INTRODUCTION:

Gastric ulcer, one of the most widespread, is believed to be due to an imbalance between aggressive and protective factors¹. The gastric mucosa is continuously exposed to potentially injurious agents such as acid, pepsin, bile acids, food ingredients, bacterial products (Helicobacter pylori) and drugs². These agents have been implicated in the pathogenesis of gastric ulcer, including enhanced gastric acid and pepsin secretion, inhibition of prostaglandin synthesis and cell proliferation growth, diminished gastric blood flow and gastric motility³.

Drug treatment of peptic ulcers is targeted at either counteracting aggressive factors (acid, pepsin, active oxidants, platelet aggravating factor “PAF”, leukotrienes, endothelins, bile or exogenous factors including NSAIDs) or stimulating the mucosal defences (mucus, bicarbonate, normal blood flow, prostaglandins (PG), nitric oxide)⁴. The goals of treating peptic ulcer disease are to relieve pain, heal the ulcer and prevent ulcer recurrence. Currently there is no cost-effective treatment that meets all these goals. Hence, efforts are on to find a suitable treatment from natural product sources.

Azima tetracantha (Salvadoraceae) is a wellknown medicinal herb, termed ‘Mulsangu’ in Tamil and 'Kundali' in Sanskrit. Root, root bark and leaves of Azima tetracantha (lam) are used with food as a remedy for rheumatism, diuretic and as stimulant⁵. Traditionally Indian medical practitioners use Azima tetracantha (lam) in inflammatory conditions, cough, asthma, small pox and diarrhoea^6,7. The major phyto-constituents reported in Azima tetracantha (lam) are azimine, azecarpin, carpine, isorhamnitine-3-O-rutinoside, friedelin, lupeol, glutinol and β-sitosterol^8,9. Azima tetracantha (lam) is reported to have antifungal¹⁰antitumour¹¹, antidiabetic ¹², antidiarrhoeal¹³ and hepatoprotective activities.

Azima tetracantha (lam) is a low, spinouts, highly branched bush, woody below but with pale green, herbaceous, almost quadrangular young branches. The leaves are in opposite to sub-opposite, decussate pairs. They are shortly petiolate, about 2x4cm long, entire, elliptic, acute, sharply mucronate, rigid, pale green with an acute base. Usually, there are two laterally placed spines in the axil of a leaf. The spines which morphologically represent the first pair of leaves of the auxiliary shoot are about three cm long, more or less, triangular in cross section, very sharp and with an indurate apex. The plant is dioeciously. The flowers are borne in the axils of leaves. Generally, there is cymes of three flowers in the axil of a leaf which is the upper branches, especially of the male plants become greatly reduced or even completely suppressed.

MATERIALS AND METHODS:

Collection of plants:

The aerial part (leaves) of Azima tetracantha (lam) was collected from the Panayur area of Madurai, Tamilnadu as raw material, during the second week of February 2015 and a voucher specimen is stored in C.L. Baid Mehta College of Pharmacy (001/ATL/CLBP) and the plant material was authenticated by a renowned botanist. About 500 g of coarse powdered leaf in 2.5 L water is boiled, cooled and filtered. The filtrate is evaporated to dryness in desiccator and stored in refrigerator (Yield- 26.5% w/w). The aqueous extract of Azima tetracantha (lam) (AEAT) was subjected to preliminary phytochemical analysis¹⁴

Various extraction methods for isolation of constituents:

The whole plant will be subjected to shade drying and extraction with petroleum ether (60-80^oC) chloroform, Ethyl acetate and 80% ethanol in soxhlet apparatus by simultaneous extraction each for 72 hours. Concentrate the solvents in vacuum. The crude solid obtained on evaporation are to be studied for preliminary qualitative phytochemical evaluation.

Phytochemical Screening:

The extract was subjected to phytochemical analysis to test the presence of carbohydrates, glycosides, alkaloids, flavonoids, tannins, sterols, and saponins in leaf extracts.

Acute toxicity study:

OECD guidelines 423 were followed to carry out acute toxicity study at dose level of 2000mg/kg. This study was carried out by administering the test solutions orally to rats, at the dose level of 2000 mg/kg for 14 days, to check whether the test solution has any toxic effects. Signs and symptoms of toxicity were observed for next 48 hrs. No toxicity or death was observed in the experimental rats when they were subjected to toxicity study.

Animals used:

Wistar albino rats of either sex weighing between 150-250 gm were used. Institutional Animal Ethics Committee approved the experimental protocol; animals were maintained under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). Albino rats were used in this thesis was obtained from the Bioneeds Animal House Dhavas Pet, Tumkur. The animals were housed in Poly propylene cages and maintained at 24˚C ± 2˚C under 12h light/ dark cycle and were feed ad libitum with standard pellet diet and had free access to water. The animals were given standard diet supplied by Pranav Agro Industries Ltd. Sangli. The composition of the diet are protein 10%, Arachis oil 4%, Fibers 1%, Calcium1%, Vitamin A 1000 IU/gm and Vitamin D 500 IU/gm.

Pyloric ligation in rats:

Animals are divided into five groups, each consisting of six rats. Control group were received distilled water orally. Second group having pyloric ligated. Third and Fourth Groups received ethyl acetate extract of Azima tetracantha (lam) in a dose of 250 and 500 mg/kg. Omeprazole, in the dose of 20 mg/kg was be administered orally for Group Fifth as a reference drug for ulcer protective studies. After 45 min of ethyl acetate extract of Azima tetracantha (lam) and Omeprazole treatment, pyloric ligation was be done by ligating the pyloric end of stomach of rats of respective groups under ether anaesthesia at a dose of 35 mg/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 postoperative period. After 4 h of surgery, rats were sacrificed and ulcer scoring was done. Gastric juice was collected and gastric secretion studies were performed. ^15,16

Ethanol induced ulcer model:

The ulcer was induced by administering ethanol. All the animals were fasted for 36 hours before administration of ethanol. The animals were divided into five groups, each consisting of six rats. One Group represented the control group, which received distilled water orally. Second group receive ethanol. Third and Fourth Groups received ethyl acetate extract of Azima tetracantha (lam) of 250 and 500 mg/kg and, Omeprazole, in the dose of 20 mg/kg were administered orally for Fifth group as reference standard drug. The gastric ulcers were induced in rats by administrating absolute ethanol (90%) (1ml/200g.) Orally, after 45 min of ethyl acetate extract and Omeprazole treatment. They were kept in specially constructed cages to prevent coprophagia during and after the experiment. The animals were anaesthetized 1h latter with anaesthetic ether and stomach was incised along the greater curvature and ulceration will be scored. A score for the ulcer was study similar to pyloric ligation induced ulcer model^17,18

Scoring of ulcer will be made as follows:

Normal stomach.......(0)

Red coloration.........(0.5)

Spot ulcer............….(1)

Hemorrhagic streak..(1.5)

Ulcers........................(2)

Perforation.................(3)

Mean ulcer score for each animal will be expressed as ulcer index. The percentage of ulcer protection was determined as follows:

Protective %= control mean ulcer index-test mean ulcer index x100/control mean ulcer index

Determination of acidity:

Acidity=

volume of NaOH X Normality of NaOH X100/0.1

Statistical analysis:

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

Histopathological evaluation:

The gastric tissue samples were fixed in neutral buffered formalin for 24 h. Sections of tissue from stomachs were examined histopathologically to study the ulcerogenic and/ or anti-ulcerogenic activity of Azima tetracantha (lam). The tissues were fixed in 10% buffered formalin and were processed using a tissue processor. The processed tissues were embedded in paraffin blocks and about 5-µm thick sections were cut using a rotary microtome. These sections were stained with hematoxylin and eosin using routine procedures. The slides were examined microscopically for Pathomorphological changes such as congestion, haemorrhage, oedema and erosions using an arbitrary scale for the assessment of severity of these changes¹⁹

RESULTS:

Pyloric ligation induced gastric ulcer:

In pyloric ligation induced ulcer model, Oral administration of Ethyl acetate extract of Azima tetracantha (lam) in two different dose showed significant reduction in ulcer index, gastric volume, free acidity, total acidity as compared to the control group It was showing protection index of 76 % and 82 % at the dose of 250 and 500 mg/kg respectively in comparison to control whereas Omeprazole as reference standard drug was reduction of ulcer 84%. (Results are tabulated in Table-1).

Ethanol-induced gastric ulcer

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, black and dark red lesions. Ethyl acetate extract of Azima tetracantha (lam) has shown significant protection index of 54% and 66% with the dose of 250 and 500 mg/kg respectively in comparison to control, Omeprazole as reference standard drug was reduction of ulcer 72%. (Results are tabulated in Table-2)

Effect of ethyl acetate extract of Azima tetracantha (lam) on various parameters in Pyloric ligation induced gastric ulcer

Group	Treatment	Ulcerindex	Protection %	PHof gastric juice	Gastric juice ml	Free acidity	Total acidity
I	Control	14.6±1.3	-	2.3±2.1	9.4±2.1	96.5±1.3	115.3±.23
II	Omeprazole (20 mg/kg)	2.4±0.4	83%	4.8±.13	2.4±.16	32.5±2.1	58.5±1.3
III	Ethylacetate extract (250mg/kg)	3.5±.04	75%	3.5±.18	4.4±.12	49.8±1.2	65.7±.32
IV	Ethylacetate extract (500 mg/kg	2.8±.07	80%	4.3±.19	3.8±.13	35.7±1.3	61.5±1.2

Macroscopical and Histopathological Evaluation:

Macroscopical change of pylorus ligation and ethanol induced models were shown in figure (1a, 1b, 1c and 2a, 2b, 2c). Histopathological changes on pylorus ligation model showed the degeneration, hemorrhage, edematous appearance of the gastric tissue, where as ethyl acetate extract of Azima tetracantha (lam) (500 mg/kg) and Omeprazole (20 mg/kg) treated groups showed regeneration and prevents the formation of hemorrhage and edema.

DISCUSSION:

The etiology of peptic ulcer is unknown in most of the cases, yet it is generally accepted that it results from an imbalance between aggressive factors and the maintenance of mucosal integrity through the endogenous defence mechanisms²⁰. To regain the balance, different therapeutic agents are used to inhibit the gastric acid secretion or to boost the mucosal defence mechanisms by increasing mucosal production, stabilising the surface epithelial cells or interfering with the prostaglandin synthesis. The causes of gastric ulcer pyloric ligation are believed to be due to stress induced increase in gastric hydrochloric acid secretion and/or stasis of acid and the volume of secretion is also an important factor in the formation of ulcer due to exposure of the unprotected lumen of the stomach to the accumulating acid.²¹Pylorus ligation induced ulcer was used to study the effect of fruit extracts on gastric acid secretion and mucus secretion. The ligation of the pyloric end of the stomach causes accumulation of gastric acid in the stomach. This increase in the gastric acid secretion causes ulcers in the stomach. The original Shay rat model involves fasting of rats for 36 hours followed by ligation of pyloric end of the stomach. The ulcer index is determined 5 hours after pylorus ligation. The lesions produced by this method are located in the lumen region of the stomach. Many authors have modified the original model. In the present study, the Shay rat model described by Kulkarni was followed. The ethyl acetate extract of Azima tetracantha (lam) and Omeprazole significantly decreased the total acidity and free acidity; this suggests that it having an antisecretory effect. Its antiulcer activity is further supported by histopathological study shows that protection of mucosal layer from ulceration and inflammation. Ethanol induced gastric ulcer was employed to study the cytoprotective effect of the extracts. Ethanol induced gastric lesion formation may be due to stasis in gastric blood flow which contributes to the development of the haemorrhage and necrotic aspects of tissue injury. Alcohol rapidly penetrates the gastric mucosa apparently causing cell and plasma membrane damage leading to increased intra cellular membrane permeability to sodium and water. The massive intracellular accumulation of calcium represents a major step in the pathogenesis of gastric mucosal injury. This leads to cell death and exfoliation in the surface epithelium^22,23. The extract shows protection against characteristic lesions produced by ethanol administration this antiulcer effect of ethyl acetate extract of Azima tetracantha (lam) may be due to both reductions in gastric acid secretion and gastric cytoprotection. Further studies are needed for their exact mechanism of action on gastric acid secretion and gastric cytoprotection.

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Received on 19.12.2018 Modified on 29.12.2018

Res. J. Pharmacology and Pharmacodynamics.2019; 11(1):27-31.

DOI: 10.5958/2321-5836.2019.00006.5