ANTIULCER ACTIVITY OF METHANOLIC LEAVES EXTRACT OF NERIUM INDICUM MILL

The objective of present study is to evaluate the anti ulcer activity of methanol extract of leaves of Nerium indicum. The methanol extract of N. indicum was investigated for its anti ulcer activity against plus pylorus ligation induced gastric ulcer and indomethacin induced ulcer in rats. The antiulcer activity was assessed by determining and comparing gastric volume, free acidity, total acidity, pH and ulcer inhibition in plus pylorus ligation induced gastric ulcer model and mean score value of ulcer inhibition in indomethacin induced ulcer model. A significant antiulcer activity was observed in all the models. Pylorus ligation model showed significant (P<0.001, P<0.05) reduction in gastric volume, free acidity and ulcer index as compared to control. Also extract showed 69.63 % ulcer inhibition in indomethacin induced ulcer. This present study indicates that Nerium indicum leaves extract have potential anti ulcer activity in the two models tested.

(Family: Apocynaceae) it is an ever green plant with a long history of traditional medicinal uses in many countries in the world, especially tropical and subtropical countries [1] . The plant has been used for treatment of several diseases . The plant in folk remedies to include the use as cardiotonic, diaphoretic, diuretic (promotes excretion), emetic and expectorant [2] . Decoction of leaves has been applied externally in the treatment of scabies and to reduce swellings [3] .The root is a powerful resolvent (power to disperse inflammatory). The oil prepared from the root bark is used in the treatment of leprosy and skin diseases of scaly nature [4] .The whole plant is believed to have anti-cancer properties [5][6] . The punded leaves and bark are used as an insecticide [7] , rat poison and parasiticide [8] . The present study was undertaken to study the effect of methanolic leaves extract of Nerium indicum on pylorus ligation and indomethacin induced ulcer in rats.

Plant material
The fresh leaves of Nerium indicum Mill was collected from Sagar,Madhya

Preparation of extract
Leaves were shade dried and powdered mechanically. The powdered plant material (100 g) was repeatedly extracted in a 500 mL round bottomed flask with 300 mL solvents of increasing polarity starting with petroleum ether and methanol. The reflux time for each solvent was 40 cycles. The extracts were cooled at room temperature, filtered, and evaporated to dryness under reduced pressure in a rotary evaporator.

Experimental animals
In bread Wistar albino rats (180 to 200 g) and Wistar mice (25-30 g) of either sex obtained from Institute of Animal Health and Vety. and Biologicals, Rasalpura, Mhow, 453 446 (Madhya Pradesh, India) (Reg. No. 5007/SAS/2006-07) were used for study. The animals were maintained in polypropylene cages of standard at a temperature of 28 ± 1 o C and standard 12 hour: 12 hour day / night rhythm. The animals were fed with standard rodent pellet diet and water ad libitum. Prior to the experiment, the animals were acclimization to the laboratory condition.

Acute toxicity study
The acute toxicity for methanolic extracts of Nerium indicum was determined in albino mice, maintained under standard conditions. The animals were fasted overnight prior to the experiment. Fixed dose (OCED Guideline No. 423) method of CPCSEA (Reg. No. -1252/AC/09/CPCSEA) was adopted for toxicity studies.
Indomethacin induced ulcer [9][10][11] Wistar rats weighing 150-200 g were used for experiment. They are randomized into four groups of six animals each. Food was withdrawn 18 h and water 1h before the experiment. Group I (control) received only indomethacin (20 mg/kg.) group II (reference or standard) received ranitidine (50 mg/kg.) and groups III & IV were pretreated with Nerium indicum leaves extract (250 and 500 mg/kg.). 30 minute later, groups III-IV were administered with indomethacin. Four hours after indomethacin administration, animals were killed by decapitation method. The stomachs were removed and open along the grater curvature. Macroscopic examination was carried out with a hand lens and the presence of lesion was scored. Scoring of ulcer will be made as follows: [12] Normal stomach…………….. Mean ulcer score for each animal will be expressed as ulcer index. The percentage of ulcer protection was determined as follows: [13] Control mean ulcer index ─ Test mean ulcer index [14][15][16][17] The animals were fasted for over night before pylorus ligation with water ad libitum. Under light ether anesthesia, the abdomen was opened by midline incision process. The pyloric portion of the stomach was slightly lifted out and ligated, avoiding damage to its blood supply. Nerium indicum leaves extract X 100 www.ijbr.ssjournals.com was administered before pylorus ligation. The stomach was placed back carefully and the abdominal wall was closed with sutures. Animals were sacrificed 6 h after pylorus ligation. The stomachs were isolated and the content of the stomachs were collected and centrifuged. The volume of the gastric juice was measured and this was used for estimation of free acidity and total acidity.1ml of centrifuged and filtered gastric secretion was titrated against 0.1N Sodium hydroxide using Topfers reagent as indicator for determination of free acidity and 1% phenolphthalein as indicator for combined acidity. The sum of the two titrations was total acidity. The stomach was open along with the greater curvature and examine for ulcers. The ulcer index was determined as mentioned above.

Pylorus ligation induced ulcer
Volume of NaOH X Normality of NaOH 0.1N

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 Dunnett's test where P<0.001, P<0.01 and P<0.05 was considered statistically significant.

RESULTS & DISCUSSION
In indomethacin-induced model, pretreatments of rats with Nerium indicum leaves extract rendered a dose dependent protection from indomethacin induced ulceration. When compared to the ulcer control animals. The results were tabulated in Table1