Pharmacological Investigation of the Plant Prunus Amygdalus (Batsch) for its Anti-ulcer

About Author: Devendra Kumar^1*, Durga Prasad Dash¹, Shushank Dixit², Pragya Seth³
Department of Pharmaceutical technology,
¹Sri Satya Sai collage of Pharmacy (Sehore),
²NH-58, Bagpath road bypass, MIET, Meerut,
³Laxmi Narayan College of Pharmacy, Bhopal

Abstract
This study has been undertaken to investigate the effect of methanolic extract of almond nuts of Prunus amygdalus (batsch.) on pylorus ligation gastric ulcers in Wistar rats. Treatment with Prunus amygdalus extract of (200 mg/Kg bodyweight, 400 mg /Kg body weight) and ranitidine (50 mg/ Kg body weight) for 1 hr to the Ethanol and  rats Pylorus ligation were given and absorb the acid parameters. The present study provides a strong evidence of antiulcer activity of Prunus amygdalus extract against gastric lesions. The antiulcer activity is recognized by a reduction in acid-secretary parameters (i.e. total and free acid), gastric volume and ulcer score suggesting that acid inhibition accelerates ulcer healing, thereby strengthening of mucosal barrier. Administration of Prunus amygdalus (200 mg/kg & 400mg/kg p.o.) increased CAT, and GSH levels as compared to the negative control group, which suggests its efficacy in preventing free radical-induced damage. However, significant increase was observed all doses but in 400mg/kg were better results. In this present study was showed that there was present a significant protection for Hexosamine in all treated groups in compare to negative control group. The LPO results in showed that there was significance level difference present in negative control group and all other group that indicated that lipid peroxides enzyme was higher in vehicle treated group. Ulcer score is the counting of spots and severity of damage in gastric part by any moiety such as ethanol. Single drug treatment (200 mg/kg and 400mg/kg of P.A.E.) was effective up to a significant level (P<0.05) in compare to negative control group. Volume of gastric juice indicate the secretions of gastric fluid and it is higher in negative control group and all drug treated group was effective in relation to negative control group (P <0.05).

Reference ID: PHARMATUTOR-ART-1118

Introduction
Plants which are the major source of drugs in Indian system of medicine have the advantage of little or no side effect. In Ayurvedic system of medicine, Polyherbal formulations were frequently used to enhance the activity or counteract the toxic effect of compounds.1
The cause of ulceration in patients is mainly due to hypersecretion of gastric juice and also due to hypersecretion of pepsin. In traditional system of medicine a number of herbal preparations have been used for the treatment of peptic ulcers. The marketed polyherbal formulation (PHF) has been used for the treatment of gastrointestinal disorders.2
There are three objectives in the treatment or management of peptic ulcer, namely the alleviation of symptoms, the healing of the ulcer and the prevention of its recurrence. A number of Ayurvedic formulations have been advocated in traditional system of medicines to overcome above disorders and diseases.3

EXPERIMENTAL MODELS
Pyloric Ligation Induced Gastric Mucosal Ulcers 4
Procedure:
Wistar rats weighing 150–170 g were fasted for 24hr with free access to water and avoidance of coprophagy.
The rats were sacrificed after four hours of pyloric ligation all rats were sacrificed under cervical dislocation.
The gastric juice was evacuated into a graduated tube by cutting along the greater curvature of the stomach, and centrifuged.
The stomach was cut along the greater curvature and the mucosa was washed under a slow stream of water. Then the parameters mentioned below were measured.

Parameter Assessed:
Physical Parameters
a. Ulcer index
b. Volume of gastric juice
c. pH

Biochemical Parameters
a. Total acidity
b. Total Acid Output
c. Antioxidants – Catalase and Reduced Glutathione
d. Lipid peroxidation – Malondialdehyde (MDA)
e. Determination of Gastric wall Mucus
f. Hexosamine assay
g. GSH determination

Experimental design:
Rats (n = 24) each group contain 6 animals were randomized into following groups:
Group 1 - Negative control group.
Group 2 - Treated with standard drug Ranitidine (50mg/kg, p.o.).
Group 3 - Treated with Prunus amygdalus extract (200 mg/kg (p.o.).
Group 4 - Treated with Prunus amygdalus extract (400 mg/kg (p.o.).

MEASUREMENT OF VARIOUS PARAMETERS
Physical Parameters:
Ulcer Index: ⁵
The following arbitrary scoring system was used to grade the incidence and severty of lesion 0 = Normal, 1 = Red colouration, 2 = Spot ulcers, 3 = Haemorrhagic streaks, 4 = Ulcers > 3 but < 5 and 5 = Ulcers > 5

The Mean Ulcer Index UI will be calculated using following formula:
UI= UN + US + UP X 10 - 1
Where,
UN= Average of number of ulcer per animal.
US= Average of severity score.
UP= Percentage of animals with ulcers

Collection of gastric juice: ⁶
After post operative period, animas were sacrificed by cervical disslocation and the stomach was dissected out as a whole by passing a ligature at the esophageal end.
Gastric content was evacuated into graduated tube by cutting along the greater curvature of the stomach, and was centrifuged at 3000 rpm for 10min.

Volume of gastric juice:
The volume of the centrifuged sample was expressed as ml/ 100 g body weight.

pH of gastric juice:
pH of gastric juice was measured with the help of pH meter.

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Biochemical Parameters:
Preparation of tissue homogenate:
Stomach was dissected out from the sacrificed animal and rinsed with distilled water. Stomach was then homogenized into ice cold 0.15 M Tris-HCl (pH 7.4) to give 10%w/v homogenate.
The homogenate was then centrifuged at 15000 rpm for 15min. at 4°C in cooling centrifuge. Supernatant was used for determination of antioxidant activity.

Lipid peroxidation measurement:
The extent of lipid peroxidation was estimated by measuring the secondary lipid peroxidation product – thiobarbituric acid-reactive substances (TBARS) formed in the lipid peroxidation processes.

Procedure:
The glandular portion of the gastric mucosa was homogenized with cold 0.15 M Tris-HCl (pH 7.4) to give a 10% (w/v) homogenate.
After 10min, 0.2 ml of tissue homogenate, 0.2 ml of 8.1% sodium dodecyl sulphate (SDS), 1.5 ml of 20% acetic acid and 1.5 ml of 8% TBA were added. The volume of the mixture was made up to 4 ml with distilled water and then heated at 95°C on a water bath for 60 min using glass balls as condenser.
After incubation the tubes were cooled to room temperature and final volume was made to 5 ml in each tube. 5.0 ml of butanol:pyridine (15:1) mixture was added and the contents were vortexed thoroughly for 2 min.
After centrifugation at 3000 rpm for 10 min, the upper organic layer was taken and its OD read at 532 nm against an appropriate blank without the sample.⁷

GSH determination
Procedure:
The supernatant (40µl) was mixed with 400µl tris and 3360µl water.. Then 0.2mL (200µl) DTNB solution was added and absorbance was measured at 412 nm.
Standard curve for GSH was prepared using glutathione.8

Catalase (CAT):
Procedure:
100µl of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0). Reaction was started by the addition of 1.0 ml of freshly prepared 30 mM H₂O₂. Decrease in absorbance was read at 240 nm for 3min at interval of 30 sec.
The activity was calculated using extinction coefficient of H₂O₂ 0.041micromole/cm². Results were expressed as micromole of H₂O₂ utilized/min/gm tissue.The rate of Decomposition of H₂O₂ was measured spectrophotometrically from changes in absorbance at 240 nm.⁹

Hexosamine assay:
Procedure:
The hexosamine concentrations in gastric tissues were assayed according to a reported method with a minor modification.
The gastric tissues were hydrolyzed in acidic medium, the hydrolysate neutralized with 3N NaOH (litmus) and diluted to 10 ml with double distilled water.
To an aliquot (1 ml) was added acetyl acetone solution (1 ml, prepared by dissolving 1 ml of acetyl acetone in 50 ml 0.5 N sodium carbonate), the solution mixed well, and heated on a boiling water bath for 15 min avoiding evaporation.
After cooling, ethanol (5 ml, 95%) was added to the mixture followed by Ehrlich’s reagent (1ml, prepared by dissolving 0.8 g para-dimethylaminobenzaldehyde in 30 ml methanol and 30 ml conc. HCl).
The mixture was diluted to 10 ml with 95% ethanol, allowed to stand for 30 min, and its absorbance at 530 nm was read.¹⁰

Gastric wall mucus determination:-
Procedure:
The glandular segments from stomachs which had been opened along their greater curvature were removed and weighed.
Each segment was transferred immediately to 10 ml of 0.1% w/v Alcian blue solution (in 0.16 M sucrose solution, buffered with 0.05 M sodium acetate pH 5).After immersion for 2 h, excess dye was removed by two successive rinses with 10 ml of 0.25 M sucrose, first for 15 and then for 45 min.
Dye complexes with the gastric wall mucus were extracted with 10 ml of 0.5 M magnesium chloride by shaking intermittently for 1 min at 30 min intervals for 2 h. Four milliliters of blue extract were then shaken vigorously with an equal volume of diethyl ether.
The resulting emulsion was centrifuged at 3600 rev./min for 10 min and the absorbance of the aqueous layer was recorded at 580 nm. The quantity of Alcian blue extracted per gram of net glandular tissue was then calculated.

PARAMETERS EVALUATED FROM GASTRIC JUICE
Total Acidity:
Procedure:
Gastric juice (1ml) was pipette into a 100ml conical flask and diluted with 9ml distilled water.
Two or three drops of Toepfer’s reagent was then added and titrated with 0.01 N sodium hydroxide until all traces of red colour disappeared and the colour of the solution was yellowish-orange.
The volume of alkali added was noted. This volume corresponds to free acidity. Two or three drops of phenolphthalein were then added and the titration was continued until a definite red ting appeared; the volume of alkali added was noted.
The volume corresponds to total acidity. Acidity was expressed in terms of mEq/L.¹¹

Statistical Analysis
Statistical analysis was carried out using Primer of biostasticals software .All the results were expressed as Mean ± standard error of the mean (SEM). Data were analyzed using one-way ANOVA followed by Bonferroni t- test. In the entire test, the criterion for statistical significance was p<0.05.

Results

Pylorus Ligation Induced Gastric Mucosal Ulcers

Table-1:- Observation of Pylorus ligation induced ulcer

Each group consists of six animals, Data is presented in mean ±SEM
Here * means no significant difference (p > 0.05) in compare to negative control group
** means significant difference (p < 0.05) in compare to negative control group

GSH determination

Graph-1 :- Bar chart of GSH determination in pylorus ligation induced ulcer

Reduced glutathione are responsible for antioxidant activity. Antioxidant activities are responsible for free radical scavenging activity to reduce the cell damage. Any kind of wound are formed by free radical damage and reduction of free radical at significant level promote the wound healing property this kind of same phenomena are responsible for ulcer protective activity. The GSH result (Table 1) showed that minimal level was present in negative control group. Protective effect of both drug was present up to a significant level because P<0.05 when compared to the all group against negative control group. Prunus amygdalus extract did not show dose depended manner due to presence of P>0.05 when compare to both dose group (200mg/kg, 400mg/kg) of Prunus amygdalus.

GWM determination

Graph-2 :- Bar chart of GWM determination in pylorus ligation induced ulcer

Gastric wall mucosa plays a main role in protection of gastric wall from aggressive factor that are responsible for gastric wall damage. Mucous layer a barrier between gastric wall and acid. When mucosa becomes less by any reason then acid contact time is increased and chances of cellular damage are increased. According data of (Table 1) concluded that among all groups level of gastric mucosa of minimum in negative control group and maximum was in ranitidine group.

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Hexosamine determination

Graph-3 :- Bar chart of  Hexosamine determination in pylorus ligation induced ulcer

Hexosamine are precursor of biochemical synthesis of glycosylated protein and lipids. Both glycosylated and lipid are basic component of cell membrane and responsible for cell rigidity. In this present study was showed that there was present a significant protection for hexosamine in all treated groups in compare to negative control group.

Catalase determination

Graph-4 :- Bar chart of  Catalase determination in pylorus ligation induced ulcer

The intracellular level of H₂O₂ is regulated by a wide range of enzymes, the most important being catalase. Catalase function through an intermediate catalyse-H₂O₂ complex and produces water and dioxide (catalase action) or can decay to the inactive compound.⁸¹ H₂O₂ are responsible for free radical generation that causes a oxidative stress at cellular level and responsible for cell death. Above data showed that all groups had a significant difference in compare to negative control and ranitidine group.

LPO determination

Graph-5 :- Bar chart of LPO determination in pylorus ligation induced ulcer

Phenolic compounds are diverse secondary metabolites (flavonoids, tannins, hydroxycinnamate esters and lignin) abundant in plant tissues. Polyphenoles possess ideal structural chemistry for radial scavenging activity.81 Above data showed that lipid peroxidation level was lowest in ranitidine group and higher was in negative control group.

Determination of pH, Volume and Total Acidity of Gastric Juice

Table-2 :- Observation of pH, Volume and total acidity of Gastric juice

Each group consists of six animals, Data is presented in mean ±SEM
Here * means no significant difference (p > 0.05) in compare to negative control group
** means significant difference (p < 0.05) in compare to negative control group

Determination of pH of Gastric Juice

Graph-6:- Bar Chart of pH Of Gastric Juice

Low PH is responsible for more damage in gastric portion. 200mg/kg of P.A.E. was effective because significant level p< 0.05 and 400 mg/kg also was effective in compare to negative control group.

Determintion of volume of Gastric Juice

Graph-7 :- Bar chart of volume of Gastric juice

Volume of gastric juice indicate the secretions of gastric fluid and it is higher in negative control group and all drug treated group was effective in relation to negative control group (P <0.05).

Determination of Total Acidity

Graph-8 :- Bar Chart of Total Acidity of Gastric Juice

Total acidity has relation with amount of HCl present in the gastric fluid and responsible for gastric environmental because it is inversely proportional to the gastric pH. This present study was show that 200 mg/kg of P.A.E. was not effective drug treatment for reduces total acidity because there was P>0.05 for both these groups in compare to the vehicle group means both these groups resembled as negative control effect. But 400mg/kg of P.A.E. was effective (P<0.05) in compare to negative control group.

Ulcer Score

Table-3:- Observation of  Ulcer Score in Pylorus Ligation induced ulcer

Each group consists of six animals, Data is presented in mean ±SEM
Here * means no significant difference (p > 0.05) in compare to negative control group
** means significant difference (p < 0.05) in compare to negative control group

Determination of Ulcer Score

Graph-9:- Bar chart of ulcer score in pylorus ligation induced ulcer

Ulcer score is the counting of spots and severity of damage in gastric part by any moiety such as Pylorus ligation. Single drug treatment (200 mg/kg and 400mg/kg of P.A.E.) was effective up to a significant level (P<0.05) in compare to negative control group.

Morphology of Stomach in Pylorus Ligation Induced Ulcer

Discussion
Although antiulcer treatment is improved, peptic ulcers with their complication still carry a significant mortality. The goal of medical therapy for bleeding ulcers has been traditionally to sustain intragastric pH greater than six, in order to promote platelet aggregation, clot formation and stability. H2-Receptor antagonists show little benefit in patients with peptic ulcer bleeding, which reflects their inadequate pH control and the rapid onset of tolerance.
Lipid peroxidation is a free radical mediated process, which has been implicated in a variety of disease states such as hepatotoxicity, atherosclerosis, hypertension etc. lipid peroxidation mediated by free radicals is believed to be an important cause of destruction of and damage to cell membrane, which has been demonstrated to play an important role in pathogenesis of gastric mucosal injury induced by ethanol. Lipid peroxidation product, malondialdehyde (MDA) was found to be significantly increased in diseased group as compared to normal group. Our study has revealed a significant decrease in lipid peroxidation by herbal extract at all the doses under investigation.
The level of CAT and GSH was significantly decreased in diseased group as compared to normal group. Administration of Prunus amygdalus (200 mg/kg & 400mg/kg p.o.) increased CAT and GSH levels as compared to the negative control animals, which suggests its efficacy in preventing free radical-induced damage. However, significant increase was observed in all doses but in 400mg/kg shows better result.
Gastric acid and pepsin are important factors for the formation of ulcers in pylorus-ligated rats. In the stomach, digestive effect of accumulated gastric juice in the induction of gastric ulcers is well documented in the pylorus ligation model. Biochemical constituents of the gastric secretion have a considerable role to play in the pathophysiological state of gastric mucosa. In addition to gastric acid secretion, reflex or neurogenic effects have also been suggested as playing an important role in the formation of gastric ulcers in the pylorus ligation model. In a recent study role of free radical in gastric ulcer induce by pylorus ligation is also reported.
This mucus consists of mucin type of glycoprotein. This high molecular weight glycoprotein are mainly responsible for viscous and gel forming capacity of mucus. Increased mucus secretion by gastric mucousal cell prevent gastric ulceration by several mechanism such as lessing of stomach wall friction during peristalsis and gastric contraction, improving the buffering of  gastric juice, and by acting as an effective barrier to back diffusion of H+ ions.

References
1. Jaikumar S, Asokan BR, Sengottuvelu S, Ramaswamy S. Anti-Ulcer Activity Of Polyherbal Formulation-Ro7d On Experimentally Induced Ulcer In Rats. Pharmacologyonline 2009; 3: 419-23.
2. Khandare RA, Gulecha VS, Mahajan MS, Mundada AS, Gangurde HH. Evaluation of Antiulcer Activity Polyherbal Formulation. International journal of pharma.Research & Development 2009; 1: 1-6.
3. Manohara KP, Raveendra RP, Savadi RV. Evaluation Of Traditional Herbal Formulation Experimentally Induced Gastric Ulcers In Rats. Pharmacology online 2009; 1:1173-77.
4. Muthusamy P, Suresh AJ, Balamurugan G, Antiulcer Activity of Azima Tetracantha: A Biochemical Study,  Res J Pharm Tech, 2009; 2: 344-48.
5. Kasture SB, A Handbook of experiments in pre-clinical pharmacology. Career publications, 2009; 1: 81-82.
6. Hawk P, Oser B, Summerson W, Gastric analysis: Practical Physiological Chemistry. Toronto: Blakinston, 1954; 378–80.
7. Ohkawa H, Oniwa N, Yagi K. Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem, 1979; 95: 351-58.
8. Sedlak J, Rudolf L, Estimation of total protein-bound and non-protein bound sulphydryl groups in tissues with Ellman's reagent, Anal Biochem, 1968; 25: 192-205.
9. Aebi H, Catalase: In Mehtods in enzymatic analysis, Academic Press: 1974; 674-84.
10. Muthusamy P, Suresh AJ, Balamurugan G, Antiulcer Activity of Azima Tetracantha: A Biochemical Study, Research J Pharm Tech, 2009; 2: 344-348.
11. Hawk P, Oser B, Summerson W, Gastric analysis, In Practical Physiological Chemistry, Toronto: Blakinston, 1954; 378–80.

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