Evaluation of the Anti-Ulcer Activity of Ethanolic Fruit Extracts of Emblica Officinalis Gaertn against Ulcer Induced Albino Rats

The oldest type of medicines is made from medicinal plants, which have been used for thousands of years in traditional medicine in many nations worldwide.  Many bioactive Phyto chemicals were identified thanks to high-throughput screening and even the recently developed reverse pharmacognosy method. Gastro esophageal illness and ulcers are the more serious conditions that call for medical intervention¹. About 4 million American’s suffer from duodenal and gastric peptic ulcers, of which 350 thousand are newly diagnosed each year, 180 thousand are hospitalized and given medication, and approximately 5,000 of these patients pass away each year as a result of their ulcer condition. The lifetime risk of peptic ulcers is around ten percent for male Americans’ and four percent for female americans². Peptic ulcers are wounds in the lesions that most commonly afflict people in their younger to older years, while they can be diagnosed in young adulthood. After the disease has been active for a few days to several months, they may heal with or without medication. They frequently show up without any clear signs or symptoms. It is also impacted by H. Pylori bacterial infections.

There are four layers, or tunics, that make up the gastrointestinal tract from the esophagus to the anal canal. In the digestive process, each layer carries out distinct tasks.

Figure1: Layers of GI Tract

Types of Peptic Ulcer

1) Gastric ulcer

2) Duodenal ulcer

The majority of gastric ulcers is solitary and has a diameter of less than 20 millimeters.

Almost always, gastric ulcers develop when there is H. pylori gastritis or chemical gastritis that damages the epithelium. The majority of gastric ulcer patients secrete less acid than people without ulcers, and even less than those with duodenal ulcers. Whereas duodenal ulcers are ordinarily located on the walls of the duodenum, on a short distance of the pylorus region.^{3, 4} Three regulating molecules—acetylcholine, histamine, and gastrin—stimulate the release of acid, whereas somatostatin inhibits it. ECL cells are responsible for the release of paracrine histamine. The hormone gastrin is released by G cells, which are endocrine cells located in the stomach epithelium. Furthermore, gastrin promotes parietal cell growth directly. Somatostatin is produced when the pH of the stomach drops too low. Somatostatin reduces acid secretion by directly hitting parietal cells and blocking the release of the positive regulators histamine and gastrin.

Figure 2: Acid secretion

H. pylori, often known as Helicobacter pylori, are a type of bacteria that can cause stomach infections and inflammation. The lining of the stomach, lower esophagus, or small intestine (the duodenum) can develop sores called peptic ulcers, which are mostly caused by erosion from stomach acids and inflammation from the H. pylori bacterium. One relatively common medical condition is peptic ulcers.
From the navel to the chest, mild to severe burning abdominal pain is the most common symptom of a peptic ulcer.

Reports have suggested that anti-ulcer activity of aqueous ethanol extract of Rheum spiciforme and its fractions in animal model was studied and suggested that plant-derived drugs hold the potential in the treatment of peptic ulcer, and Rheum spiciforme (family: Polygonaceae) has been known in the folk medicine for possessing medicinal effect on ulceration and concluded that the aqueous ethanol extract of R. spiciforme and its butanol fraction exhibited gastro-protective effect.⁵

Antiulcer activity of methanolic seed extract o Citrullus lanatus seeds (CLS) in albino rats was studied. CLS extract has showed a significant (p<0.05) anti-ulcer effect at both 200mg/kg and 400mg/kg dose level in a dose dependent manner as well as significant (p<0.05) reduction in the ulcer index when compared to control group and finally conclude that the CLS extracts possess antiulcer potential which may contribute to its ethno medicinal use.⁶

The aim of the study is to prepare the extract then isolate the fractions from natural source and screen for ulcer protective activity. We have selected this topic as most of the synthetic non-steroidal anti-inflammatory drugs produces ulcer as main side effect. So we aimed at find out a new compound that is having activity against ulcer protective.

MATERIALS AND METHODS:

The designing of methodology involves a series of steps from field trip to the observation including selection and collection of the medicinal plant, selection of dose value, standardization of protocol, usage of instruments, preparation of reagents, selection of specific solvents for extraction, formation of protocols and final execution of the standardized protocol.

Drugs, Chemicals and Reagents:

Ranitidine and Omeprazole were obtained as gift samples from Cipla Labs, Hyderabad. Absolute Alcohol was obtained from Merck.

Preparation of Ethanolic Extract:

The Wild Root of Emblica Officinalis Gaertn was collected from the Botanical Garden and was identified and authenticated from Botanical Department. The plant material was cleaned, reduced to small fragments, air dried under shade at room temperature and coarsely powdered in a mixer. The powdered material was stored or taken up for extraction process. Shade dried plant material was coarsely powdered and subjected to extraction with petroleum ether by maceration. The extraction was continued till the defatting of the material had taken place. Defatted powdered of Emblica Officinalis Gaertn has been extracted with ethanol solvent using maceration process for 48 hrs, filtered and dried using vacuum evaporator at 40ºC (yield of extract was 9.40% with respect to dry material). Just prior to use, the substance was dissolved in physiological saline solution.

Experimental animals⁷

Wistar rats (150-200 g) and were procured from Animals were housed in appropriate cages in uniform hygienic conditions and fed with standard pellet diet (Amrul Laboratory Animal Diet) and water ad libitum. All the animals were maintained under standard conditions, that is room temperature 26 ± 1°C, relative humidity 45 - 55% and 12:12 h light – dark cycle. The animals were housed in large spacious hygienic cages during the course of the experimental period.  Animal studies had approval of IAEC.

Selection of dose for animal study

The dose considered for the experiment on rats was obtained from conversion of human dose of Emblica Officinalis Gaertn (3-5 g/kg). The conversion factor of human dose (per 200 g body weight) is 0.018 for rats. Hence the calculated dose for the rats (considering human dose 3 and 5 g/kg) is 200 mg/kg. Acute toxicity was done at dose of 2000mg/kg body weight as per OECD guidelines No 423.⁸

Acute oral toxicity:

The acute oral toxicity of ethanolic extracts of Emblica Officinalis Gaertn was determined by using rats which were maintained under standard conditions. The animals were fasted 12 hour prior to the experiment, up and down procedure OECD guideline no. 423 were adopted for toxicity studies. Animals were administered with single dose of individual extract up to 2000mg/kg and observed for its mortality during 14days and 21days study period (long term) toxicity and observed up to 14days for their mortality, behavioral and neurological profiles.⁹

Screening for Anti-Ulcer Activity:

The Ethanolic extracts of Emblica Officinalis Gaertn were tested for antiulcer activity using various methods like pyloric ligation induced gastric ulcer and Ethanol-induced gastric ulcer.

Pyloric Ligation in Rats¹⁰

The animals were divided into 5 groups, each consisting of six rats. Control group received distilled water only. Second group of rats are pyloric ligated. Third and fourth groups received EEEOG in a dose of 150 and 300 mg/kg. The fifth group of animals received Ranitidine in the dose of 20mg/kg as a reference drug for ulcer protective studies. After 45 min of the treatment, pyloric ligation was done by ligating the pyloric end of stomach of rats of respective groups under ether anesthesia at a dose of 35mg/kg of body weight. Ligation was done without causing any damage to the blood supply of the stomach. Animals were allowed to recover and stabilize in individual cages and were deprived of water during post-operative period. Rats were sacrificed after 4hr of surgery and ulcer scoring was done. Gastric juice was collected and gastric secretion studies were performed according to the standard procedure.

Ethanol induced Ulcer Model

The ulcer was induced by administering absolute ethanol (1ml/200g). All the animals were fasted for 36 hours and then ethanol was administered to induce ulcer. The animals were divided into five groups, each consisting of six rats. The Group I - Normal group received Distilled water, second group received Ethanol. Third and fourth groups received EEEOG in a dose of 150 and 300 mg/kg. The fifth group of animals received Omeprazole in the dose of 20 mg/kg as a reference drug. They were kept in specially constructed cages to prevent coprophagia during and after the experiment. The animals were anaesthetized 1 hr later with anaesthetic ether and stomach was incised along the greater curvature and ulceration was scored. A score for the ulcer was studied to pyloric ligation induced ulcer model.

Ulcers (>2 < 4 mm) perforation         -3 Ulcers (< 4mm)      -4

Mean ulcer score for each animal was expressed as ulcer index. The percentage of ulcer protection was determined by

Control mean ulcer index – Test mean ulcer index

% of ulcer protection =-------------------------------------------------------------------------100

Control mean ulcer index

Determination of free acidity

Volume of sodium hydroxide x Normality x 100mEq/L/100g

Acidity = ------------------------------------------------------------------------ 0.1

Statistical Analysis:

The values are represented as mean ± S.E.M, and statistical significance between treated and control groups was analyzed using of one-way ANOVA, followed by Dennett’s test where P<0.05 was considered statistically significant.

RESULTS AND DISCUSSION:

Phytochemical Screening Test:

The freshly prepared extract of the leaves of Emblica Officinalis Gaertn was subjected to phytochemical screening tests for the detection of various active constituents. The results are depicted in Table 1.

Table1: Result of chemical group tests of the ethanolic Extract of Emblica Officinalis Gaertn

Alcoholic extract; (+): Present; (-): Absent; (+++); Reaction intensity is high; (++): Reaction intensity is medium; (+): Reaction intensity is normal;

Acute Toxicity Study:

Administration of the Emblica Officinalis Gaertn extracts in rats at doses of 2000 mg/kg by oral gavage did not reveal any adverse effects or signs of toxicity.

Observations twice daily for fourteen days also did not reveal any drug related observable changes or mortality. Accordingly, the acute oral LD50 of the extractives was concluded to exceed 2000 mg/kg body weight, the highest dose tested in the study.

Pyloric Ligation in Rats:

Pyloric ligation induced gastric ulcer in pyloric ligation induced ulcer model, oral administration of EEEOG in two different doses showed significant reduction in ulcer index, gastric volume, free acidity, total acidity compared to the central group. EEEOG exhibited a protection index of 71.5% and 83.3% at the dose of 150 and 300 mg/kg respectively, whereas Ranitidine as reference standard exhibited a protection index of 88.2%.

In control animal, oral administration of absolute ethanol produced characteristic lesions in the glandular portion of rat stomach which appeared as elongated bands of thick, blackish red lesions. EEEOG has shown significant protection index of 66.1% and 73.6% with the dose of 150 and 300 mg/kg respectively whereas Ranitidine as reference standard showed protection index of 82.5%.