ARTICLE IN PRESS Phytomedicine 17 (2010) 569–574
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Gedunin and Photogedunin of Xylocarpus granatum show significant anti-secretory effects and protect the gastric mucosa of peptic ulcer in rats V. Lakshmi b,1, N. Singh a,1, S. Shrivastva b, S.K. Mishra b, P. Dharmani a, V. Mishra a, G. Palit a,n a b
Division of Pharmacology, Central Drug Research Institute, Lucknow 226001, U.P, India Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226001, U.P, India
a r t i c l e in fo
Keywords: Peptic ulcer Proton pump Gastric acid secretion Xylocarpus granatum Medicinal plant
abstract In the present study, the gastroprotective mechanism of Xylocarpus granatum fruit and its active constituents gedunin and photogedunin was investigated. Chloroform fraction (Fr-CHCl3) of X. granatum fruit was evaluated against cold restraint (CRU), aspirin (AS), alcohol (AL) and pyloric ligation (PL) induced gastric ulcer models in rats and histamine (HA) induced duodenal ulcer model in guinea pigs. Potential anti-ulcer activity of Fr-CHCl3 was observed against CRU (58.28%), AS (67.81%), AL (84.38%), PL (65.66%) and HA (61.93%) induced ulcer models. The standard drug omeprazole (10 mg/kg, p.o.) showed 68.25% protection against CRU, 57.08% against AS and 69.42% against PL model and 70.79% against HA induced duodenal ulcer. Sucralfate, another standard drug (500 mg/kg, p.o.) showed 62.72% protection in AL induced ulcer model. Fr-CHCl3 significantly reduced free acidity (51.42%), total acidity (30.76%) and upregulated mucin secretion by 58.37% respectively. Phytochemical investigations of Fr-CHCl3 yielded gedunin (36%), photogedunin (2%). Further, Fr-CHCl3 and its compounds gedunin and photogedunin significantly inhibited H + K + -ATPase activity in vitro with IC50 of 89.37, 56.86 and 66.54 mg/ml respectively as compared to the IC50 value of omeprazole (30.24 mg/ml) confirming their anti-secretory activity. Conclusively, Fr-CHCl3 of Xylocarpus granatum was found to possess anti-ulcerogenic activity which might be due to its anti-secretory activity and subsequent strengthening of the defensive mechanism. This study is the first of its kind to show significant anti-secretory effect of gedunin and photogedunin. Therefore it could act as a potent therapeutic agent against peptic ulcer disease. & 2009 Elsevier GmbH. All rights reserved.
Introduction Peptic ulcer disease (PUD) is one of the major gastrointestinal disorders which occur due to an imbalance between offensive (acid, pepsin and Helicobacter pylori) and defensive (mucin, prostaglandin and bicarbonate) factors. Consequently reduction of gastric acid production as well as reinforcement of gastric mucosal protection has been the major therapeutic approaches of peptic ulcer disease (Hoogerwerf and Pasricha 2006). A number of anti-ulcer drugs including proton pump inhibitors (PPI) and H2 receptor antagonists are available for the treatment of PUD, but clinical evaluation of these drugs has shown incidence of relapse, side effects and drug interactions. This has been the rationale for the development of new anti-ulcer drugs and thus the search for novel molecules has been extended to medicinal plants that can offer better protection and decrease relapses.
n Corresponding author: Tel.: + 91 522 2612411–418x4303; fax: + 91 522 2623405, + 91 522 2623938. E-mail addresses:
[email protected] (N. Singh),
[email protected] (G. Palit). 1 These authors contributed equally to this work.
0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.10.016
Several Indian medicinal plant species like Allophylus serratus (Dharmani et al., 2005b), Desmodium gangeticum (Dharmani et al., 2005a), Ocimum sanctum (Dharmani et al., 2004), Hemidesmus indicus (Anoop and Jegadeesan, 2003), Asparagus racemosus (Sairam et al., 2003) etc. have been reported to possess anti-ulcer activity. Studies on different biological activities of X. granatum in general are also available. But there is less information available regarding its pharmacological effect on the gastrointestinal system. Keeping these facts in considerations, we have assessed the anti-ulcer activity of the plant species, X. granatum. X. granatum Koen. belongs to the Natural Order Meliaceae. It is a mangrove plant and is commonly known as pussur in Hindi language. X. granatum. Koen (Bengali- Dhundul) is a moderate sized evergreen tree with their grey barks, usually grows in coastal forests of Bengal, Andaman’s, Burma, the Malay peninsula and island of Australia and Africa. (Ghani 1998; Kirtikar and Basu 1980). Some mangroves have shown insecticidal activity (Miki et al., 1994). The barks are astringent and are used for dysentery, diarrhea, and other abdominal troubles and as febrifuge (Ghani 1998). The seed ash mixed with sulphur and coconut oil is applied as ointment for itch (Ghani 1998). Fruit is used as a cure for swelling of the breast and elephantiasis (Ghani 1998). The
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purpose of the present study, however is to evaluate the antiulcerogenic effect of the X. granatum fruit and its chloroform fraction against different experimental gastric ulcer models in rats and duodenal ulcers in guineapigs and also to identify the active constituents responsible for these gastroprotective effects along with its mechanism of action.
Materials and methods
hours light and dark cycle). Animals were fed with standard laboratory food pellets and water was provided ad libitum. Guinea pigs of either sex, weighing 300–350 g were used for histamineinduced ulcer model, which were also housed under standard conditions as described above. All experimental protocols were approved by our Institutional Ethical Committee following the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) which complies with International norms of INSA (Indian National Science Academy).
Plant material
Materials
X. granatum plant grows naturally in tidal forests along the East and West coastal areas up to Maharastra and in Andaman Island. The Xylocarpus fruits were collected in the month of January from South Andaman Coast and were identified by the Division of Botany, Central Drug Research Institute (CDRI), Lucknow. The voucher of this specimen is kept at the herbarium of CDRI (acquisition number 328).
Sucralfate was obtained from Meranani Pharmaceuticals, India, whereas omeprazole and other chemicals were obtained from M/ s. Sigma Chemicals, St Louis, MO, USA.
Extraction/Fractionation Procedure Air dried powdered fruits (1.0 Kg) were extracted with 50% aqueous ethanol 5 5.0 lit. and the combined extracts were filtered, concentrated under reduced pressure below 50 1C to minimum volume of 1.0 lit. It was further dried in hot air vacuum oven at 45 1C to brown powder (yield 15%). The brown powder was further fractionated into chloroform soluble (yield 8.5% of the 50% aq. ethanol extract) and chloroform insoluble fractions by maceration with chloroform. The chloroform fraction on repeated column chromatography over silica gel and final purification by HPLC on reverse phase C18 R.P columns using acetonitrile-water 55:45, v/v, flow rate-1.0 ml/min using UV detector (l 230 nm) yielded compounds namely Gedunin (36%) Taylor (1974), Photogedunin (2%) (Burke et al., 1969) as shown in Fig. 1. All these were characterized using IR, NMR, mass, derivetization and comparing the data with those given in literature for these compounds. These were also compared with authentic samples on thin layer plates as well as their spectral data. Experimental Animals Adult Sprague Dawley rats of either sex, weighing 180–200 g were housed in raised bottom mesh cages to prevent coprophagy and were kept in environmentally controlled rooms (25 7 2 1C, 12
Treatment Schedule Ethanolic extract (E-EtOH) of X. granatum fruits and its chloroform fraction (Fr-CHCl3), omeprazole (Omz) (10 mg/kg) and sucralfate (SUC) (500 mg/kg) were prepared in 1% carboxymethyl cellulose (CMC) as suspension and administered orally 45 mins prior to exposure of ulcerogens to the animals at a volume of 1 ml/200 g of body weight. All animals were deprived of food for 16 h before ulcerogens exposure and were divided into three groups, (n=6). 1. Control group of animals were treated with vehicle 1% CMC. 2. Graded doses of E-EtOH of X. granatum fruit (200, 400 and 800 mg/kg, p.o.) and its Fr-CHCl3 (10, 20 and 40 mg/kg, p.o.) were tested against Cold restraint ulcer (CRU) model to identify the effective dose and selected for further studies in other ulcer models. 3. Experimental group was treated with standard anti-ulcer drugs such as Omz (10 mg/kg, p.o.) in (CRU), aspirin (AS), pyloric ligation (PL), histamine induced duodenal ulcer (HA) and SUC (500 mg/kg, p.o.) in Alcohol (AL) induced ulcer model. Anti-ulcer studies Cold restraint induced gastric ulcer (CRU) Animals were subjected to cold restraint stress after 45 mins of treatment with E-EtOH, Fr-CHCl3 and Omz. All the animals were immobilized in restraint cage and kept at 4 1C in an environmental chamber (Suleyman et al. 2001). Two hours later the animals were
Fig. 1. Chemical structure of gedunin and photogedunin.
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sacrificed and stomachs were observed and scored under Magnascope for ulcers. Aspirin induced gastric ulcer model (AS) Aspirin at a dose of 150 mg/kg was administered to induce ulcer after 45 mins of treatment of Fr-CHCl3 and Omz. The animals were sacrificed 5 hours after aspirin treatment (Goel et al. 1985) and the stomach was dissected out, incised along the lesser curvature and the lesion was scored.
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Measurement of ulcer index Ulcer formed due to treatment with different ulcerogens were observed under Magnascope (5X magnification) and were scored according to the arbitrary scoring system as described by Srivastava et al. (1991). The severity and intensity of the lesions were graded as following: i) Shedding of epithelium = 10; (ii) Petechial and frank hemorrhages = 20; (iii) one or two ulcers = 30; (iv) more than two ulcers = 40; and (v) Perforated ulcers = 50.
In vitro assay of H + K + -ATPase activity Alcohol induced gastric ulcers in rats (AL) Gastric ulcer was induced in rats by administering chilled absolute alcohol (1 ml/200 g, body weight of animals) (Suleyman et al. 2004). The E-EtOH, Fr-CHCl3 and sucralfate were administered 45 minutes before alcohol treatment. After 1 hour of alcohol administration, the animals were sacrificed and stomach was cut open along the greater curvature to observe the gastric lesions which appear as hemorrhagic bands along the mucosal ridges of the stomach. The lengths of the lesions were measured using Biovis image analyzer software and summated to give a total lesion score. Pyloric ligation induced ulcer model (PL) After 45 mins of administration of Fr-CHCl3 and Omz, ulcer was induced in rats by pyloric ligation. Under Chloral hydrate anesthesia (300 mg/kg, i.p.), the abdomen was opened and the pyloric end of the stomach was ligated avoiding any damage to the adjacent blood vessels (Shay et al. 1945). Stomach was replaced carefully and the animals were allowed to recover with free access to water. After 4 hours the animals were sacrificed and the stomach was dissected out. Lesions were scored and gastric fluid was collected and centrifuged at 2000 rpm for 10 mins. The collected supernatant was used for the estimation of gastric secretion studies, mucin estimation and peptic activity. Gastric secretion study Free and total acidity was measured from the collected gastric juice by titrating against 0.01 N NaOH, using phenolphthalein as an indicator and expressed in terms of mequiv./ml (Anoop and Jegadeesan 2003). Peptic activity was determined by measuring the amount of liberated tyrosine by the action of pepsin on hemoglobin as substrate and expressed in terms of units/ml (Debnath, Gode, Das, Sanyal 1974) and expressed in terms of units/ml. Mucus content was expressed in terms of mg% Varley, Gowenlock, Bell (1980).
H + K + -ATPase activity was assayed in gastric microsomes isolated from normal fasted rat stomach (Berglindh 1990). For the enzyme assay, gastric microsomes incubated with or without different concentrations of Fr-CHCl3, gedunin, photogedunin as well as standard drug Omz for 10 min at 37 1C, were added to an assay buffer containing (in mM) 150 KCl, 10 PIPES, 1 MgSO4, 5 Mg ATP, 1 EGTA and 0.1 ouabain, at pH 7.2 and 10 mg/ml valinomycin, 2.5 mg/ml oligomycin. The reaction was carried out at 37 1C for 20 min and was stopped by adding 10% ice-cold trichloroacetic acid. After centrifugation (2000 g for 1 min), inorganic phosphate release was determined from the resulting supernatant spectrophotometrically at 310 nm wavelength (Sanui 1974) and expressed as mM/h/mg protein.
Statistical analysis All values shown in the figures and tables represent the means7S.E.M. IC50 values with 95% confidence limits were estimated using Maximum Likelihord Iterative Procedure (Finney 1952). Statistical analysis was performed with Prism version 3.0 software using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Po0.05 was considered to be statistically significant.
Results Anti-ulcer effect of E-EtOH against cold restraint and alcohol induced ulcer in rats In our preliminary study, graded doses of E-EtOH (200, 400 and 800 mg/kg, p.o.) of X. granatum showed percentage protection of 24.98, 33.32 (P o0.05) and 41.63 (Po0.05) respectively whereas standard drug, omeprazole showed a percentage protection of 68.25 (Po0.01) in comparison to control against CRU model. In addition, E-EtOH (400 mg/kg, p.o.) showed 38.88% protection against alcohol induced gastric ulcer model, whereas standard drug sucralfate showed 62.72% protection (Fig. 2).
Histamine induced duodenal ulcer in guinea pigs (HA) Duodenal ulcers were induced in guinea pigs by intramuscular application of histamine acid phosphate at a dose of 0.25 mg/kg at every 30 mins interval for 4 hours and the animals were sacrificed after 30 mins of the last dose (Parmar and Desai 1993). Animals were treated with Fr-CHCl3 and Omz 45 mins prior to histamine administration. Promethazine hydrochloride at a dose of 2.5 mg/ kg of body weight was injected intraperitoneally to each animal 15 mins prior to administration of histamine, in order to protect the animals from histamine toxicity. Stomach was cut along the lesser curvature down to the duodenum to observe the formation of ulcer on the anterior and posterior wall of duodenum.
Anti-ulcer effect of Fr-CHCl3 against cold restraint induced ulcer in rats Graded doses of Fr-CHCl3 (10, 20 and 40 mg/kg, p.o.) showed percentage protection of 41.65, 58.28 (Po0.01) and 61.07 (Po0.01) respectively whereas standard drug, omeprazole showed a percentage protection of 68.25 (P o0.01) in comparison to control against CRU model. From this observation 20 mg/kg dose of Fr-CHCl3 was identified as the effective dose and selected for further studies. The results are graphically represented in Fig. 3.
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standard drug, sucralfate, showed 62.72% protection (P o0.05) as depicted in Fig. 3. Anti-ulcer effect of Fr-CHCl3 against pyloric ligation induced ulcer in rats Anti-ulcer activity of Fr-CHCl3 was also observed against pyloric ligation induced ulcer in rats where it showed protection of 65.66% (Po0.01) and omeprazole showed 69.42% (P o0.01) protection (Fig. 3). Effect of Fr-CHCl3 on gastric secretion
Fig. 2. Effect of E-EtOH of Xylocarpus granatum and standard drugs (Omz and SUC) on percentage protection of ulcer against cold restraint and alcohol induced gastric ulcer models in rats. Data expressed as mean % protection7 S.E.M. Statistical analysis was done by One Way ANOVA followed by Dunnett’s Multiple Comparison Test. nStatistically significant at P o 0.05 and nnPo0.01, in comparison to control. n = 6 in each group.
The antisecretory effect of Fr-CHCl3 was evaluated by estimating free and total acidity of gastric juice and by estimating the activity of pepsin and mucin as shown in Table 1. This fraction has reduced free acidity (51.42%, Po0.01), total acidity (30.76%, Po0.05) which was comparable with standard drug omeprazole (67.41%, Po0.001) and (42.69%, Po0.01) respectively. It significantly upregulated mucin secretion by 58.37% (Po0.01) whereas omeprazole increased mucin secretion by 39.35% (Po0.05) in comparison to control. Effect of Fr-CHCl3 against histamine induced duodenal ulcer in guinea pigs In order to confirm whether Fr-CHCl3 was effective against duodenal ulcer, its efficacy was tested against histamine induced duodenal ulcer. It showed significant anti-ulcer activity with 61.93% protection (P o0.01) whereas omeprazole showed percentage protection of 70.79 (P o0.01) as shown in Fig. 3. Effect of Fr-CHCl3, gedunin and photogedunin on H + K + -ATPase activity The antisecretory mechanism of action of Fr-CHCl3 (10–100 mg/ ml), gedunin (10–100 mg/ml) and photogedunin (30–70 mg/ml) has been confirmed through the inhibition of gastric H + K + ATPase activity in comparison with control with an IC50 value of 89.37 mg/ml, 56.86 mg/ml and 66.54 mg/ml respectively. Omeprazole (10–50 mg/ml) used as positive control reduced the enzyme activity with an IC50 value of 30.24 mg/ml (Table 2). Physicochemical data of pure compounds
Fig. 3. Effect of Fr-CHCl3 of Xylocarpus granatum and standard drugs (Omz and SUC) on percentage protection of ulcer against cold restraint, Aspirin, pyloric ligation and alcohol induced gastric ulcer models in rats and histamine induced duodenal ulcer in guineapigs. Data expressed as mean % protection7 S.E.M. Statistical analysis was done by One Way ANOVA followed by Dunnett’s Multiple Comparison Test. nStatistically significant at P o 0.05 and nnPo0.01, in comparison to control. n =6 in each group.
Effect of Fr-CHCl3 against aspirin induced ulcer in rats Potential anti-ulcer activity of Fr-CHCl3 was observed when its efficacy was tested against aspirin induced ulcer model. 67.81% protection (Po0.01) was observed when Fr-CHCl3 was administered whereas omeprazole showed 57.08% protection in comparison to control as shown in Fig. 3.
Gedunin m.p. :215–216 1C IR (KBr)cm 1 :2950, 1750, 1681, 1508, 1380, 1020, 870. FAB MS (m/z) :482(M + ), 422(M + -60), 407(M + -75), 438(M + 44). Photogedunin m.p. :290–293 1C : +151(c 1.9, py) [a]27 D IR (KBr) cm 1 :3417(OH), 3023(C= C stretching), 1745 (lactone & ester C =O str.), 1656 (cyclohexenone), 1218, and 1023. FAB-MS (m/z) :515[M+ H] + , 537[M+Na] + , 454(M-60); 439(M -75).
Effect of Fr-CHCl3 against alcohol induced ulcer
Discussion
Fr-CHCl3 showed significant anti-ulcer activity against ethanol induced ulcer having 84.38% protection (Po0.01), whereas the
In our contemporary times, the use of medicinal plants and natural products has become universal. The discovery of new and
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Table 1 Effect of Chloroform fraction (20 mg/kg, p.o.) of X. granatum and Omeprazole (10 mg/kg, p.o.) on free acidity, total acidity, pepsin and mucin contents in pyloric ligation model (n= 6 in each group). Treatment
Free acid lequiv./ml
Total acid lequiv./ml
Peptic Concentration lmol/ml
Mucin mg%
Control Chloroform fraction Omeprazole
56.007 3.22 27.2 73.988nn 18.25 7 1.284nnn
65.00 74.686 45.00 76.205n 18.2957 1.309nnn
38.10 7 3.519 40.62 7 2.34 37.25 7 3.46
735.9 757.06 17687149.7nn 1212733.41n
Data expressed as mean7 S.E.M. Statistical analysis was done by One Way ANOVA followed by Dunnett’s Multiple Comparison Test. nStatistically significant at Po 0.05 and nn Po 0.01, nnn Po 0.001 in comparison to control.
Table 2 Effect of Chloroform fraction (10–100 mg/ml) of X. granatum, its active compounds gedunin (10–100 mg/ml) and photogedunin (30–70 mg/ml) and standard drug Omeprazole (10–50 mg/ml) on H + K + ATPase isolated from rat gastric microsomes. Data expressed as mean 7 S.E.M. of experiments performed in triplicates (n = 3 in each group). Treatment (mg/ml)
Control Chloroform fraction Gedunin Photogedunin Omeprazole
mean
H+ K + ATPase
95% Confidence limit of IC50
% inhibition
IC50 (mg/ml)
Lower limit
Upper limit
35.14 45.03 54.06 80.07
89.37 mg/ml 56.86 mg/ml 66.54 mg/ml 30.24 mg/ml
86.78 53.63 62.28 27.52
92.86 60.36 70.18 33.25
novel pharmaceutical products from plants used in traditional system of medicine or folklore for the treatment or amelioration of the incidence of gastric ulcers (Barros et al. 2008). The antiulcer activity of Fr-CHCl3 of X. granatum fruit has been studied against various models of experimentally induced gastric and duodenal ulcer in order to evaluate its mechanism of action involved in prevention of ulcer formation. The finding receives an impetus by considering the fact that Fr-CHCl3 of X. granatum showed anti-ulcerogenic activity in all the models, each of which induced ulcer through a different mechanism. Peptic ulcer is postulated to develop when there is a disbalance of aggressive and defensive factors either because of increased secretion of acid or pepsin or because of impairment of mucosal resistance. So we select two models one of antisecretory and other of cytoprotective for the preliminary gastroprotective study of E-EtOH of X. granatum fruit. We performed a dose dependent anti-ulcer study of E-EtOH in CRU model. CRU is a well-accepted model for the induction of gastric ulcers, in which peripheral sympathetic activation and increased acid secretion play important roles (Djahanguiri et al. 1973). E-EtOH exhibited significant protection in a dose dependent manner in the CRU model, with maximum protection observed at 400 mg/kg, p.o. In addition, E-EtOH exerted a protective effect against ethanol-induced gastric lesions in contrast to standard drug, sucralfate. Since ethanol damages the superficial epithelial layers and inhibit the release of mucosal prostaglandins Miller and Henagan (1984) and depresses the gastric defensive mechanisms, these agents appear to augment the gastric mucosal defense (Kinoshita et al. 1995) indicating the cytoprotective potentials of E-EtOH of Xylocarpus fruit. For the further identification of the phytochemical constituents responsible for the above mentioned anti-ulcer effect, E-EtOH was fractionated into Fr-CHCl3 and tested in different gastric ulcer models. Graded doses of Fr-CHCl3 exerted anti-ulcer effect in the CRU model, offering maximum protection at much lower dose 20 mg/kg than E-EtOH (400 mg/kg) indicating concentration of active constituents in this fraction. Hence, 20 mg/kg dose was considered to be the optimal dose for evaluation in further
studies. Fr-CHCl3 was highly effective in decreasing the hemorrhagic lesions induced by ethanol in contrast to standard drug, sucralfate, reflecting its cytoprotective activity. Furthermore, gastric acid is an important factor for the genesis of ulceration in pyloric-ligated model (Shay et al. 1945). In this model, auto-digestion of mucosa by gastric acid and pepsin results in the development of ulcers (Goel and Bhattacharya 1991). FrCHCl3 significantly reduced free and total acidity and pepsin level in this model, which suggests its anti-secretory potency. Duodenal ulcer is caused mainly by an increase in acid and pepsin load and gastric metaplasia in the duodenal cap (Dore and Graham 2000). Induction of experimental duodenal ulcers by histamine administration is mediated through both enhanced gastric acid secretion and vasospastic action of histamine (Cho and Pfeiffer 1981). Protective effect of Fr-CHCl3 against histamine induced duodenal ulcer in guinea pigs signifies its role in control of injury mediated by gastric acid secretion suggesting its antisecretory activity. In an attempt to clarify the mode of action of Fr-CHCl3, through the anti-secretory pathway, its influence on gastric secretion was studied using inhibition of H + K + ATPase (Proton pump). This proton pump is a membrane bound enzyme that catalyses H + transport at the expense of ATP hydrolysis. Thus the inhibition or the blockade of H + K + -ATPase may account for suppressed acid secretion observed in the in vivo studies. The results obtained with gastric microsomes isolated from rat stomach showed that FrCHCl3 potently inhibited the H + K + -ATPase activity comparable to the positive control, Omeprazole, thus suggesting that Fr-CHCl3 might be imparting anti-ulcer activity through decrease in acid secretion via proton pump inhibition. The cytoprotective ability of Fr-CHCl3 was evident with increase in mucin content in pyloric ligation model and protection against ethanol induced ulcer model in comparison with the standard drugs. To further substantiate the cytoprotective potency of Fr-CHCl3, its effect against NSAIDs induced ulcer model was explored. Studies suggest that NSAIDs induces ulcers due to their effect on cyclooxygenase enzyme leading to reduced prostaglandin production and increase in acid secretion (Goel
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and Bhattacharya 1991; Bhargava et al. 1973). Fr-CHCl3 significantly reduced ulcer incidence, which further supports cytoprotective effect of Fr-CHCl3, which may be mediated by prostaglandins. Phytochemical investigations of the Fr-CHCl3 demonstrated the presence of pure compounds namely Gedunin and Photogedunin. The gastroprotective activity of the compounds gedunin and photogedunin is not well established. Thus, we investigated the effect of gedunin and photogedunin on H + K + -ATPase inhibitory activity in isolated gastric microsomes from rat stomach. Gedunin and photogedunin inhibited the proton pump activity with an IC50 comparable to that observed with Fr-CHCl3 signifying that antisecretory activity of the fraction might be attributed to the presence of these compounds. Though different biological activities of the plant Xylocarpus granatum has been reported earlier, anti-ulcer activity of this plant has not been reported till date. Our study is the first of its kind to show significant anti-secretory effect of gedunin and photogedunin isolated from the Fr-CHCl3 of Xylocarpus granatum fruit. Thus, the present study demonstrated that the Fr-CHCl3 of X. granatum and its active constituents gedunin and photogedunin impart gastroprotective effects through the inhibition of H + K + ATPase (proton pump) activity. Thus, Fr-CHCl3 of X. granatum may emerge as a more potent therapeutic agent in treating gastric ulcer incidences since they possess both anti-secretory and cytoprotective potentials.
Acknowledgements We are grateful to the Ministry of Earth Sciences, Government of India, New Delhi for providing financial assistance. One of the authors (Neetu Singh) gratefully acknowledges ICMR, New Delhi for providing financial support. Mrs. Shibani Sen Gupta for her technical work and Dr. H.K. Singh for correcting the manuscript. References Anoop, A., Jegadeesan, M., 2003. Biochemical studies on the anti-ulcerogenic potential of Hemidesmus indicus R.Br. var. indicus. J. Ethnopharmacol. 84, 149– 156. Barros, M.P., Lemosd, M., Maistrob, E.L., Leite, M.F., Sousac, J.P.B., Bastos, J.K., 2008. Evaluation of antiulcer activity of the main phenolic acids found in Brazilian Green Propolis. J. Ethnopharmacol. 120, 372–377. Burke, B.A., Chan, W.R., Magnus, K.E., Taylor, D.R., 1969. Extractives of Cedrela odorata L.—III, The structure of photogedunin. Tetrahedron 25, 5007–5011. Berglindh, T., 1990. Gastric glands and cells: Preparation and in vitro methods. Methods enzymol. 192, 93–107. Bhargava, K.P., Gupta, M.B., Tangri, K.K., 1973. Mechanism of ulcerogenic activity of indomethacin and oxyphenbutazone. Eur. J. Pharmacol. 22, 191–195.
Cho, C.H., Pfeiffer, C.J., 1981. Gastrointestinal ulceration in the guinea pig in response to dimaprit, histamine, and H1- and H2-blocking agents. Dig. Dis. Sci. 26, 306–311. Debnath, P.K., Gode, K.D., Das, D., Sanyal, A.K., 1974. Effects of propanolol on gastric secretion in albino rats. Br. J. Pharmacol. 51, 213–216. Dharmani, P., Mishra, P.K., Maurya, R., Chauhan, V.S., Palit, G., 2005a. Desmodium gangeticum: a potent anti-ulcer agent. Indian J. Exp. Biol. 43, 517–521. Dharmani, P., Mishra, P.K., Maurya, R., Singh, C.V., Palit, G., 2005b. Allophylus serratus: a plant with potential anti-ulcerogenic activity. J. Ethnopharmacol. 99, 361–366. Dharmani, P., Kuchibhotla, V.K., Maurya, R., Srivastava, S., Sharma, S., Palit, G., 2004. Evaluation of anti-ulcerogenic and ulcer-healing properties of Ocimum sanctum Linn. J. Ethnopharmacol. 93, 197–206. Djahanguiri, B., Taubin, H.L., Landsburg, L., 1973. Increased sympathetic activity in the pathogenesis of restraint ulcer in rats. J. Pharmacol. exp. ther. 184, 163–168. Dore, M.P., Graham, D.Y., 2000. Pathogenesis of duodenal ulcer disease: the rest of the story. Baillie re’s Best Pract. & Res. Clin. Gastroenterol. 14, 97–107. Finney, D.J., 1952. In: A statistical treatment of the sigmoidal response curve 2nd ed. Cambridge Univ. Press, New York London 318. Ghani, A., 1998. Medicinal Plant of Bangladesh with Chemical Constituents and Uses, Asiat. Soc. Bangladesh 2nd ed., pp. 431–432. Goel, R.K., Bhattacharya, S.K., 1991. Gastroduodenal mucosal defence and mucosal protective agents. Indian J. Exp. Biol 29, 701–714. Goel, R.K., Das, D.G., Sanyal, A.K., 1985. Effect of vegetable banana powder on changes induced by ulcerogenic agents in dissolved mucosubstances of gastric juice. Indian J. Gastroenterol. 4, 249–251. Hoogerwerf, W.A., Pasricha, P.J., 2006. Pharmacotherapy of gastric acidity, peptic ulcers, and gastroesophageal reflux disease. In: Brunton, L.L., Lazo, J.S., Parker, K.L. (Eds.), The Pharmacol. Basis Ther.. Mc Graw Hill, New York, pp. 967–981. Kinoshita, M., Tsunehisa, N., Tamaki, H., 1995. Effect of a combination of ecabet sodium and cimetidine on experimentally induced gastric-lesions and gastricmucosal resistance to ulcerogenic agents in rats. Biol. Pharmaceut. Bull. 18, 223–226. Kirtikar, K.R., Basu, B.D., 1980. In: Indian Med. Plants 2nd ed., pp. 551–553. Miki, T., Sakaki, T., Shibata, M., Inukai, Y., Hirosue, H., Ikema, Y., Yaga, S., 1994. Soxhlet extraction of mangrove and biological activities of extracts. Kyushu Kogyo Gijutsu Kenkyusho Hokoku 53, 3347–3352. Miller, T.A., Henagan, J.M., 1984. Indomethacin decreases resistance of gastric barrier to disruption by alcohol. Dig. Dis. Sci. 29, 141–149. Parmar, N.S., Desai, J.K., 1993. A review of the current methodology for the evaluation of gastric and duodenal anti-ulcer agents. Indian J. Pharmacol. 25, 120–135. Sairam, K., Priyambada, S., Aryya, N.C., Goel, R.K., 2003. Gastroduodenal ulcer protective activity of Asparagus racemosus: an experimental, biochemical and histological study. J. Ethnopharmacol. 86, 1–10. Sanui, H., 1974. Measurement of inorganic orthophosphate in biological materials: Extraction properties of Butyl Acetate. Anal. Biochem. 60, 489–504. Shay, M., Kamarov, S.A., Fels, D., Meraaze, D., Grueinstein, H., Siplet, H., 1945. A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology 5, 43–61. Srivastava, S.K., Nath, C., Gupta, M.B., Vrat, S., Sinha, N.J., Dhawan, N.K., Gupta, G.P., 1991. Protection against gastric ulcer by verapamil. Pharmacol. Res. 23, 81–86. Suleyman, H., Demirezer, L.O., Kuruuzum-Uz, A., 2004. Effects of Rumex patientia root extract on indomethacine and ethanol induced gastric damage in rats. Pharmazie 59, 147–149. Suleyman, H., Demirezer, L.O., Buyukokuroglu, M.E., Akcay, M.F., Gepdiremen, A., Banoglu, Z.N., Gocer, F., 2001. Antiulcerogenic effect of Hippophae rhamnoides L. Phytother. Res. 15, 625–627. Taylor, D.A.H., 1974. 13C Nuclear Magnetic Resonance spectra of some limonoids Part-1 The structure of Procerin, an extractive from Carapa procera. J Chem. Soc. Pt 1, 437–441. Varley, H., Gowenlock, A.H., Bell, M., 1980. In: Pract. Clin. Biochem. 5th ed. The Whitefrairs Press, London 535–595.
