Study of the protective effect of Pomegranate peel Ethanolic Extract on Gastric ulcer caused by stress on rats

 

Rana Alsamaan*, Farah Alhakim

College of Pharmacy, Al-Wataniyah University, Hama, Syria.

 

ABSTRACT:

Lots of herbal recipes are used Syria for treating and preventing gastric ulceration, especially the pomegranate peel powder, so the aim of this study was to investigate whether the pretreatment with pomegranate peel ethanolic extract PPEE in two doses (250, 500mg/kg) has any prophylactic effect on gastric mucosal damage caused by cold-restraint ulcer (CRU) in rats. It was noticed that pretreatment for four weeks was enough to protect the stomach, the ulcer index (UI) was macroscopically calculated and the stomach wall layers were histologically studied, the higher dose (500mg/kg) was more effective comparing with the lower one (250 mg/kg). These results suggest that the pomegranate peel ethanolic extract PPEE has special characteristics that make it useful for preventing from the gastric ulcers caused by stress.

 

 

 

INTRODUCTION:

Peptic ulcer disease (PUD) is one of the most common GI disorders¹ and it is caused by many factors including H.pylori infection, NSAIDs use, alcohol consumption, and stress². stress produces free radicals which are chemical species containing unpaired electrons in their outer orbital which are generally reactive. If a radical reacts with a non-radical, another free radical must be produced. This implication is reflected continuously in cells either during phagocytosis or pathological condition. The most important free radicals include: superoxide anion (O₂^-), alkoxyl, hydroxyl radical (OH^-), and alkoxyl singlet oxygen (¹O₂)³.

 

These are highly reactive species capable of wide spread, indiscriminate oxidation and peroxidation of proteins, lipids and DNA which can lead to significant cellular damage and even tissue and/or organ failure⁴.  Stress-related mucosal damage (SRMD) is the broad term used to describe the spectrum of pathology attributed to the acute, erosive, inflammatory insult to the upper gastrointestinal tract associated with critical illness⁵ and it often occurs in seriously ill patients who have an acute pathophysiological disturbance, such a hypotension, hypoxia, sepsis, uremia or ischemia⁶.

 

 

Medicinal plants play an important function in the field of treatment and cure of diseases⁷. Pomegranate (Punica granatum) is a famous plant in Syria, and it is an important medical plant that was used in ancient times in folk medicine to prevent or cure many gastric bowel diseases, diabetes, lung, prostate cancer, cardiac and kidney diseases, dental infection^8,9, pomegranate also has antibacterial activity¹⁰, anti-leukoderma activity and antioxidant activity¹¹. Also, of the peel, pulp and seed, pomegranate peel has the highest antioxidant activity¹². The peels, the waste part of it, are used to treat many diseases, some studies improved that pomegranate peels have antioxidants¹³, antibacterial, anti-fungal and anti-inflammatory effects⁹. The therapeutic value of pomegranate belongs to its constituents, that phenolic compounds (gallic acid), tannins, flavonoids (catechins and quercetin), quinones, saponins, cardinolites, steroids, terpinoids¹⁴, alkaloids, vitamin C and polyphenols such as anthocyanin (cyanidin) and pelargonidin which gives pomegranate its red color⁸.

 

So, our study aimed to investigate the protective effect of pomegranate peel ethanolic extract against stress ulcer induced in rats.

 

MATERIALS AND METHODS:

Preparing the plants:

Pomegranate fruits were picked in October 2021 form Misayf, Syria. They were cleaned and washed and then the peels were separated from fruits, dried and grinded for further extraction.

 

Pomegranate peels extraction:

50 gr powdered pomegranate peel was extracted with 50% ethanol in ultrasonic bath during 40 min at 60°C, obtained extract was filtered and evaporated to dryness by rotary evaporator¹³.

 

Experimental animals:

50 male wistar rats weighing 100-125gr (from Linn factory, Atomic Energy Commission for Syrian Arab Republic) were used in this study.

The animals were fed under normal conditions in 5 separate groups consisting of 10 rats (n=10)

1.     1^st group: the control group.

2.     2^nd group: the model group: was exposed to stress ulcer.

3.     3^rd group: the standard group: has been treated with ranitidine at dose of 50mg/kg daily for 4 weeks via oral gavage prior to inducing the stress ulcer ¹⁶

(Ranitidine was brought from Medico Labs for pharmaceutical products, Homs, Syria)

4.     4^th group: the PPEE1 group: has been treated with pomegranate peel ethanolic extract at dose of (250 mg/kg) daily for 4 weeks via oral gavage prior to inducing the stress ulcer.

5.     5^th group: the PPEE2 group: has been treated with pomegranate peel extract at dose of (500 mg/kg) daily for 4 weeks via oral gavage prior to inducing the stress ulcer.

 

Stress ulcer was induced according to Mohamed and Remon method (cold-restraint ulcer)¹⁷.

 

Animals were allowed just to drink water with no food access for 24 hours and then were put in separate small cages at 2°C for 3 hours.

 

After inducing ulcers, the animals were killed using an overdose of diethyl ether, and stomachs were isolated and opened along the greater curvature, washed with saline, and then the gastric mucosal was visually examined.

 

Ulcer Index (UI) was determined according to Robert method ¹⁵:

UI = a + b + c

a : the percentage of infected animals in each group divided to 10

b : the average of ulcer acuity, using numbers from (0-6)

0 : no erosions or blood

1 : some blood or one small erosion

2: many small erosions or one intermediate erosion (1-2mm)

3: many intermediate erosions or one big erosion (more than 2mm)

4,5,6: according to the severity of erosions

c : average of number of ulcers in the group's animals

After that, samples of gastric tissues were taken for the histological study.

 

Statistical Analysis:

The results were analyzed by one-way ANOVA using Tukey's Multiple Comparison test for comparing parametric data, and non-parametric data were compared by Kruksal-Wallis using Dunns test: Compare all pairs of columns.

In all comparisions, P<0.05 was considered as statistically significant

 

RESULTS AND DISCUSSION:

Macroscopic examination results:

Table 1. results of the macroscopic examination of the isolated stomachs

The values were expressed using mean ± SEM

*** there is a very important statistical difference comparing with the model group (P<0.001)

** there is an important statistical difference comparing with the model group (P<0.01)

* there is an important statistical difference comparing with the model group (P<0.05)

 

 

Figure 1. Effects of pomegranate peel extract (250,500 mg/kg) on ulcer acuity (1) and number of ulcers (2) in rats comparing with the normal, stress and standard groups

 

Figure 2. Macroscopic study of the isolated stomachs

(1)Normal stomach, (2)Many big erosions, (3)Bleeding areas

 

Our experiment showed a high UI of the model group comparing with the normal group, while the UI values of the standard group and the pomegranate (500 mg/kg) group were almost similar and very close to the value of the normal group.

 

When we statistically compared the ulcer acuity and number of ulcers between groups (fig 1), a very significant statistically difference was observed when comparing the model group with the normal group, the standard group and the PPEE2 group at the level of significance (P<0.001), while a significant statistically difference was observed when we compared the PPEE1 group with the model group at the level of significance (P<0.05)

 

The histological study result:

The thickness of the gastric mucus layer was studied and grades were scored for the histopathological

 

Changes as follows:

 

Thickness of mucus layer	Normal thickness	Grade (0)
	Simple erosions	Grade (1)
	Intermediate thickness	Grade (2)
	Almost complete erosion	Grade (3)
	Complete erosion	Grade (4)

 

The results were as follows:

Table 2. Results of the histopathological study of the isolated stomachs

The values were expressed using mean ± SEM

*** There is a very important statistical difference comparing with the model group (P<0.001)

** There is an important statistical difference comparing with the model group (P<0.01)

* There is an important statistical difference comparing with the model group (P<0.05)

 

Figure 3. Effects of pomegranate peel extract (250 ,500 mg/kg) on the histopathological changes of the gastric mucus in rats comparing with the normal, stress and standard groups

 

 

Figure 4. A microscopic section of a rat from the normal group the gastric mucus layers were:

a.      Cylindrical epithelial cells from the lamina mucosa

b.     Deep layer of the lamina mucosa with the gastric glands

c.      Muscularis mucosa

 

Figure 5. Microscopic sections of:

(1) A rat from the stress group, obvious ulceration was found, the erosion was almost within the muscularis mucosa

(2) A rat from the PPEE1 group, Many erosions were found

(3) a rat from the PPEE2 group, normal thickness of the mucus layer

 

The statistical comparison between the groups (fig 3) showed a very significant statistically difference was observed when comparing the model group with the normal group, the standard group and the PPEE2 group at the level of significance (P<0.001), while a significant statistically difference was observed when we compared the PPEE1 group with the model group at the level of significance (P<0.05)

 

DISCUSSION:

We observed a decrease in the UI values for the PPEE1 group rats comparing with the model group (the UI decreased from 20.6 to 14.5), but when we compared it with the standard group we found that this protective effect wasn’t as strong as the ranitidine (the UI was just 6.4)

 

The same results were observed for the number of infected animals and the number of ulcers in the groups' rats, but the prophylactic effect of the pomegranate peel ethanolic extract was better when we increased the dose (from 250mg/kg to 500mg/kg)

 

Rats with dose of (500mg/kg) were completely protected against stress ulcer, the UI was 6.1 which is so close to the UI of the normal group and the standard group.

 

The number of infected animals in this group was just 3 which is almost similar to the normal group with a very important statistical difference comparing with the model group for both number of ulcers and the ulcer acuity (P<0.001)

 

These macroscopic results agreed with the histological results, partial erosions were observed in the mucus layer (fig 5) with a statistical difference between the model group and the PPEE1 group (P<0.05)

 

While a more important statistical difference was observed for the PPEE2 animals (P<0.001) and the thickness of the mucus layer was very similar to the normal group (fig 5)

 

After all, we can say that pomegranate peel ethanolic extract has an excellent protective effect against stress ulcer induced in rats.

 

Stress ulcer refers to an acute mucosal lesion of the stomach or even the duodenum that occurs during shock, trauma, post-operation and severe bacterial infections ⁽¹⁸⁾. Gastric mucosal ischemia, excessive gastric acid secretion, imbalance between aggressive factors (such as gastric acid, pepsin) and defense factors (such as mucosal blood flow and prostaglandin E₂, PGE₂) accounts for the pathophysiology of stress ulcer. However, the precise mechanism still remains       unclear ^19,20.

 

It has been suggested that in stress-induced peptic ulcer disease, as a result of insufficient blood microcirculation to the gastric mucosa, an accumulation of reactive oxygen species (ROS) will occur with accompanying lipid peroxidation  which leads to membrane fluidity, resulting in reduced membrane integrity of surface epithelial cells, thereby causing gastric ulcers²¹ and subsequent loss of normal cellular functions²².

 

As we know, plant polyphenols are important antioxidants which they are substances that neutralize free radicals or their actions, which protect the body against free radicals–unstable molecules that cause damage to cell membranes, lipids, proteins, and DNA²³. Polyphenols comprise flavonoids (flavonols, flavanols, and anthocyanins), phenols, phenolic acids, lignins, and condensed tannins (pro-anthocyanidins)²⁴. Flavonoids prevent superoxide anion production by inhibiting XO. In addition, they inhibit COX, LOX, GST, microsomal monooxygenases, and NADH oxidase. Many flavonoids chelate free Fe and Cu that could otherwise increase ROS generation, and also reduce ROS such as O2^·, and HO².

 

So, our study suggests that polyphenols in pomegranate peel can participate in protecting the mucosa of the upper gastrointestinal tract from stress ulcer damage.

 

CONCLUSION:

The present study indicated that pomegranate peel ethanolic extract (PPEE) has a protective effect of stress ulcer induced in rats by visual and histological examination at the dose of 250mg/kg, and a much better protective effect was observed at the dose of 500 mg/kg due to its active components.

 

ACKNOWLEDGMENT:

Medico Labs for pharmaceutical products provided ranitidine. This work was supported by AlWataniya Private University, Hama, Syria.

 

DECLARATION OF CONFLICTING INTERESTS:

The author (s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article

 

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Accepted on 08.06.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):86-90.