THE INTERACTION BETWEEN NIGELLA SATIVA FIXED OIL AND RANITIDINE ON STRESS-ETHANOL INDUCED GASTRIC MUCOSAL DAMAGE IN RABBIT : A POTENTIAL HERB-DRUG INTERACTION

Background: The use of herbal preparations has increased dramatically, making drug interactions with these preparations a major health concern, especially as herbal medications are usually not subjected to the same regulations as prescription drugs Aim: as a potential drug-herb interaction is possible, this study was designed to investigate the interaction between Nigella sativa (NS) and ranitidine (R) on absolute ethanol induced gastric mucosal damage in rabbit Materials and Methods: Five groups of rabbits (6 each) were used. Acute gastric ulcerations were induced by ethanol through a stomach tube. The oil of NS was given orally, ranitidine by (IM), combination of NS+R or normal saline were given 1 hour before ethanol. Ulcer index, serum and stomach tissue MDA, gastric volume and pH, and histopathology were evaluated. Results: Monotherapy of NSoil or R reduced the mean ulcer index from 91.7±19.4mm in the control group to 43.3±8.7 and 22.5±9.4mm for NS and R treatment respectively. There were significant reductions in serum and stomach tissue MDA and in gastric secretion. When NS and R were given in combination the anti-ulcer effect of both disappeared. This was associated with increased MDA levels in stomach tissue, but not serum. The pH of stomach content was also changed toward ethanol treated values. Conclusion: These findings document the gastro-protective potential of NS against ethanol-induced gastric ulcer. There was a significant NS-R interaction manifested as failure of the combination to inhibit ulcers formation. Until further wider studies are available to confirm such interaction, the simultaneous use of Nigella sativa and ranitidine should be discouraged.


INTRODUCTION
he likelihood of herb-drug interactions could be higher than drug-drug interactions, because drugs usually contain single chemical entities, while almost all herbal medications (even a single herb) contain mixtures of pharmacologically active constituents [1] and usually the herbal supplements are not subject to the same regulations as prescription drugs.Nigella sativa, a frequently used herbal preparation and as food supplement has a number of pharmacologic effects such as immunomodulative, [2] antibacterial, [3] hepatoprotective, [4] and gastroprotective effects. [5]Nigella sativa is increasingly used in the folk medicine for alleviating various gastric symptoms either alone or in combination with principal anti-ulcer drugs, thus a possible herb-drug interaction may be seen when the drugs and herbs are taken together.
This study, therefore, was designed to investigate a possible interaction between the oil of Nigella sativa and ranitidine on gastric mucosal lesion induced by ethanol and stress in rabbits.

MATERIALS AND METHODS
The study was approved by the institutional Ethical Committee and carried out between August 2006 and April 2007.

Plant materials and procedure of extraction
The seeds of Nigella sativa were purchased from a local market in Basrsh, south of Iraq.The seeds were identified and authenticated by an expert local pharmacist.Voucher specimens were kept in the Department of Pharmacology.The seeds of Nigella sativa were powdered mechanically using an ordinary blender for 6 minutes.Eighty grams of the powder were T dissolved in petroleum ether, and then the fixed oil of Nigella sativa was extracted by soxhlet at 60-90 o C for 18 hours.The extract was then concentrated using rotary evaporator for 30 minutes.

Treatment of rabbits
The experiments were carried out on 30 locally bred sexually mature male rabbits.They were maintained on free excess to food and drinking water.Prior to the day of the experiment each rabbit was kept in a restrain cage for 48 hours.During restrain the rabbits had free access to drinking water only.On the day of the experiment the rabbits were allocated randomly to receive either Nigella sativa oil orally in a dose of 10ml/kg or ranitidine 10mg/kg I.M.; (ranitidine, Glaxo-Smith-Kleine, UK) physiological saline administered orally or the combination as shown in (Table-1).A child stomach tube was used for administration of oral treatments.It was advanced into the stomach through a wooden tongue depressor with a hole in the center to prevent the animal chewing the tube.The rabbits were divided into five groups, six each.Treatments were given as presented in (Table -1).

Table 1. Study design
Four hours after starting the experiment and before sacrificing, the rabbits were exposed to a short chloroform anesthesia.Two milliliters of blood were taken directly from the heart by an ordinary syringe for the measurement of serum MDA.The rabbits were then sacrificed by cervical dislocation.The stomach was exposed, the lower esophagus and the first part of the duodenum were ligated, and the stomach contents were collected and stored in a deep freezer (-10 o C).The stomach was then opened along the greater curvature, gently washed in a tap water and then fixed on a piece of cork for macroscopical examination.Ulcers were scored using ordinary ruler with the help of magnifying lens; ulcer index which is the sum of length of all ulcers was calculated for each animal.A piece of stomach tissue was taken from the largest ulcerated area for the measurement of tissue MDA.The stomach then fixed with 10% formalin for histopathological examination.thiobarbituric acid assay method. [6]Statistical analysis was carried out using SPSS (version 11), analysis of variance (ANOVA) was used to compare means of groups followed by unpaired t-test to compare different groups means.Pvalue less than 0.05is considered significant.

Effect on ulcer index
Ethanol administration induced gastric ulceration in all treated rabbits, with a mean ulcer index of 91.7±19.4mm.The administration of the oil of Nigella sativa in a dose of 10ml/kg orally one hour before ethanol administration reduced the mean ulcer index to 43.3±8.7mmas compared to the ethanol treated value, p<0.01.Ranitidine 10mg/kg administered intramuscularly one hour before ethanol administration had reduced ulcer index to 22.5±9.4mm which was significantly lower than that of the ethanol treated group (p<0.01).The mean ulcer index of the combination of Nigella sativa oil (10ml/kg orally) and ranitidine (10mg/kg I.M) given one hour before ethanol administration was 95±13.7,which was not statistically different from the ulcer index of the ethanol treated group (91.7±19.4mm) (Table -2).

Effect on gastric pH
The administration of Nigella sativa oil had resulted in an elevation of gastric pH from 1.36 ± 0.4 in the ethanol treated group to 3.5±2.1 in the Nigella sativa treated group (p<0.01).
In the ranitidine group there was complete inhibition of gastric acid secretion, and therefore, it was not possible to measure the pH.
The combination of Nigella sativa extract with ranitidine had resulted in a pH value of 1.97±0.86which was slightly higher than 1.36±0.4 in the ethanol treated group but the difference was statistically not significant.

Histopathological examination
Histopathological examination of gastric sections revealed that absolute ethanol elicited gastric ulceration, necrotic mucosal lesions with wide areas of polymorph nuclear cell infiltration in all rabbits treated with ethanol.One hour before ethanol, the administration of Nigella sativa extractor ranitidine demonstrated a clear reduction of mucosal damage and reversal of histological changes, while the combination of Nigella sativa and ranitidine had failed to reduce ethanol induced gastric damages.These changes are presented in (Figure -1).

Figure 1 .
Figure 1.Histopathological examina on of gastric mucosa of the rabbits; figure 1A.Normal gastric mucosa in a rabbit treated with normal saline (control) (X 100); figure 1B.Gastric mucosa from a rabbit treated with absolute ethanol administered orally.Large gastric ulceration and necrotic mucosal lesions with wide areas of polymorphonuclear cells infiltra on are seen; figure 1C.Small superficial gastric ulcera on in a rabbit treated with I.M. rani dine one hour before ethanol; figure 1D.Normal gastric mucosa in a rabbit treated with the oil of Nigella sa va one hour before absolute ethanol treatment; figure 1E.Large gastric mucosal ulcera on with inflammatory cell infiltration in a rabbit treated with combination (Nigella sativa orally and ranitidine I.M.) one hour before absolute ethanol treatment.
Gastric acidity was measured by a digital pH meter.Serum and stomach tissue MDA level were measured spectrophotometrically by