Gastroprotective effect of anti-cancer compound rohitukine: possible role of gastrin antagonism and
H
+K
+-ATPase inhibition
Neetu Singha†, Pratibha Singha†, Shishir Shrivastvab, Sunil Kumar Mishrab, Vijai Lakshmib* Rolee Sharmac and Gautam Palita*
aDivision of Pharmacology, bDivision of Medicinal and Process Chemistry, Central Drug Research Institute, C.S.I.R.,Lucknow – 226001, U.P, India
cDepartment of Biotechnology, Integral University, Lucknow - 226026, India.
*Corresponding Author Name: Dr. Gautam Palit
Postal Address: Division of Pharmacology, Central Drug Research Institute, Lucknow – 226001, Uttar Pradesh, India
Tel No: +91-522-2612411-418, Ext: 4303 Fax No: +91-522 2623405, +91-522 2623938 Email: [email protected]
† These authors contributed equally to this work.
Abstract
The present study was designed to evaluate the anti-ulcerogenic properties of an alkaloid chromane, rohitukine from Dysoxylum binecteriferum. Anti-ulcer potential of rohitukine was assessed in cold restrained, pyloric ligated and ethanol induced ulcers in rats. Also, rohitukine was tested in vitro for H+ K+-ATPase inhibitory activity in gastric microsomes.
Moreover, we studied the role of rohitukine on the cytosolic concentration of Ca2+ in parietal cell enriched cell suspension in order to ascertain its mechanism of action.
Cytoprotective activity was evaluated through PGE2 level. Rohitukine significantly attenuated the ulcers in CRU model in a dose-related manner. Moreover, it significantly lowered the free acidity and pepsin activity in pyloric ligated rats while improved the depleted level of mucin. Also, rohitukine significantly reversed the cold restrained- induced increase in gastrin level. Our in vitro study revealed that rohitukine moderately inhibited the microsomal H+ K+-ATPase activity with respect to positive control omeprazole. Furthermore, rohitukine potently antagonized the gastrin-elicited increase in cytosolic Ca2+ level in parietal cell enriched suspension. In ethanol induced gastric lesions in rats, rohitukine significantly inhibited the formation of erosions and increased PGE2 content showing more potency than reference drug sucralfate. Our results thus suggest that rohitukine possess significant anti-ulcer and anti-gastrinic activity in rats. It is likely that gastro-protective influences of rohitukine are dependent partly on its acid- lowering potential and partly on cytoprotective property. The acid-reducing effect of rohitukine might be attributed to its lowering effect on gastrin production and/or antagonism of gastrin evoked functional responses of parietal cells. Thus, rohitukine represent a useful agent in the treatment of peptic ulcer disease.
Keywords Rohitukine · gastric ulcer · Gastrin · intracellular [Ca2+] · H+ K+-ATPase · CCK2 receptor
Introduction
Peptic ulcer being the most prevalent gastrointestinal disorder continues to occupy the key position in concern of both clinicians and researchers. As a result more and more drugs, both herbal and synthetic, are coming up offering newer and better options for the treatment of peptic ulcer, but their critical clinical evaluation have recorded several discrepancies including incidence of relapses, adverse side effects and drug interactions.
To avoid these adverse effects, investigation has been extended to exploit medicinal plant derived novel molecules as new leads which can offer better protection and lower incidence of side effects.
Rohitukine (C16H19O5N), a chromane alkaloid, was first reported from Amoora rohituka (Roxb.) Wight & Arn. (Harmon et al., 1979) and later from Dysoxylum binectariferum Hook. f., both belongs to the family Meliaceae. Rohitukine, from D.
binectariferum has proved to be a good disease modifying anti-rheumatic agent apart from having a CNS depressant action. Besides acting as an anticancer compound rohitukine also exhibits both anti-inflammatory as well as immuno-modulatory properties (Naik et al., 1988). This alkaloid has also provided the chemical basis for synthesis of flavopiridol, a novel flavone that is currently in clinical trials against a broad range of tumors (Carlson et al., 1999).
Regulation of inflammatory response is an essential element in the pathogenesis of a variety of inflammatory disorders including gastric ulcer and compound rohitukine is reported to have anti-inflammatory properties therefore, we evaluated the anti-ulcer potential of the rohitukine in various rat models of gastric ulcer. Moreover, we carried out in vitro studies of the effect of rohitukine on H+ K+ -ATPase activity in gastric microsomes and intracellular Ca2+ level in parietal cell in order to ascertain its mechanism of action.
Materials and Methods
Extraction/Fractionation Procedure
The stem bark of Dysoxylum binectariferum was collected and identified by the Botany Division of the Institute from the Andaman coast of India. The voucher specimen (No.
8091) has been kept in the herbarium of the Institute. The extraction and fractionation from 1.0 kg of plant material was carried out adopting the procedure described previously (Lakshmi et al., 2007). Briefly, air-dried powdered plant material was extracted with distilled ethanol, concentrated under reduced pressure and further fractionated into four fractions (n-hexane, chloroform, soluble n-butanol and insoluble n-butanol fraction).
From chloroform fraction, a known alkaloid rohitukine {5,7-dihydroxy-2-methyl-8-[4-(3- hydroxy-1-methyl)-piperidinyl]-4H-1-benzopyran- 4-one)} was isolated by repeated column chromatography over silica gel and further purification by HPLC-LC-20AD using methanol solvent 55:45 v/v, flow rate 1.0ml/min. The characterization of compound was performed using IR, NMR (Table 1), mass, derivatization and comparison with available literatures. The yield of rohitukine was 1% and its purity was 99.6%.
Structure of rohitukine (Fig. 1) was also compared with authentic samples on thin layer plates as well as their spectral data.
Table 1 1NMR data of Rohitukine at 300MHz in CD3OD
H δ values
6 3 3’
4’
2’a 6’a 5’a 2’b N-Me
2-Me 6’b 5’b
6.2630 (1H, s) 6.0760 (1H, s) 4.2591 (1H, s) 3.7258 (1H, d, J=11.4)
3.4794 (1H, m) 3.5894 (1H, d, J=9.12)
3.2611 (1H, m ) 3.3092 (1H, s) 2.9242 (3H, s) 2.4274 (3H, s) 3.2192 (1H, m) 1.8410 (1H, d, J=12.6)
δ value at 4.83 represents for solvent CD3OD Experimental Animals
Experimental protocols were approved by the Institutional Ethical and Usage Committee of Central Drug Research Institute, Lucknow, following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Adult Sprague Dawley rats, weighing 130-180g 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 ± 20C, with a 12h/12h 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.
Fig. 1 Structure of Rohitukine
Treatment schedule
Rohitukine, standard anti-ulcer drug omeprazole (Omz) (Sigma Chemicals, USA) and sucralfate (SUC) (Meranani pharmaceutical, India) were prepared in 1% sodium carboxymethylcellulose (CMC) suspension and administered orally, 45 min prior to exposure to ulcerogens to the animals at a volume of 1ml/200g of body weight. All animals were deprived of food for 16 h before ulcerogens exposure and were divided into three groups each group comprising of six animals (n=6).
1. Control group of animals were treated with vehicle 1% CMC.
2. Graded doses of compound rohitukine (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), pyloric ligation (PL) and SUC (500 mg/kg, p.o.) in alcohol (AL) induced gastric ulcer model.
Anti-ulcer studies
Cold restraint induced gastric ulcer (CRU)
Animals were subjected to cold and restraint stress after 45 min of treatment with graded doses of rohitukine (10, 20 and 40 mg/kg, p.o.) and omeprazole (10 mg/kg, p.o.). Rats were immobilized in a restraint cages and kept at 4ºC in an environmental chamber for two hours (Suleyman et al., 2001). The animals were then sacrificed and stomachs were observed under Magnascope for ulcers and scored.
Pyloric ligation induced ulcer model (PL)
Pyloric ligation was done (Shay et al., 1945) under chloral hydrate anesthesia (300mg/kg, i.p.). After 45 min pretreatment with rohitukine (20 mg/kg, p.o.) and omeprazole (10 mg/kg, 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.
Gastric secretion study
Free and total acidity were measured from the collected gastric juice by titrating against 0.01N NaOH, using phenolphthalein as indicator and expressed in terms of μequiv./ml (Anoop and Jegadeesan, 2003). Peptic activity was determined by measuring the amount of liberated tyrosine by the action of pepsin on hemoglobin as substrateand expressed in terms of units/ml (Debnath et al., 1974). Mucin level in gastric juice was quantified with a fluorometric assay and expressed as µg of mucin/ml of gastric juice (Crowther and Wetmore, 1987).
Alcohol induced gastric ulcers in rats (AL)
Gastric ulcer was induced in rats by administering absolute alcohol (1ml/200g) (Suleyman et al., 2004). The rohitukine (20 mg/kg, p.o.) and SUC (500 mg/kg, p.o.) were administered 45 min before alcohol treatment. After 1h, the animals were sacrificed and stomachs were excised to observe the gastric lesions.
Measurement of ulcer index
Ulcers formed in stomach of CRU and pyloric ligated rats were scored according to the arbitrary scoring system (Srivastava et al., 1991) and 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 AL model the length of the lesions were measured using Biovis image analyzer software (Expert Vision Lab Private Ltd., Mumbai, India) and summated to give a total lesion score.
Gastrin measurement
Gastrin level was measured in the blood samples of normal, ulcer control (cold restraint induced) and treated rats (omeprazole & rohitukine). The blood samples were collected by cardiac puncture and drawn into tubes containing chilled EDTA (1mg/ml blood).
Samples were then centrifuged at 1,600 g for 15 min at 0ºC 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 and its concentration was measured following kit’s protocol and results were expressed as pg/ml.
In vitro assay of H+ K+-ATPase activity
H+ K+-ATPase activity was analyzed in gastric microsomes isolated from rat stomach (Berglindh, 1990, Reenstra and Forte, 1990) by measuring the inorganic phosphate released after hydrolysis of ATP. For the enzyme assay, 50µg of gastric microsomes were added to an assay buffer (pH 7.2) containing (in mM) 150 KCl, 10 PIPES, 1 MgSO4, 5 Mg ATP, 1 EGTA and 0.1 ouabain, 10µg/ml valinomycin and 2.5µg/ml oligomycin.
Further, the microsomes were incubated with or without different concentrations of rohitukine as well as standard drug omeprazole for 30 min at 37°C after which the reaction was stopped by adding 10% ice-cold trichloroacetic acid. The inorganic phosphate release was determined from the resulting supernatant spectrophotometrically at 310 nm wavelength(Sanui, 1974) and expressed as µM/hr/mg protein.
Intracellular Ca2+ monitoring
Preparation of isolated parietal cells from rat stomach
Gastric cell isolation was performed as described by (Berglindh, 1990) with some modifications. Media of the following compositions were used:
Medium A (mmol/l) NaH2PO4 (0.5), Na2HPO4 (1.0), NaHCO3 (20.0), NaCl (70.0), KCl (5.0), glucose (11.0), EDTA (2.0), HEPES (50.0), and BSA (2%).
Medium B was of same composition as A but was EDTA -free and contained CaCl2 (1.0), MgCl2 (1.5), and BSA (1%).
Medium C differed from B in having 0.1% BSA.
Hanks’ balanced salt solution (HBSS) with 10 mM glucose was used for final suspension of the isolated cells.
After anaesthetization, the rats were given in situ perfusion through the heart with normal saline. Stomach was removed and washed with normal saline, mucosa was minced. The minced mucosa was incubated in 5 ml of buffer A and was digested with 1 mg/ml pronase with continuous stirring at 37C. After 30 min, the supernatant was collected into a fresh tube and the undigested mucosa was digested with fresh enzyme solution (0.5 mg/ml) and incubated for 15 min. It was then centrifuged at 600 g for 5 min and the pellet was resuspended in 5 ml buffer B along with 0.5 mg/ml collagenase.
Another digestion for 30 min with continuous stirring was given and later it was centrifuged at 600g for 10 min and the supernatant was collected. The pellet was resuspended in buffer C and was washed twice with buffer C at 600g for 10 min.The
parietal cells were then separated from the final cell suspension by density-gradient centrifugation through linear Optiprep gradient.
Enrichment in parietal cells was obtained by the counterflow centrifugal elutriation technique (Soll, 1978). The apparatus for continuous-flow elutriation consists of a centrifuge containing a rotor with four separation chambers (Curame 3000, Haereus) and a peristaltic pump fitted with loading equipment (Cole Parmer Masterflex easyload).
The cell suspension (5 ml/chamber) was injected into the elutriator chambers and the cells were separated on the basis of varying sedimentation velocity in counterflow (McEwen et al., 1968). Cells were purified in four fractions (the rotor was set at 2000 rpm, the flow rate at 26, 37 and 41 ml/min for the first three fractions, respectively), the fourth fraction, collected at 1800 rpm and 59 ml/min of counter-flow rate, was enriched in parietal cells. Purity was 82 ± 5% and viability was > 95%, evaluated by exclusion of trypan blue. Normally 60-80 X 106 cells were obtained from about 7.5 g of gastric mucosa.
Ca2+ measurement
Cytosolic free Ca2+ concentration, [Ca2+]i, was monitored with the fluorescent Ca2+
indicator fura-2, as described by Grynkiewicz et al (Grynkiewicz et al., 1985). Cells were incubated with fura-2/AM (final concentration 4 µM) for 20 min at 37°C in Earle’s medium. After loading, the cell suspension was diluted with 10 vols. of Earle’s medium, centrifuged for 5 min at 200 X g, finally resuspended in 10 ml of Hepes-buffered saline (HBS composition in mM: NaCl 145, KC1 5, MgCl2, 1, CaCl2 1, Hepes 10, glucose 10, pH 7.4) at 5-10 X 106 cells/ml concentration, and kept at room temperature in the dark until use. The suspension (0.8 X l06 cells/l.5 ml) was added to a glass cuvette thermostated at 37°C under continuous stirring. Controls received either Ca2+ free HBS or dimethyl sulfoxide as vehicle. Fluorescence was measured by a dual excitation fluorimetry in a fluorescence spectrophotometer (Varian, Cary Eclipse).
Wavelengths were set at 340 nm and 380 nm for excitation, 505 nm for emission;
data points were collected at 0.2-0.4 s intervals. [Ca2+]i was estimated from the ratios of 340 and 380 nm signals according to the following equation (Grynkiewicz et al., 1985).
[Ca2+]i =Kd x β x R-R min/Rmax – R where Kd, is the dissociation constant for fura- 2-Ca2+ complex (224 nM), β = F(380)min/F(380)max, R is the ratio of fluorescence at 340 over 380 nm; Rmax and Rmin (fluorescence ratios at saturating and zero Ca2+ concentration respectively) were obtained in separate experiments by adding digitonin at 50 µM followed by 2 mMEDTA,and Tris and adjusted to pH 8.3.
Statistical analysis
Results are expressed as the mean ± S.E.M. from six rats per group. IC50 values with 95% confidence limits were estimated using Maximum Likelihood 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.
Probabilities of less than 5% (P < 0.05) were considered significant.
Results
NMR spectra of the rohitukine
The 1H NMR spectra of the rohitukine displayed the aromatic proton of H-6 at δ 6.2630 as a singlet and H-3 proton at δ 6.0760 as a singlet. Methyl protons at 1’ was found at δ
2.9242 as a singlet for 3 protons and methyl at 2 was found at δ 2.4274 as a singlet for 3 protons. The other aliphatic protons were seen at their usual positions as given in the Table 1 of NMR of rohitukine.
Experiment 1
Effect of rohitukine against various ulcer models in rats
Fig. 2 Effect of Rohitukine (10, 20 and 40mg/kg, p.o.) and standard drug omeprazole on percentage protection against cold restraint induced gastric ulcer in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by One Way ANOVA followed by Dunnett's Multiple Comparison Test. **P< 0.01, in comparison to control. n = 6 in each group.
As shown in Fig. 2, graded doses of Rohitukine (10, 20 and 40mg/kg, p.o.) significantly prevented the ulcers formation in CRU rats showing percentage protection of 66.63, 74.97 and 80.53 respectively in comparison to control. Also standard drug, omeprazole significantly protected against lesion formation in CRU rats showing 77.73 percentage protection compared to control group. From our this preliminary study we identified the 20 mg/kg dose of rohitukine as effective and selected for further studies in pyloric ligation and ethanol induced gastric lesion model in rats.
As represented in Fig. 3, rohitukine pretreatment significantly reduced the ulcer formation in pyloric ligated rats offering 50.0% protection in comparison to control which was comparable to protection afforded by omeprazole (69.42%). Further, in ethanol induced gastric lesion model, rohitukine showed 82.0% protection, whereas standard drug sucralfate showed 62.55% protection over control group (Fig. 3).
Experiment 2
To understand the biological basis of anti-ulcerogenic effect of rohitukine, we examined the alterations in the following parameters.
(i) The involvement of rohitukine on the gastric secretions we examined by estimating the free/total acidity, pepsin activity and mucin content of the accumulated gastric juice in pyloric ligated rats. As evident from the Table 2, rohitukine significantly reduced free acidity, total acidity and pepsin activity showing 47.92%, 25.46% and 51.69% inhibition respectively compared to control group. In addition, standard drug omeprazole showed significant lowering of free acidity, total acidity and pepsin activity offering 57.02%, 46.81%, and 60.09% inhibition respectively over control group, which was comparable to the values observed with rohitukine. Besides lowering acid secretion, rohitukine also significantly upregulated the gastric mucin contents by 68.12% in comparison to control
while omeprazole showed 53.42% increase in the gastric mucin levels with respect to control values.
Fig. 3 Effect of Rohitukine (20mg/kg, p.o.) and standard drug omeprazole and sucralfate against pyloric ligation and alcohol induced ulcer in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by One Way ANOVA followed by Dunnett's Multiple Comparison Test. **Statistically significant at P< 0.01, in comparison to control. n = 6 in each group.
Table 2 Effect of Rohitukine (20mg/kg) and Omeprazole (10mg/kg) on free acidity, total acidity, pepsin and mucin contents in pyloric ligation model (n= 6 in each group).
*Statistically significant at P<0.05 and **P< 0.01, in comparison to control. n = 6 in each group.
(ii) The in-vivo anti-secretory effects of rohitukine 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, rohitukine (10–100 μg/ml) moderately
inhibited the microsomal H+ K+-ATPase activity in comparison to the control with an IC50 value of 83.19 μg/ml. While omeprazole (10–50 μg/ml), used as a positive control, reduced the H+ K+-ATPase activity with an IC50 value of 30.24 μg/ml.
(iii) The involvement of gastrin hormone in the anti-secretory mechanism of rohitukine was examined by estimating the plasma gastrin levels in CRU model. As represented in Fig. 5, CRU control group exhibited significantly increased plasma gastrin levels compared to normal group. Pretreatment with rohitukine significantly reduced the plasma gastrin level compared to CRU control group. In addition, omeprazole used as reference drug showed significantly reduced plasma gastrin level over CRU control rats.
These results revealed that rohitukine possess significant anti-secretory properties at the dose of 20mg/kg, p.o. that may underlie its gastroprotective effects. However, it seems that rohitukine 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 rohitukine on the gastrin elicited functional response of isolated parietal cells in suspension.
Fig. 4 Effect of Rohitukine and standard drug omeprazole on H+ K+-ATPase activity in the rat gastric microsomes.
Dots and lines are mean ± S.E.M. of experiments performed in triplicates (n=3).
Experiment 3
As the functional responses of parietal cells to gastrin is directly coupled to the change in the cytosolic level of [Ca] ions, we examined the alterations in the intracellular [Ca] ions by employing the fura-2AM method. As illustrated in Fig. 6, the basal value of [Ca2+]i in the resting parietal cells was 162.7±8.76 nM (n = 3). Gastrin dose dependently (0.1 nM - 4 µM) increased the intracellular Ca2+ levels in parietal cells showing 50% increase over resting levels at 50nM (effective concentration) with 244.7±7.96 nM. Antagonism studies were thus performed using gastrin at 50 nM concentration. Rohitukine dose dependently (0.008 µM, 0.016 µM and 0.032 µM) reduced the [Ca2+]i with 163.3±9.28 nM, 153.7±5.78 and 71.0±6.65 nM respectively. Positive control, benzotript (gastrin receptor antagonist) used at 10µM concentration, inhibited the effect of gastrin stimulated [Ca2+]i
with 68.8±9.86 nM.
Fig. 5 Effect of Rohitukine on plasma gastrin level in comparison to omeprazole in cold restraint 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<0.05, in comparison to Ulcer control group (n = 6 in each group).
Fig. 6 Effect of Rohitukine (dose dependent) on intracellular Ca2+ level in comparison to Cholecystokinin receptor antagonist Benzotript. Data were obtained using parietal-enriched cell suspension, loaded with 4µM fura-2/AM. Cells were stimulated with 4µM rat gastrin I (agonist). ## Statistically significant at P<0.01, in comparison to basal group (n
= 3 in each group). **Statistically significant at P< 0.01, in comparison to agonist (rat gastrin). n = 3 in each group.
Experiment 4
The PGE2 generation in the ulcer control group was 2442.0±61.87 pg/mg tissue protein.
The PGE2 value of rohitukine and sucralfate treated group was found to be 2871.0±64.47 (P<0.05), 3868.0±66.62 (P<0.01) respectively (Table 3).
Discussion
Our present study demonstrated that rohitukine, a chromane alkaloid isolated from Dysoxylum binecteriferum possess remarkable anti-ulcer activity in rats. The
characterization of compound was performed using NMR (Table 1) and its structure (Fig.
1) was determined based on ESIMS, 1H NMR spectra and literature.
Table 3 Effect of compound rohitukine (20mg/kg,p.o.) and standard drug sucralfate (500mg/kg,p.o.) on gastric PGE2 level in ethanol induced gastric lesion model.
*Statistically significant at P<0.05 and **P< 0.01, in comparison to control. n = 6 in each group.
The anti-ulcer mechanism of rohitukine seems to be mediated by its significant acid inhibitory potential. The acid lowering action of this compound was evident not only in pyloric ligated rats; it also blocked gastric microsomal H+ K+ -ATPase activity under in vitro experimental conditions. Moreover, it normalizes the increased gastrin level in CRU model. The gastroprotective influences of rohitukine was not merely restricted to its acid lowering potential but may also involve reinforcement of gastric mucosal defense mechanism as suggested by significant protection against ethanol elicited gastric lesion and increased PGE2 level. Further evaluation of its molecular mechanism of acid inhibition at cellular level, revealed that rohitukine functions as gastrin receptor antagonist as it potently inhibited the gastrin elicited increase in cytosolic [Ca2+]i in gastric parietal cells.
In present study, rohitukine given orally inhibited the formation of gastric ulcer in rats caused by cold and restraint stress, in a dose-related manner. The compound was noted to be as efficient as reference drug, omeprazole in reducing the gastric lesions in CRU model. Cold and restraint stress alters the regulation of acid secretion, and it increases the acidity of gastric juice through vagal activation (Kitagawa et al., 1979).
Thus, the significant protection imparted by rohitukine in this model reflected the possibility of its involvement in the regulatory mechanism of gastric acid secretion. This interesting finding in CRU model intrigued us to further explore its effect on gastric acid secretions in rats.
Gastric acid plays a central role in ulcer induction through pyloric ligation model (Shay et al., 1945). Rohitukine significantly inhibited the secretion of gastric juice in pyloric ligated rats, an effect that is accompanied by a reduction in both free/total acidity and pepsin activity. Our this data further complemented our hypothesis that anti-
ulcerogenic effect of rohitukine is related to its acid inhibitory influences on gastric mucosa of rats.
As the molecular action of acid secretagogues such as histamine, gastrin and PGE2 finally converge to the activation of enzyme, H+ K+ -ATPase, we next examined the effect of rohitukine on the H+ K+ -ATPase activity in gastric microsomal preparation. It was observed that rohitukine moderately affects the H+ K+ -ATPase activity under in vitro condition compared to positive control omeprazole. In contrast, the anti-ulcerogenic potential of rohitukine 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 acid (Larsson et al., 1983) while rohitukine 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.
As early as 1938, MacIntosh (MacIntosh, 1938) had proposed that vagal stimulation resulted in the release of histamine and later this idea was extended to gastrin (Code, 1965, Kahlson and Rosengren, 1972) 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 rohitukine on the plasma level of gastrin in rats subjected to cold and restraint stress. The choice of CRU model for measurement of gastrin 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 stomach (Erin et al., 2000). It is well known that gastrin is a key hormonal inducer of acid secretion. The formation of ulcers in animals subjected to cold and restraint stress is mainly associated with vagal stimulation of gastrin release, followed by increased acidity of gastric juice (Debas and Carvajal, 1994). Concurringly we also found significantly increased gastrin levels and gastric acidity in CRU rats. However either omeprazole or rohitukine pretreatment in these rats reversed this increase in gastrin level, which reaches towards nearly basal levels. The possible explanation of these findings might be that noticeable increase in gastrin productions were seen only after prolonged use of acid suppressant (PPIs), however, omeprazole was administered acutely, it may not bring any visible increase in gastrin levels in CRU rats under given time period.
So far the gastroprotective activities of gastrin has been demonstrated against necrotizing agents, which produces stomach injuries independently of acid secretions (Konturek et al., 1995). Our study outcomes however differed from these previous investigations possibly since we are demonstrating the changes in gastrin level following ulcer induction by cold and restrain treatment in rats. As it is already known that incidence of stomach injuries in CRU rats were mainly dependent on acid secretion and gastrin is reported to be involved in the stimulation of gastric acid secretion under vagal activation in CRU model (Kitagawa et al., 1979). Thus it might be possible that gastrin can exerts its gastroprotective effects only during undisturbed acidic circumstances (as in ethanol induced stomach lesions), while under agitated acidic settings (CRU induced gastric injury), it act to stimulate acid secretions and in this manner contributes to the pathomechanism of mucosal injury.
As our preliminary invivo investigations showed that rohitukine reduced the acid secretion and normalizes the gastrin levels. Thus we next moved to identify the possible pathway through which rohitukine is inhibiting acid secretion. Parietal cells have been
used extensively as a tool to study the anti-secretory effect of any test compound in vitro.
The anti-secretory substances may act to inhibit gastric acid secretion by limiting the secretory responses to either histamine or gastrin. Therefore we used parietal cells as a tool to identify the mechanism behind rohitukine’s anti-secretory functions. Recent advances identified that functional responses of gastrin on acid secretion from parietal cells can be measured by monitoring the variations in cytosolic Ca2+ levels as gastrin receptor/CCK2 activation is directly coupled to cytosolic increase in Ca2+ ions (Letari et al., 1996). As rohitukine failed to show significant protection against histamine induced duodenal ulcers in guineapig during our initial study (data not shown), we specifically targeted gastrin pathway seeking as an answer to rohitukine’s potent anti-secretory activity seen invivo.
We observed that incubation of parietal cell with rat gastrin significantly elevated the intracellular Ca2+ level over basal values. This agonist-elicited amplification of Ca2+
level was significantly reversed by rohitukine. Comparing the lowering effect of rohitukine on intracellular Ca2+ ions, with reference CCK2 receptor antagonist benzotript revealed that rohitukine achieved significant attenuation of gastrin augmented level of cytosolic Ca2+ at much lower concentration than benzotript. Taken together our results revealed that rohitukine inhibits the acid secretion from parietal cells by antagonising gastrin elicited increase in Ca2+ ions. As Ca2+ blockers such as nifedipine and verapamil have been shown to inhibit gastrin release from cultured rat antrum preparations (Harty et al., 1981, Harty et al., 1984), its inhibition by rohitukine might be the explanation due to which we are getting reduced gastrin levels following its treatment in CRU rats.
With regard to the production of ethanol-induced gastric lesions, pathogenic factors include the presence of gastric acid, the increase in gastric motility and the depletion of mucosal prostaglandins (Lippmann, 1974, Kasuya et al., 1979, Takeuchi et al., 1986). Our result showed that rohitukine increased gastric PGE2 level and exerted significant protection in ethanol induced gastric lesions in rats, which was even more than protection offered by reference drug sucralfate. The possible explanation for this finding might be that inhibitory mechanisms of sucralfate in ethanol induced gastric lesion model is related to the activation of defensive mechanisms such as an increase in mucosal glycoprotein content rather than antisecretory mechanism. In contrast, it is likely that rohitukine exerted protection in this model, may be due to its potent antisecretory activity that goes along with its stimulatory effects on PGE2 production and mucin release. The mucin up-regulatory response of rohitukine was evident in the gastric secretions of pyloric ligated rats. Thus it seemed that rohitukine imparted protection in ethanol induced gastric lesion model, was partially acid-dependent and partly related to its stimulatory influence on gastric mucus glycoprotein production and PGE2 release.
Although antisecretory drugs such as histamine H2 receptor antagonists and proton pump inhibitors greatly reduce peptic ulcers, however ulcers frequently relapse even in the presence of maintenance therapy with these drugs (Wormsley, 1986).
Moreover, long-term treatment with antisecretory agents caused hypergastrinemia and a consequence hyperplasia of ECL cells and development of carcinoids in gastric mucosa in rats (Poynter et al., 1985). In view of the present clinical scenario, it is desirable that new drugs be developed which are affordable, less toxic and more effective. In developing countries, people are almost completely dependent on traditional medical practices for their primary health care and higher plants remain the main source of drug
therapy in traditional medicine. Rohitukine, isolated from Dysoxylum binecteriferum may represent a promising agent as it possess several medicinal properties and offers advantage over existing anti-secretory drugs, in that it act as a anti-gastrinic agent with a cytoprotective potential, and thus would call for more detailed investigation in the different stages of drug development.
In conclusion, our results demonstrated that rohitukine inhibits the formation of gastric lesions in rats by inhibiting acid secretion and through cytoprotective effects. It was likely that rohitukine mediate its anti-secretory effects by lowering the release of acid-stimulatory hormone gastrin and inhibition of its stimulatory responses on parietal cell’s acid secretion. Thus, rohitukine may emanate as a potent anti-ulcer drug.
Acknowledgements
Authors gratefully acknowledge ICMR and CSIR, New Delhi, India for providing financial support. Mrs. Shibani Sengupta was acknowledged for her technical assistance.
CDRI Communication No. 8158.
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