SYNERGISTIC EFFECT OF CHRYSIN WITH PIPERINE AGAINST ETHANOL-INDUCED ULCERS IN RATS

Abstract

The present invention was designed to investigate the comparative efficacy of various gastroprotective effects with chrysin and piperine together using ethanol induced ulcers in rats. In India, chrysin and piperine have long been used to heal ulcers, skin damage, frostbite, wounds, herpes, scars, and blood purification. The purpose of the current study was to assess the antiulcer activity in experimental rats. Chrysin and Piperine (10 and 40 mg/kg, p.o administered) were assessed. Utilizing an absolute ethanol-induced ulcer model, the gastroprotective effect was assessed. Chrysin and piperine significantly inhibited the ulcer index and were comparable to standard. We hypothesize that the antiulcer properties of piperine and chrysin have significant synergistic gastroprotective effects on the rats. The ethanol group exhibited the most severe damage, with numerous ulcers, extensive necrosis, and hemorrhages in the stomach tissue. Treatment with piperine and chrysin reduced ulcer incidence at 10 and 40 mg/kg doses. The 10 mg/kg dose limited ulcers to the upper glands, while the 40 mg/kg dose showed fewer ulcers and preserved glandular structure. The synergistic group reduced overall quantity and glandular abnormalities. The control group displayed normal tissue structure. Our findings indicate that the synergistic effect of piperine and chrysin has significant anti-ulcerogenic action and can effectively lower the occurrence of gastric ulcers and their consequences.

Specification

Description:The following describes the invention and its preferred embodiment by way of understanding and not by way of any limitation. Furthermore, the results provided here in after are all exemplary and should not be construed as any sort of restriction.
The present invention describes a synergistic herbal extract formulation for the treatment of peptic ulcer. Accordingly, the said herbal formulation contains herbs selected from group of herbs available for peptic ulcer. The selected herbs are chrysin and piperine.
EXPERIMENTAL WORK
EXPERIMENTAL WORK
Material and Method
Reagents and Chemicals
The chyrsin and piperine from BLD Pharmatech PVT Ltd (India).
Experimental Animals
The department's animal house provided Wistar albino rats of both sexes. They were kept in a climate-controlled room with free access to food and water, and they were on a 12-hour light/dark cycle at a temperature of 25±2 °C. The Institutional Animal Ethical Committee (IAEC) of the Institute authorized the experimental procedure, and the studies were carried out in compliance with the rules on the use and care of experimental animals published by CCSEA, India (Ref no. CCSEA/1205/2024/05).
Acute oral toxicity study
In compliance with Organization for Economic Cooperation and Development (OECD) guideline 423, various doses of chrysin (55, 175, 550, 1750, and 2000 mg/kg, p.o.) were administered to the animals for acute toxicity. During the test, three female rats were employed, each receiving a dosage at a 48-hour interval. Observations from the cage side once included alterations to the skin, fur, eyes, mucous membrane (nasal), autonomic nervous system (drowsiness, gait, tremors, convulsions), and autonomic nervous system (salivation, lacrimation, sweat, piloerection, urine incontinence, and defecation). Over a two-week period, the mortality, if any, was assessed.
Selection of doses
A low dose, equal to 50% of the one-tenth dose (10, 40 mg/kg, p.o.), and a high dose, approximately one-tenth of the highest dose during acute toxicity tests, were chosen for the activity assessment.
Pharmacological evaluation
The in-vivo antacid and antisecretory effects of piperine and chrysin were evaluated using an ulcer model induced by indomethacin. We tested the gastroprotective effect using a model of ulceration induced by 100% ethanol. The stomach mucosa's integrity was evaluated by measuring glutathione (GSH), measuring the amount of mucus in the stomach, and histologically analyzing the gastric mucosal cells. The ability of the in-vitro antacid was evaluated using the titration method. We assessed the extract's effect on the liver using measurements of serum biochemicals.
Evaluation of anti-ulcer activity of chrysin and piperine in rats
Ulcer index
Scoring of ulcer was done as follows: No ulcer?=?0; Superficial ulcers?=?1; Deep ulcers?=?2; Perforation?=?3. The ulcer index will be the mean ulcer score for every animal. Ulcer index will be measured by using following formula:
UI = UN + US + UP × 10-1
Where, UI = ulcer index, UN = mean of ulcer number, US = mean of ulcer score, UP = ulcer probability (incidence %) for each group.
The percentage of ulcer protection was determined by formula:
% Protection?=?[(Control mean ulcer index - Test mean ulcer index)/Control mean ulcer index] 100
pH
pH of gastric content will be determined by dipping electrode of pH meter in beaker containing gastric contents.
Volume of gastric content
Volume of gastric contents will be measured by pouring gastric contents carefully in graduated cylinder.
Total and free acidity
One millilitre of centrifuged and filtered gastric secretion will be titrated against 0.1?N NaOH using the Topfers reagent as indicator for determination of free acidity and 1% phenolphthalein as indicator for combined/total acidity. The sum of the two titrations will be total acidity.
Lipid peroxidation
The glandular part of the stomach tissue will be homogenized in trichloroacetic acid (TCA) and the homogenate will be used to estimate malondialdehyde. Briefly, lipid peroxidation will be induced by adding ferric chloride (10?µl, 400?mM) and l-ascorbic acid (10?µl, 400?mM) to a mixture containing stomach homogenate (0.3?ml) in phosphate buffer solution (5?ml, pH 7.4, 0.2?M). After incubation for 1?h?at 37?°C, the reaction will be stopped by adding hydrochloric acid (2?ml, 0.25?N) containing trichloroacetic acid (1?ml, 15% w/v) and thiobarbituric acid (0.5?ml, 0.375% w/v) boiled for 15?min, cooled, and centrifuged, and the absorbance of the supernatant will be measured at 532?nm.

Ethanol-induced ulcer model
In this study rats will be divided into six groups. Each group contains six animals.
Group 1: Animal receives normal saline and served as control.
Group 2: Animal receives normal saline and served as ulcer control.
Group 3: Animal receive Chrysin respectively.
Group 4: Animal receive Piperine respectively.
Group 5: Animal receive Chrysin and piperine (synergistic) respectively.
Group 6: Animals are given oral Lansoprazole which is used as the standard medication for analysis.
The rats receive all treatments orally. Before the trial, the rats were fed nothing for eighteen hours, but they were allowed unlimited access to water. The medications were given to the rat's half an hour before one millilitre of 100% ethanol was given to them. The rats are killed by overdosing on diethyl ether one hour after the ethanol is administered. After the stomachs were removed, they were carefully cleaned with 0.9% saline, inflated with 10 mL of 1% formalin solution, then submerged in the same solution to seal the stomach's outer layer. Each stomach is then opened along its greater curvature and examined under a dissecting microscope after 10 minutes to determine whether any ulcers have formed. The following parameters will be estimated: lipid peroxidation, total and free acidity, pH of the stomach content, and volume of the stomach content.
RESULTS
Effect of chrysin and piperine on Ethanol-Induced Gastric Lesions
The rat was administered 96% ethanol, which caused large, long-lasting, hemorrhagic stomach ulcers, as seen in Figure 1. The rat was administered 96% ethanol, which caused large, long-lasting, hemorrhagic stomach ulcers, as seen in Figure 1. A dose-dependent reduction in the quantity and length of gastric lesions was seen after oral pretreatment with piperine (40 mg/kg). The number and duration of stomach ulcers were similarly affected by lansoprazole (20 mg/kg) and chrysin (10 mg/kg) (Figure 2). Furthermore, a significant reduction was observed on stomach tissue when piperine (40 mg/kg) and chrysin (10 mg/kg) were administered alone. Regarding the quantity and duration of stomach ulcers, lansoprazole (20 mg/kg) and chrysin (10 mg/kg) had comparable results. Synergistic (CP) has significantly different results when compared with Chrysin (10 mg/kg) or piperine (40 mg/kg) gastroprotective effects (Figure 2). Percentage inhibition of ulcer index due to chrysin and piperine and their Synergistic (CP) on the length of gastric ulcers are presented in the figure 3.

Effect of chrysin and piperine Malondialdehyde (MDA) Level in Gastric Tissue
As shown in (Figure 4), ethanol increased the gastric MDA level compared to the normal saline. Administration of chrysin and piperine at the dose of (10 and 40 mg/kg), as similar to the lansoprazole group, exhibited a significant reduction in MDA level in stomach tissue.

Effect of chrysin and piperine on Nitric Oxide (NO) Level in Gastric Tissue
The results (Figure 5) showed that ethanol administration significantly decreased the gastric nitric oxide level in rats. Administration of chrysin and piperine (10 and 40 mg/kg) restored the nitric oxide level in the stomachs exposed to ethanol.
, Claims:1. A synergistic herbal formulation comprises chrysin and piperine.
2. A synergistic herbal formulation as claimed in claim 1, wherein invitro activities are determined from the said herbal formulation, whereas the said activities are anti-ulcer activity.
3. A synergistic herbal formulation as claimed in claim 1, wherein the process for preparation of antiulcer composition comprising chrysin and piperine with other pharmaceutical acceptable excipients.
4. The process for preparation of antiulcer composition as claimed in claim 3, wherein evaluate antiulcer properties of chrysin and piperine in ethanol induced ulcer model in experimental animals.
5. The process for preparation of antiulcer composition as claimed in claim 1, wherein it indicates that significant inhibition of gastric acid secretion and ulcer index in ethanol induced gastric ulcers in rats.

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