A study of the anti-ulcer activity of the ethanolic extract of the leaves of Psidium guajava on experimental animal models

Swarnamoni  Das; Sarmistha  Dutta; Saurav  Deka

A study of the anti-ulcer activity of the ethanolic extract of the leaves of Psidium guajava on experimental animal models

S Das, S Dutta, S Deka

Citation

S Das, S Dutta, S Deka. A study of the anti-ulcer activity of the ethanolic extract of the leaves of Psidium guajava on experimental animal models. The Internet Journal of Pharmacology. 2008 Volume 7 Number 1.

Abstract

The aim of the study is to study the anti-ulcer activity of the ethanolic extract of theleaves of Psidium guajava on experimental animal models. Four groups of albino rats weighing 130-180grams were taken for the study (n=6).Group I : control (3%gum acacia 5ml/kg/day orally for 7days).Group II :Experimental control(Aspirin 400mg/kg orally as single dose on 7th day).Group III :Test (Psidium guajava extract 200mg/kg/day orally for 7days and Aspirin 400mg/kg orally on 7th day) and Group IV:Standard(Ranitidine150mg/kg orally for 7days and Aspirin 400mg/kg orally on 7th day).The stomachs of the sacrificed rats were removed and (1)volume of gastric juice (2)ulcer index (3)pepsin activity(4)free acidity(5)total acidity (6)gastric mucus secretion were studied. The ulcer index, pepsin activity, free and total acidity and volume of gastric juice in group III and IV showed significant decrease in comparison to group II whereas there is increase in gastric mucus secretion (p<.01).

Sources and support- Animals-Assam Medical College Animal House
Drugs and Equipment- Department of Pharmacology,Assam Medical College

Introduction

Peptic ulcer is a major health problem with multifactorial etiology. The

development of gastric ulcer occurs with acid at the breakdown of mucosal defense 1. The

prevalence rate of peptic ulcer is approximately 1 in 54 or 1.84% in USA2 .In the last

few years,efforts have been taken to identify new antiulcer drugs from natural sources. Plants are

the source of certain known anti-ulcer drugs3.

Psidium guajava Linn. is a large small evergreen or sub-deciduous tree with smooth pinkish brown bark exfoliating in thin flakes. Leaves are opposite,2-5.6 inch long, oblong or elliptic oblong and is faintly aromatic, lateral nerves prominent, petiole upto 0.3 inch long. Flowers -1.5 inch across white on 1-3 flowered axillary peduncles, calyx urceolate, lower portion adnate to the ovary,upper portion free and irregularly lobed. Flowers and fruits almost throughout the year but flowers mainly hot season and fruits rainy and cold season4. The leaves of Psidium guajava is known to be useful in stomach upsets, gastric disorders,gastroenteritis5 and in gastric ulcer6. Guava is rich in tannins, phenols, triterpenes, flavonoids, essential oils, saponins, carotenoids, lectins, vitamins, fiber and fatty acids. . The leaves of guava are rich in flavonoids, in particular, quercetin. Guava also has antioxidant properties which is attributed to the polyphenols found in the leaves5.

Literature reviews indicated that the ulcer protective activity of this plant has not been evaluated so far. Thus, the aim of the study was to evaluate the protective role of P. guajava on ulceration induced by aspirin on albino rats.

Materials and methods

Collection,identification and extraction of plant materials-

Fresh tender leaves of P. guajava, approximately one kilogram collected in the month of April- May ,2008 were used for the study. The plant was authenticated by Dr.M.Islam, Professor of Life Science, Dibrugarh University , Assam, India. The plant material was air dried at room temperature. The dried leaves were grounded to fine powder and stored in air tight container. Preparation of extract- The powder obtained was soaked in ethanol for 24 hours

in percolator. After 24 hours it was allowed to percolate slowly and extract was collected in Petri

dishes7. The extract was concentrated in vacuum using rotary flash evaporator.

Animals- The experiments were carried out in albino rats of the species Rattus norvegicus of either sex weighing 130-180 gms. The animals were procured from Chakraborty Enterprise,Kolkata. The study was conducted in accordance with CPCSEA(Committee for the Purpose of control and supervision of Experiment on Animals) guidelines and the study was approved by the Institutional Animal Ethical Committee(Registration no.-634/02/a/CPCSEA). The animals were acclimatized for one week under laboratory conditions. They were fed with standard diet and water ad libitum was provided.

Acute toxicity studies- Acute oral toxicity test for the ethanolic extract of leaves of

Psidium guajava was carried out as per OECD Guidelines 4258 . Mortality in the acute

oral toxicity test was not seen in the limit test up to dose 2000mg/kg . One-tenth of the

upper bound dose of the extract from the limit test was decided to be considered for the

experiments 9.

Materials required for the study

a)Ethanolic extract of leaves of P. guajava extract.

b)3 % gum acacia as vehicle for all preparations

c)Aspirin(obtained from BD Pharmaceutical Works)

d)Ranitidine(obtained from Ranbaxy Laboratories)

Experimental design-

4 groups of albino rats of either sex of species Rattus norvegicus weighing 130-180gms

were taken for the study taking six animals in each group.

Group I - control(3%gum acacia 5ml/kg/day orally for 7days).

Group II - Aspirin(400mg/kg ) orally as a single dose on the 7th day.

Group III -P. guajava extract(PGE) (200mg/kg/day orally for 7days)and Aspirin(400mg/kg orally on the 7th day) and

Group IV- Ranitidine(150mg/kg orally for 7days)and Aspirin(400mg/kg orally on the

7th day).

On the 7th day, the groups II,III,IV were given aspirin (400mg/kg)as single dose. After

24 hrs pyloric ligation was done in all the animals and kept for 4hrs. Thereafter, the rats were

sacrificed and stomachs were removed and the following biochemical estimation were done-

1) Pepsin activity 2)free acidity 3)total acidity 4) ulcer index 5)gastric

mucus secretion 6) Volume of gastric juice.

1)Pepsin activity - By the method of Debnath PK(1974)10 and Lowry OH(1951)11

One ml of diluted gastric juice mixed with 2% haemoglobin solution in 0.06M HCl and

incubated for 20 mins .Then 0.6 M ice cold Trichloroacetic acid was added. Later

solution centrifuged and supernatant mixed with Reagent C (alkaline copper sulphate

solution), Reagent E (diluted Folin reagent) and optical density was measured at 610 nm

against a blank distilled water.

2)Free acidity and Total acidity-By the method of Kulkarni SK (1999)12 .Briefly 2 drops of

Topfers reagent was added and diluted supernatant of gastric juice in a conical flask.

0.01 N NaOH was taken in a burette and allowed to titrate till flask change to yellow

colour. Then 2 drops of phenolphthalein added and titration continued till orange colour is
reached.

3) Ulcer index -By the method of Goyal RK (2002)15. The dissected stomachs of the

sacrificed rats were opened along the greater curvature and the ulcer index calculated

from the glandular portion of the stomach. The ulcer index was calculated as,

Ulcer index = 10/x

where x = Total mucosal surface/Total ulcerated area

Each lesion was measured along the greatest length. In case of petechiae , five of these

were considered to be equivalent to 1mm2 of ulcer area. The total area of the glandular

portion of the stomach and that of ulcerated mucosa were measured for determination of

the ulcer index.

4)Gastric mucus - As described by Crone SJ (1974)13.Briefly excised glandular portion

of stomach was soaked in 0.1% alcian blue solution buffered with 0.05M sodium acetate

and HCl .Uncomplex dye adhered to tissue washed with 0.025M sucrose again soaked in

MgCl2 and resulting blue solution mixed with ether and optical density measured against

605nm.

5)Volume of Gastric Juice: By the method of Deshpande SS (2003)14.Contents of the

resected stomachs of the rats were taken in graduated test tubes and allowed to centrifuge

at 2000 rpm for 10 mins. The supernatant fluid was measured for volume of gastric juice

and expressed as ml/4 hrs. Then the juice was subjected to the biochemical tests.

Statistical analysis-For all the above methods, the results were expressed as mean ±SEM.

Statistical analysis was done using one way ANOVA followed by multiple

comparison test using the Graph pad prism software.

Results

a)Effect on pepsin activity- Pepsin activity showed significant fall in Group III and IV

whereas in Group II there was a rise as shown in Table 1.