EVALUATION OF GASTROPROTECTIVE EFFECTS OF METHANOL EXTRACT OF TEPHROSIA VILLOSA AGAINST EXPERIMENTALLY INDUCED ULCERS IN WISTAR RATS

Objective : The present research work was designed to investigate the gastroprotective potentials of methanol extract of Tephrosia villosa . Methods : The aerial parts of Tephrosia villosa were dried under shade, powdered and deffated with petroleum ether and then marc leftover was subjected to methanol extraction using soxhlet apparatus using soxh-let apparatus. Antiulcer activity of methanol extract was determined against stress-induced and aspirin-induced ulcers in experimental animal models. The total number of ulcers formed, ulcer index, percentage inhibition, ulcerated area, protected area, pH and Total acidity were parameters in the study. Results: Methanol extract of Tephrosia villosa at medium (200 mg/kg) and high (200 mg/kg) could significantly (P<0.01) reduced the total number of ulcers formed, ulcer index, ulcerated area and total acidity in therapeutic groups compared to vehicle control and thereby significantly (P<0.01) increased percentage inhibition of ulcers and protected area which was evident by the significant rise in pH of gastric content. The effect of extracts was dose-dependent and results were comparable to that of standard drug omeprazole. Conclusion: The results obtained from the present work suggest that the methanol extract of Tephrosia villosa possess significant anti-ulcer potentials against experimentally induced ulcers in albino rats. The methanol extract of Tephrosia villosa possess significant antiulcer property against stress and aspirin-induced ulcers. The further investigation should be performed to isolate and evaluate specific constituents responsible for the antiulcer activity. The present study was undertaken for the investigation of antiulcer activity of methanol extracts of Tephrosia villosa against stress and aspirin-induced ulcers in animal model. From the results obtained from the study, it can be concluded that methanol extract of the plant exhibited a strong gastroprotective activity against experimentally induced ulcers. However further investigation required establishing the clear mechanism of action of the extract and also to isolate individual phytoconstituents present in Tephrosia villosa that may be responsible for these beneficial therapeutic effects.


INTRODUCTION
There is increased incidence of peptic ulcer worldwide due to stressful lifestyle and eating habits which includes both gastric and duodenal ulcers. It is one of the most prevalent gastrointestinal tract diseases that affect a wide range of people globally [1]. Due to its high morbidity and mortality rates, peptic ulcer disease has been one of the leading causes of gastrointestinal surgery for over a century. The pathophysiology of peptic ulcer disease was attributed to the imbalance between the offensive factors (e. g. acid, pepsin, Helicobacter infection) and the defensive ones (e. g. bicarbonate, mucin, prostaglandins, nitric oxide and growth factors) [2]. The use of non-steroidal anti-inflammatory drugs (NSAIDs), irregular diet, emotional stress, excessive alcohol use and smoking are all the principal etiological factors associated with the peptic ulcer [3]. Nowadays, the drug treatment of ulcers is commonly focused on the suppression of acid secretion and the enhancement of gastric protection [4]. However, more and more clinical evaluations on the drug treatment showed that tolerance was developed and also the incidence of relapses as well as side effects were increased, which made the efficacy of the treatment be arguable. Many of the existed medicines have limitations, especially when they were used against ulcers with complex etiologies [4].
The main approach to treat peptic ulcer disease is to relieve pain, heal the ulcer and prevent ulcer recurrence. Currently, efforts are on the research of a suitable treatment from natural product sources. A large number of species and herbs have been evaluated by various researchers for their anti-ulcer effects to achieve a favourable outcome [5]. A wide range of drug is currently available for the treatment of gastric ulcer which includes proton pump inhibitor, H2 blocker, antacid, and anti-cholinergic. The most common adverse effect of these drugs are hypergastrinemia, hypersensitivity, gynecomastia, impotence, arrhythmia and blood dyscaris such as thrombocytopenia and enteric infection (Clostridium deficile) [6]. Hence till now there is no truly satisfactory medicine for the management of peptic ulcer and plant drugs are proven as effective and safe drugs for the management ulcers [6].
About 600 commercial preparations with claimed liver-protecting activity are available all over the world. About 100 Indian medicinal plants belonging to 40 families are used for herbal formulation [7]. The Tephrosia villosa Linn. is native to India and it is medicinally important and used in the traditional system for the treatment of liver ailments [8]. The Tephrosia villosa Linn. is commonly known as Sarapunkha and used in ayurvedic system of medicine as a memory enhancer, neuroprotective [9]and treatment many ailments. The leaves of this plants contains alkaloids, flavonoids, tannins and phenols [10] and are scientifically proved for its anti-diabetic [11] antiulcer [9], anti-anxiety [12], antioxidant [13] and many other pharmacological activities. The Phyto-constituents of plant leaves are capable of reducing liver toxicity due to their antioxidant properties but, the plant has not been scientifically investigated for evaluation of hepato-protective activity [14]. The Tephrosia is a genus of plant, pantropical taxa with about 400 species distributed chiefly in Asia, Africa, Australia and America 7 . About twenty-four species of Tephrosia were recorded in India. The genus is well known for its richness in prenylated flavonoids and is considered to possess insect repellant, larvicidal, piscicidal, antimicrobial and anticancer properties [8][9][10]. The Tephrosia villosa belongs to the genus was essentially used for the management of diabetes, ulcers, cancer, hyperlipidemia, hepatotoxicity and renal problems in the folklore medicine but doesn't have the scientific evidence for the same [12][13][14]. In view of this, the present study was undertaken to investigate the anti-ulcer properties of aqueous extracts of Tephrosia villosa (TVME) leaves against experimentally induced ulcers in wistar rats.

Collection and authentication of plant material
The areal parts of Tephrosia villosa have been collected from Sri Venkateshwara university, Tirupati, India and dried under shade. The leaves were in the Tirupathi forest and authenticated by Dr. Madhava chetty Asst. Prof. Dept. of Botany and specimen herbarium (Voucher No.-907) was preserved at institute herbarium library. The leaves were separated from other parts, washed, cleaned and dried for further use.

Preparation of methanol extract of Tephrosia villosa
The shade dried leaves were pulverized into powder and sieved through No. 22 mesh. About 350 g (appx.) of coarse powder was subjected to successive solvent extraction using petroleum ether, benzene, chloroform and methanol in soxhlet apparatus [15].

Preliminary phytochemical investigation of methanol extract of Tephrosia villosa
The preliminary phytochemical investigation for the methanol extract of Tephrosia villosa had been conducted as per the procedure prescribed by Khandelwal [16].

Drugs and chemicals
All the chemical and reagents used in the present study were of analytical grade and procured from following sources. The ethanol and asprin procured from Sigma-aldrich chemical Pvt. Ltd., Bangalore and Tween80 was obtained from Nice chemicals Bangalore. All the other solvents and chemicals used for extraction and physiochemical investigation were as of analytical grade purchased from SD fine chemicals Pvt. Ltd. Bangalore.

Animals
The healthy albino wistar male rats were procured from Sri Venkateshwara Enterprises, Bangalore housed under standard conditions of temperature (22±10C), relative humidity (55±10%), 12 hr light/dark cycles and fed with a standard pellet diet (Amrut, Pranav Agro Industries Ltd., Sangli, India) and water ad libitum. After randomization into various groups and before initiation of the experiment, the rats were acclimatized for a period of 7 d under above said environmental conditions. The experimental protocol has been approved by the Institutional Animals Ethics Committee, IJAHSM, Bangalore

Acute oral toxicity studies
The OECD guidelines 423 (up and down procedure) were used to determine acute oral toxicity for methanol extract of Tephrosia villosa. A starting dose used was 2000 mg/kg body weight p. o. of extract (TVME) was administered to 3 male rats, observed for 14 d. The experiments were repeated again with the same dose level, 2000 mg/kg body weight p. o. of extracts for 3 d more, and observed for 14 d [17].

Evaluation of anti-ulcer activity
The methanol extract of Tephrosia villosa was evaluated against stress and aspirin induced ulcers and study design in both studies consisting of six groups of six animals in each group as follows.

Evaluation of anti-ulcer activity against stress ulcers
Stress ulcers were induced by forcing the Wistar albino rats of either sex to swim in the glass cylinder containing water to the height of 35 cm maintained at 25 °C for 3 h. After the drug treatment animals were allowed to swim in cold water for 4 h. After this, the animals were killed with a high dose of anesthetic ether. Stomach of each rat were opened along the greater curvature and examined macroscopically for gastric erosions under a dissecting microscope (10×). Gastric juice collected into centrifuge tubes and centrifuged at 1 000 r/min for 10 min and the volume were noted. The number of ulcers were scored and percentage of protection and ulcer index were calculated [18][19][20].

Evaluation of anti-ulcer activity against stress ulcers
All the experimental animals were kept for overnight fasting and gastric ulcers were by administering absolute cold aspirin (150 mg/kg p. o). All the extracts and standard drug omeprazole were administered orally prior to the administration of aspirin. One hour later, the animals were sacrificed by cervical dislocation and the stomachs were removed. Gastric acid was collected and its pH was determined. Stomachs were opened along the greater curvature and gently rinsed with water for subsequent scanning. The number of ulcers was scored and the percentage of protection and ulcer index were calculated [18,[20][21][22].

Preliminary phytochemical study
The percentage yield of the TVME was found to be 8.15 % w/w. The preliminary phytochemical investigation of the methanol extract of Tephrosia villosa reveals the presence of alkaloids, glycosides, poly phenols, flavonoids, tannins, steroids, and carbohydrates in the plant.

Acute toxicity studies
The methanol extract of Tephrosia villosa was safe up to dose of 2000 mg kg -1 b.w. and caused neither mortality nor any signs of clinical abnormality in the tested animals during the observation period of 14 d after administration of the highest dose. There was no considerable change in body weight before and after treatment of the experiment and no signs of toxicity were observed. When the experiments were repeated again with the same dose level, 2000 mg/kg body weight p. o. of extracts for 3 d more, no changes were observed for 14 d. As per the results obtained in acute oral toxicity study doses were selected as 100, 200 and 400 mg/kg on the ratio 1/20 th , 1/10 th and 1/5 th respectively.

Anti-ulcer activity against stress induced ulcers
In the present study of stress-induced ulcer model, control animals have shown significant (P<0.001) no of ulcers and ulcer index compare to normal animals which have shown no ulcers and ulcer index. Administration of Standard drug omeprazole and TVME at medium and high dose have significantly (P<0.001) reduced number of ulcers formed and ulcer index when compare to vehicle control. Due to the reduction in number of ulcers and ulcer index, percentage of protection was significantly increased in therapeutic groups treated with standard drug and TVME. Effect of TVME at 100 mg/kg was not significant (table 1) The significant increase in formation of ulcerated area and total acidity were observed in vehicle control animals due to stress. But there was significant (P<0.001) decrease in ulcerated area and total acidity found in omeprazole and TVME (200 mg/kg and 400 mg/kg) treated animals compare to vehicle control group. Hence the percentage of protected area was significantly increased in animals treated with omeprazole and TVME (table 1)

Anti-ulcer activity against aspirin-induced ulcers
Administration of aspirin has shown significant (P<0.001) no of ulcers and ulcer index in-vehicle control animals in the present study compare to normal animals, which have shown no ulcers and ulcer index. Administration of Standard drug omeprazole and TVME at medium and high dose have significantly (P<0.001) reduced number of ulcers formed and ulcer index when compared to vehicle control. Due to the reduction in number of ulcers and ulcer index, percentage of protection was significantly increased in therapeutic groups treated with standard drugs and TVME. (table 2) The formation of significant ulcerated area was observed and total acidity was significantly increased in vehicle control animals due to the administration of aspirin. But there was significant (P<0.001) decrease in ulcerated area and total acidity found in omeprazole and TVME (200 mg/kg and 400 mg/kg) treated animals compare to vehicle control group. Hence the percentage of protected area was significantly increased in animals treated with omeprazole and TVME (table 2). Values are mean±SEM, n=6 symbols represent statistical significance, ns p>0.05, * p<0.05, ** p<0.01, ***p<0.001 vs diabetic control. ns p>0.05,+p<0.05,+ + p<0.01,+ ++ p<0.001 normal control vs positive control. Values are mean±SEM, n=6 symbols represent statistical significance. ns p>0.05, * p<0.05, ** p<0.01, ***p<0.001 vs diabetic control. ns p>0.05,+p<0.05,+ + p<0.01,+ ++ p<0.001 normal control vs positive control.

DISCUSSION
The causative factors that may produce ulcer in the human being are several they are stress, chronic use of anti-inflammatory drugs and continuous alcohol ingestion, spicy food among others. In most of cases, the exact causative factor of ulcer is unknown, but it is generally accepted that it is the result of an imbalance between aggressive factors and defensive factors that mucosal maintenance integrity through the several endogenous mechanisms. Peptic ulcer is leading cause of mortality and morbidity in developing countries, characterized by an imbalance between aggressive gastric luminal factor and defensive mucosal barrier. This disease is mainly associated with an increase in gastric acid secretion. Numerous factors like diet, smoking, drugs like aspirin and infection are responsible for the augmentation of ulcers. Still, no therapeutic intervention has been found successful. So, in the present study, efforts has been made to review and to explore various animal models to find out a suitable medication for the treatment of peptic ulcer [24][25][26].
Tephrosia villosa are employed in the treatment and management of ulcers in folklore medicine, but still no complete curative treatment is available. So this review has been designed to explore the effects of Tephrosia villosa extracts for the treatment of peptic ulcers against various ulcer models like pyloric ligation; ethanol and aspirin-induced ulcer models.
In the present study, oral administration of methanol extract Tephrosia villosa at 200 mg/kg and 400 mg/kg significantly inhibited gastric ulcer formation in both selected ulcer models when compared control and effect was comparable to standard omeprazole.
Aspirin is commonly used for inducing ulcer in experimental rats' due to its intense gastric mucosal damage. Studies suggest that the Aspirin damage to the gastrointestinal mucosa starts with microvascular injury, namely disruption of the vascular endothelium resulting in increased vascular permeability, edema formation and epithelial lifting [27][28][29].
Aspirin is an NSAID its abuse will induce gastric ulceration by inhibition of gastric cyclo-oxygenase resulting in the formation of prostacyclin, which is the predominant prostanoid produced in the gastric mucosa. The ulcers produced can be prevented by exogenous PGE2 and PGI2 [30,31].
The significant decrease in gastric ulcer, ulcer index, ulcerated area and total acidity was observed animals in pretreated methanol extract compared to the vehicle control group. In the present study, we also found that there is a significant rise in percentage inhibition of ulcer formation, protected area and pH of gastric content. The antiulcer potentials of plant extracts were almost similar to the standard drug omeprazole used in the present study.
The stress produces stimulate vagus that increases the release of acetylcholine which ultimately produces nitric oxide responsible for the development of ulcers in the stomach due to free radical nature.
The other ulcerogenic aspirin used in the present study directly irritate GIT mucosa and acts as free radical results in the formation of peptic ulcers. Hence the drugs those possess antioxidants can be good approaches for the treatment of ulcers due to the presence flavonoids and phenolic compounds. In this regard, the study can be performed to evaluate the antioxidant properties of the plant to determine the possible mechanism [32,33].
The methanol extract of Tephrosia villosa possess significant antiulcer property against stress and aspirin-induced ulcers. The further investigation should be performed to isolate and evaluate specific constituents responsible for the antiulcer activity.

CONCLUSION
The present study was undertaken for the investigation of antiulcer activity of methanol extracts of Tephrosia villosa against stress and aspirin-induced ulcers in animal model. From the results obtained from the study, it can be concluded that methanol extract of the plant exhibited a strong gastroprotective activity against experimentally induced ulcers. However further investigation required establishing the clear mechanism of action of the extract and also to isolate individual phytoconstituents present in Tephrosia villosa that may be responsible for these beneficial therapeutic effects.

ACKNOWLEDGEMENT
The authors of manuscript are thankful to The Principal and management of East-West College of Pharmacy, Bangalore for providing facilities to conduct this research work.

AUTHORS CONTRIBUTIONS
The present research work was designed by Dr. Ramesh C. The experiment was performed by Mrs. Pinkey Rawal and Ms. Shabana S under the supervision of Dr. Soma Pramanik.