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Pharmacologia
Year: 2013  |  Volume: 4  |  Issue: 2  |  Page No.: 70 - 76

Role of Gastrin and Histamine in the Anti-ulcerogenic Potential of Compounds-gedunine and Photogedunine

Neetu Singh, Pratibha Singh, Gautam Palit and Vijai Lakshmi    

Abstract: Aim: Preliminary study revealed that gedunin and photogedunin, from Xylocarpus granatum exhibit anti-ulcer property. In an attempt to clarify the mechanism involved in its anti-ulcerogenic potential, an advanced studies to examine their effects on potent gastric acid secretagogues i.e., plasma gastrin and gastric mucosal histamine in pyloric ligation induced ulcer model. In addition, an evaluation of the cytoprotective function of these compounds by assessing the gastric PGE2 levels in alcohol induced gastric ulcer model. Methods: Adult Sprague Dawley rats, weighing 180-220 g procured from National Laboratory Animal Centre, CDRI, were used in the study. Rats were housed three to four per cage, in a room with temperature regulated at 22±2°C, with a 12 h/12 h light/dark cycle (lights on 07:00 h, lights off 19:00 h). Standard chow pellets and water were given ad libitum, except during the period when food deprivation was applied. Compounds of Xylocarpus granatum and standard drugs like omeprazole (10 mg kg-1) and sucralfate (500 mg kg-1) were prepared in 1% carboxymethyl cellulose (CMC) as suspension and administered orally 45 min 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). Control group of animals were treated with vehicle 1% CMC. Compound gedunin and photogedunin (20 mg kg-1, p.o.) were tested against alcohol and pyloric ligation induced gastric ulcer model. Standard anti-ulcer drugs, omeprazole (10 mg kg-1, p.o.) were treated in pyloric ligation model and sucralfate (500 mg kg-1, p.o.) in alcohol induced ulcer model. Results: Gedunin and photogedunin exerted significant acid lowering activity in pyloric ligated rats as evident through reduced free acidity and total acidity as well as in vitro H+K+-ATPase activity. Also, both compounds normalized the plasma gastrin and tissue histamine level compared to ulcerated rats. Contrastingly, both compounds illustrated no significant increase in gastric PGE2 level and mucin content compared to ulcer control rats. Conclusion: Thus, the study suggested that gedunin and photogedunin imparts gastro-protection through antisecretory mechanism.

1,2,3,4,5. Similarly, it has been observed that plants rich in polyphenols and various natural polyphenolics4 demonstrate dual anti-inflammatory and anti-ulcer activities, through the inhibition/blockade of ROS generation5 Helicobacter pylori (HP) is an ubiquitous gram-negative bacterium. Its specific microbiological features allow its survival in the stomach and the colonisation of the antrum. HP expresses high levels of urease enzyme, producing and CO2 from the urea being present in the gastric juice6. This mechanism called acid acclimation regulates the cytoplasmic pH of the bacterium and guarantees the mainte-nance of a periplasmic pH near neutrality, despite the acidic environment7. Once HP has colonised the stomach, an immune response begins; as a result, normal acid secretion and gastric architecture are modified. Consequences are gastritis, peptic ulcer disease, functional dyspepsia, cancer. HP was first identified in 1982 and by 1989 had been associated with gastric infiammation and ulcers in adults and children8. During the 1990s evidence emerged of its etiologic role in stomach cancers in adults. HP was classified in 1994 among the group I carcinogens by the International Agency for Research on Cancer9 . There are also a lot of associations between HP and other extra intestinal diseases such as cardiovascular diseases, diabetes mel litus, lu ng diseases, hematolog ic diseases, neurolog ical diseases and glaucoma. Nevertheless, more studies are necessary to delineate the cause-effect relationship for any of these issues10. Helicobacter pylori infects approximately 50% of the adult population6,7,8,9,10,11. Only 20% of HP-infected people a resymptomatic where as 80% of them are symptom-free.

Modern approaches to this disease includes various pharmacological interventions like H2-receptor antagonists, Proton pump inhibitors, sucralfate, rebamipide, antimicrobial agents, alone or in combination have proved benefits in the treatment of peptic ulcer disease in several studies but there are reports of development of tolerance and incidence of relapses and side effects on clinical evaluation make their efficacy arguable. Now days, various approaches have been made to study herbal drugs for treatment of various gastrointestinal disorders.

In previous study, it has been reported that gedunin and photogedunin of Xylocarpus granatum exhibit anti-ulcer property12, however, its exact mechanism remain unexplored. In this investigation, mechanism involved in its anti-ulcerogenic potential has been explored.

**ad1**

MATERIALS AND METHODS

Chemicals: All the chemicals used in the study were obtained from M/s. Sigma Chemicals, St Louis, MO, USA unless otherwise mentioned.

Experimental animals: Experimental protocols were approved by the Institutional Ethical and Usage Committee of Central Drug Research Institute (CDRI), Lucknow, following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Adult Sprague Dawley rats, weighing 180-220 g procured from National Laboratory Animal Centre, CDRI, were used in the study. Rats were housed three to four per cage, in a room with temperature regulated at 22±2°C, with a 12 h/12 h light/dark cycle (lights on 07:00 h, lights off 19:00 h). Standard chow pellets and water were given ad libitum, except during the period when food deprivation was applied.

Treatment schedule: Compound gedunin, photogedunin of Xylocarpus granatum (Fig. 1) and standard drugs like omeprazole (10 mg kg-1) and sucralfate (500 mg kg-1) were prepared in 1% carboxymethyl cellulose (CMC) as suspension and administered orally 45 min 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). Control group of animals were treated with vehicle 1% CMC. Compound gedunin and photogedunin (20 mg kg-1, p.o.) were tested against alcohol and pyloric ligation induced gastric ulcer model. Standard anti-ulcer drugs, omeprazole (10 mg kg-1, p.o.) were treated in pyloric ligation model and sucralfate (500 mg kg-1, p.o.) in alcohol induced ulcer model.

Anti-ulcer studies
Pyloric ligation induced ulcer model: Pyloric ligation was done under chloral hydrate anesthesia (300 mg kg-1, i.p.)21. After 45 min pretreatment with compound gedunin and photogedunin (20 mg kg-1, p.o.) and omeprazole (10 mg kg-1, p.o.), pyloric end of the stomach was ligated and abdomen was closed by suturing. After 4 h of surgery, rats were sacrificed and the stomach was dissected out and the accumulated gastric juice was collected. Ulcers were also scored after examining the dissected stomach under Magnascope and gastric fluid was collected and centrifuged at 2000 rpm for 10 min.

Fig. 1: Structure of compound gedunin and photogedunin

The collected supernatant was used for the estimation of gastric secretion studies, mucin estimation and peptic activity.

Gastric secretion study: Free and total acids in the gastric juice were titrated with 0.01 N NaOH, using Topfer’s reagent and phenolphthalein as indicators respectively and were expressed in terms of μ eq./mL13. Mucin level in gastric juice was quantified with a fluorometric assay and expressed as μg of mucin mL-1 of gastric juice14.

Alcohol induced gastric ulcer in rats: Gastric ulcer was induced in rats by administering absolute alcohol15.

Measurement of ulcer index: Ulcers were scored with the help of magnascope under 5X magnification using the ulcer scoring criteria16. The following scoring system was used to grade the incidence and severity of the lesions: (I) shedding of epithelium = 10 (ii) petechial and frank hemorrhages = 20 (iii) one or two ulcers = 30 (iv) more than two ulcers = 40 (v) Perforated ulcers = 50. Length of hemorrhagic band is measured in AL model using Biovis Image Analysis Software.

Percentage protection is calculated as follows:

% protection = (Uc-Ut)x 100/Uc

where, Uc = ulcer index in control group; Ut = ulcer index in treated group.

In vitro assay of H+, K+-ATPase activity: H+-K+-ATPase-containing microsomes were prepared from fasted rat stomach as previously reported with minor modifications17,18.

For the enzyme assay, gastric microsomes incubated with or without different concentrations of gedunin and photogedunin as well as standard drug omeprazole for 10 min at 37°C. Buffer containing 150 mM KCl, 10 mM PIPES, 1 mM MgSO4, 5 mM MgATP, 1 mM EGTA and 0.1 mM ouabain, at pH 7.2 and 10 μg mL-1 valinomycin, 2.5 μg mL-1 oligomycin was added. The reaction was carried out at 37°C for 20 min and was stopped by adding 10% ice-cold trichloroacetic acid. After centrifugation at 2000xg for 1 min, inorganic phosphate (Pi) released in the supernatant was determined spectrophotometrically at 310 nm wavelength and expressed as μM/(h mg) protein.

Gastrin measurement: Gastrin level was measured in the blood samples of normal, ulcer control (pyloric ligation induced) and treated rats (gedunin, photogedunin and omeprazole). The blood samples were collected by cardiac puncture and drawn into tubes containing chilled EDTA (1 mg mL-1 blood). Samples were then centrifuged at 1,600 g for 15 min at 0°C for plasma separation. Further, for the accurate determination of Gastrin, plasma was subjected to extraction procedure described in manufacturer’s instruction catalogue in which gastrin was extracted from plasma samples using 1% TFA (Assay designs, Hines Drive Ann Arbor, USA). The purified gastrin thus recovered was reconstituted in assay buffer provided in kit, its concentration was measured following kit’s protocol, and results were expressed as pg mL-1.

Estimation of gastric prostaglandin PGE2 estimation: PGE2 assay was performed as described by manufacturer instructions (Amersham Pharmacia Biotech, USA). Briefly, the gastric mucosa of different groups was excised and homogenized in an ice-cold Tris/HCl buffer containing 50 mM Tris/HCl (pH 7.4), 100 mM sodium chloride, 1 mM calcium chloride, 1 mg mL-1 d-glucose and 28 FM indomethacin. PGE2 recovery and purification was conducted according to protocols provided with the PGE2 EIA kit. Purified PGE2 samples were stored at -80°C until assayed. Samples were dissolved in 0.5-1.0 mL of sample buffer and assayed in 96-well plates. The quantities of PGE2 were determined by PGE2 standards ranging from 2.5 to 320 pg mL-1. Total protein concentration was determined using Lowry protein assay. Results were expressed as pg of PGE2 mg-1 of protein.

Estimation of gastric histamine content: Histamine in gastric tissue was estimated flourimetrically by the method19. Gastric tissues were homogenized in 9 volumes of 0.4 N perchloric acid. Tissue homogenate is allowed to stand for 10 min and is then centrifuged at 10000 g at 4°C for 20 min. Supernatant was treated with 250 μL 5N NaOH with 750 mg of solid NaCl. Total mixture was extracted with 5 mL of n-butanol and centrifuged at 2500 g for 10 min. Aqueous phase was separated by aspiration. Organic phase was separated and shaken with 2.5 mL of salt saturated 0.1 N NaOH. Butanol layer was collected and 3 ml of 0.1 N HCl and 5 mL of n-heptane. Tubes were shaken for 1 min and centrifuged at 2500 g for 10 min. About 2.5 mL of aqueous phase was collected and histamine was estimated flourimetrically after condensation with 1% O-phthalaldehyde reagent. Intensity of fluorescence was read at 450 nm after excitation at 360 nm.

Statistical analysis: All values shown in the figures and tables represent the means±S.E.M. IC50 values with 95% confidence limits were estimated using Maximum Likelihood Iterative Procedure 20. Statistical analysis were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Significance was ascertained at p<0.05. All the data are presented as means±SEM (standard error of the means).

RESULTS

Effect of compound gedunin and photogedunin against various ulcer models in rats: As shown in Fig. 2, gedunin (20 mg kg-1, p.o.) significantly prevented the ulcers formation in Pyloric Ligated (PL) rats showing percentage protection of 62.50 in comparison to control. Also standard drug, omeprazole significantly protected against ulcer formation in PL rats showing 72.80 percentage protection compared to control group. In ethanol induced ulcer model, gedunin showed 78.60% protection whereas standard drug sucralfate showed 64.50% protection over control group.

Compound photogedunin showed 53.20% protection in pyloric ligation model, whereas standard drug omeprazole showed 72.80% protection in comparison to ulcer control group.

Fig. 2:
Effect of gedunine (20 mg kg-1, p.o.), standard drug omeprazole and sucralfate on percentage protection against pyloric ligation and alcohol induced gastric ulcer model in rats. Data expressed as mean % protection±SEM. Statistical analysis was done by One Way ANOVA followed by Dunnett’s Multiple Comparison Test. *p<0.05, **p<0.01, in comparison to control, n = 6 in each group

Further, this compound showed 60.60% protection in ethanol induced gastric ulcer model, whereas standard drug sucralfate showed 64.50% protection over control group (Fig. 3).

In order to understand the biological basis of anti-ulcerogenic effect of gedunin and photogedunin, we examined the changes occurring in the following parameters:

The involvement of gedunin and photogedunin on the gastric secretions was examined by estimating the free/total acidity and mucin content of the accumulated gastric juice in pyloric ligated rats. As evident from the Table 1, gedunin and photogedunin significantly reduced free acidity and total acidity showing (32.92 and 24.25%) and (30.76 and 13.55%) inhibition, respectively compared to control group. In addition, standard drug omeprazole showed significant lowering of free acidity and total acidity offering 67.41 and 71.86% inhibition, respectively over control group. Besides lowering acid secretion, gedunin and photogedunin also up regulated the gastric mucin contents but the upregulation is not significant in comparison to control while omeprazole showed 39.28% increase in the gastric mucin levels with respect to ulcer control values.

The in-vivo anti-secretory effects of gedunin and photogedunin were further ascertained by examining its H+ K+-ATPase (proton pump) inhibitory activity under in-vitro conditions in gastric microsomal preparations. As shown in Fig. 4, gedunin and photogedunin (10-100 μg mL-1) moderately inhibited the microsomal H+ K+-ATPase activity in comparison to the control with an IC50 value of 56.86 and 66.54 μg mL-1.

Fig. 3:
Effect of photogedunine (20 mg kg-1, p.o.) and standard drug omeprazole and sucralfate on percentage protection against pyloric ligation and alcohol induced gastric ulcer model in rats. Data expressed as mean % protection±SEM. Statistical analysis was done by One Way ANOVA followed by Dunnett’s Multiple Comparison Test. *p<0.05, **p<0.01, in comparison to control, n = 6 in each group

Table 1:
Effect of gedunine, photogeunine (20 mg kg-1) and Omeprazole (10 mg kg-1) on free acidity, total acidity and mucin contents in pyloric ligation model (n = 6 in each group)
*Statistically significant at p<0.05 and **p< 0.01, ***p<0.001 in comparison to control, n = 6 in each group

Fig. 4:
Effect of gedunine, photogedunine and standard drug omeprazole on H+ K+-ATPase activity in the rat gastric microsomes. Mean±SEM of experiments performed in triplicates (n = 3)

While omeprazole (10-50 μg mL-1), used as a positive control, reduced the H+ K+-ATPase activity with an IC50 value of 30.24 μg mL-1.

The involvement of gastrin hormone in the anti-secretory mechanism of gedunin and photogedunin was examined by estimating the plasma gastrin levels in PL model. As represented in Fig. 5, PL control group exhibited significantly increased plasma gastrin levels compared to normal group. Pretreatment with gedunin and photogedunin significantly reduced the plasma gastrin level compared to PL control group. In addition, omeprazole used as reference drug showed significantly reduced plasma gastrin level over PL ulcer control rats.

These results revealed that gedunin and photogedunin possess significant anti-secretory properties at the dose of 20 mg kg-1, p.o. that may underlie its gastroprotective effects. However, it seems that gedunin and photogedunin mediated effects were more dependent on its influences on the action of gastric acid secretagogue, gastrin rather than proton pump activity, as it weekly inhibited the in-vitro H+K+-ATPase activity. Thus, we next examined the role of gedunin and photogedunin on the gastric histamine level and we found that these compounds reduced the gastric histamine level as compared to the ulcer control group Fig. 6.

Fig. 5:
Effect of gedunine, photogedunine on plasma gastrin level in comparison to omeprazole in pyloric ligation induced gastric ulcer model. ##Statistically significant at p<0.01, in comparison to Normal control group (n = 6 in each group). **Statistically significant at p<0.01, ***p<0.001 in comparison to Ulcer control group (n = 6 in each group).

Fig. 6:
Effect of gedunine, photogedunine on gastric histamine level in comparison to control and histamine receptor antagonist ranitidine. **Statistically significant at p< 0.01, *p<0.05in comparison to control, n = 3 in each group

The results were comparable to the H2 receptor antagonist ranitidine.

DISCUSSION

In our earlier studies, we have shown that compound gedunin and photogedunin of Xylocarpus granatum exerts anti-ulcer activity12, but the mechanism of actions through which it mediates gastroprotective effects were not elucidated. In this attempt, we advanced our studies to examine their effects on potent gastric acid secretagogues i.e., plasma gastrin and gastric mucosal histamine in Pyloric Ligation (PL) ulcer model, respectively. In addition, we evaluated the cytoprotective function of these compounds by assessing the gastric PGE2 levels in alcohol model.

In our previous report, we have shown that chloroform fraction of Xylocarpus granatum showed significant anti-ulcer activity at a dose of 100 mg kg-1, p.o. In this study, we select 1/5th of the dose i.e., 20 mg kg-1, p.o. for its compounds in order to screen anti-ulcer activity.

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. Therefore, we select two models one of antisecretory i.e., pyloric ligation and other of cytoprotective i.e., alcohol model for the gastroprotective study of compound gedunin and photogedunin.

Gastric acid plays a central role in ulcer induction through pyloric-ligation model21. In this model, gastric mucosal auto-digestion via increased gastric acid secretion and pepsin activity was believed to propagate the ulcer formation22. Significant inhibition of free acidity and total acidity was observed with compound gedunin and photogedunin in this model, reflecting its potent anti-secretory activity in vivo.

As the molecular action of these secretagogues finally converge to the activation of enzyme, H+ K+ -ATPase, we next examined the effect of gedunin and photogedunin on the H+ K+ -ATPase activity in gastric microsomal preparation. It was observed that gedunin and photogedunin moderately affects the H+ K+ -ATPase activity under in vitro condition compared to positive control omeprazole. In contrast, the anti-ulcerogenic potential of gedunin and photogedunin under in vivo experiments was found as effective as omeprazole. The possible explanation for these observations might be the fact that the antisecretory functions of omeprazole is not dependent on secretagogues of gastric acid23, while gedunin and photogedunin may exert its effect on gastric acid secretion in vivo by its collective influences on the actions of gastric acid secretagogue and H+ K+ -ATPase enzyme. MacIntosh23, had proposed that stimulation of the vagus resulted in the release of histamine and Code19 and later Kahlson24 extended that idea to gastrin, making histamine the final common chemostimulant. As the release of acid secretagogue, histamine is also controlled by hormone gastrin, in the next series of experiment we evaluated the effect of gedunin and photogedunin on the plasma level of gastrin and gastric tissue histamine level in rats subjected to pyloric ligation. The choice of PL model for measurement of gastrin and histamine level was based on the reports demonstrating the regulatory role of vagus in the release of gastrin and accompanying increase in formation of ulcer in rat stomach25. It was noted that gedunin and photogedunin significantly abolished the increase in plasma gastrin level induced by pyloric ligation in rats. We have found that gedunin and photogedunin significantly reduced the gastric histamine level as compared to the control group suggesting that observed decrease in acid secretion of gedunin and photogedunin both in-vitro and in-vivo might be via decreased gastric histamine level.

Moreover, in order to understand the cytoprotective efficacy of compound gedunin and photogedunin, it was tested against ethanol-induced gastric lesions and further checked for gastric PGE2 level. Since ethanol directly causes mucosal damage by inhibiting the release of mucosal prostaglandins26 and depressing the gastric defensive mechanisms27, both of these compounds failed to reverse the alcohol induced gastric mucosal damage, gastric PGE2 level and mucin content compared to control rats. Conclusively, the present study suggested that gedunin and photogedunin imparts gastro-protection through antisecretory mechanism rather than cytoprotective mode of action.

ACKNOWLEDGMENTS

Authors gratefully acknowledge to Council of Scientific and Industrial Research, HRDG, Government of India, New Delhi for providing to VL emeritus scientistship and ICMR New Delhi, India for providing financial support to NS. Mrs. Shibani Sengupta was acknowledged for her technical assistance.

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