and ulcer disease. Gastroenterology 84, 114–119. Nalkami, 1989. Indian Materia Medica 2, 947. Okabe, S., Roth, J.L.A., Pfeiffer, C.J., 1971. Differential healing periods of the acetic acid ulcer model in rats and cats. Experientia 27, 146–148. Pitiphanpong, J., Goto, M., Sasaki, M., Shotipruk, A., 2005. Extraction of charantin from Momordica charantia. In: Symposium of Separation Science and Technology, Osaka, Japan June 3–4, p. 2005. Ross, I.A., 1999. Medicinal Plants of the World. Humana Press, New Jersey, USA, pp. 213–219. Sarkar, S., Pranava, M., Marita, R.A., 1996. Demonstration of the hypoglycemic action of Momordica charantia in a validated animal model of diabetes. Pharmacological Research 33, 1–4. Sathishsekar, D., Subramanian, S., 2005a. Beneficial effects of Momordica charantia seeds in the treatment of STZ-induced diabetes in experimental rats. Biological and Pharmaceutical Bulletin 28, 978–983. Sathishsekar, D., Subramanian, S., 2005b. Antioxidant properties of Momordica Charantia (bitter gourd) seeds on streptozotocin induced diabetic rats. Asia Pacific Journal of Clinical Nutrition 14, 153–158. Schmourlo, G., Mendonca-Filho, R.R., Alviano, C.S., Costa, S.S., 2005. Screening of antifungal agents using ethanol precipitation and bioautography of medicinal and food plants. Journal of Ethnopharmacology 96, 563–568. Shay, H., Komarov, S.A., Fele, S.S., Meranze, D., Gruenstein, H., Siplet, H., 1945. A simple method for uniform production of gastric ulceration in rat. Gastroenterology 5, 43–46. Shibib, B.A., Khan, L.A., Rahman, R., 1993. Hypoglycaemic activity of Coccinia indica and Momordica charantia in diabetic rats: depression of the hepatic gluconeogenic enzymes glucose-6-phosphatase and fructose-1.6-bisphosphatase and elevation of both liver and red-cell shunt enzyme glucose-6-phosphate dehydrogenase. Biochemical Journal 292, 267–270. Soll, A.H., 1990. Pathogenesis of peptic ulcers and implication for therapy. New England Journal of Medicine 322, 909–916. Surendra, S., 1999. Evaluation of gastric anti-ulcer activity of fixed oil of tulsi and possible mechanism. Indian Journal of Experimental Biology 36, 253– 257. Suzuki, Y., Ishihara, M., Segami, T., Ito, M., 1998. Anti-ulcer effects of antioxidants, quercetin, alpha-tocopherol, nifedipine and tetracycline in rats. Japan Journal of Pharmacology 78, 435–441. Szabo, S., 1978. Animal model for human disease: duodenal ulcer disease. American Journal of Pathology 73, 273–276.
S. Alam et al. / Journal of Ethnopharmacology 123 (2009) 464–469 Vedavyasa, S., 1999. Gastric mucosal cellular changes induced by indomethacin male albino rats. Indian Journal of Experimental Biology 37, 365–369. Vincent, G.P., Galvin, G.B., Rutkowski, J.L., Pare, W.P., 1977. Body orientation, food deprivation and potentiation of restraint induced gastric lesions. Gastroenterology and Clinical Biology 1, 539–543. Vogel, H.G., 2002. Drug Discovery and Evaluation, 2nd ed. Springer-Verlag, Berlin, Germany, p. 871. Wang, J.Z., Wuy, J., Rao, C.M., Gao, M.T., Li, W.G., 1999. Effect of recombinant human basic fibroblast growth factor on stomach ulcers in rats and mice. Acta Pharmacologica Sinica 209, 763–768. Xiao, M., Yang, Z., Jiu, M., You, J., Xiao, R., 1992. The anti-gastroulcerative activity of beta-sitosterol-beta-d-glucoside and its aglycone in rats. Hua Xi Yi Ke Da Xue Xue Bao 23, 98–101.
469
Yesilada, E., Gurbuz, I., Shibata, H., 1999. Screening of Turkish anti-ulcerogenic folk remedies for anti-Helicobacter pylori activity. Journal of Ethnopharmacology 66, 289–293. Yıldırım, O.F., 1994. Momordica charantia L. (Kudret Narı) Bitkisinin Cesitli Ekstrelerinin Sicanlarda Deneysel Olarak Olusturulan Peptik Ulserde Etkisinin Incelenmesi (Studies on the Effect of Momordica charantia L. on the Experimentally- Induced Peptic Ulcer in Rats). Yuksek Lisans tezi (Master thesis), Gazi Univ. Saglık Bilimleri Enstitusu (Gazi University, Institute of Medical Sciences). Zheng, Z.X., Teng, J.Y., Liu, J.Y., Qiu, J.H., Ouyang, H., Xue, C., 2005. The hypoglycemic effects of crude polysaccharides extract from Momordica charantia in mice. Wei Sheng Yan Jiu 34, 361–363.