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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 16  |  Issue : 1  |  Page : 60-70

Microanatomical evaluation of flavonoid-rich fraction of Musa paradisiaca in Aspirin-induced gastric lesion


1 Department of Anatomy, College of Medicine, Ekiti State University, Ado Ekiti, Nigeria
2 Department of Anatomy and Cell Biology, College of Health Sciences, Obafemi Awolowo University, Ile Ife, Nigeria
3 Department of Physiology, College of Medicine, Ekiti State University, Ado Ekiti, Nigeria

Date of Web Publication9-Aug-2017

Correspondence Address:
Margaret Olutayo Alese
Department of Anatomy, College of Medicine, Ekiti State University, Ado Ekiti
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jeca.jeca_5_17

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  Abstract 

Aim: This study investigated the healing effects of a flavonoid-rich fraction of Musa paradisiaca (MP) fruit on the histomorphology and histomorphometry of the gastric corpus in Wistar rats following aspirin-induced gastric lesion. This was with a view to providing information on the alternative therapies of treating gastric ulcers using unripe fruits of MP.
Materials and Methods: Ninety adult male Wistar rats were randomly assigned into 6 groups of 15 rats each. Aspirin at a dose of 400 mg/kg was dissolved in distilled water and administered as a single oral dose to induce gastric lesions in the test groups of rats. After 24 h, flavonoid fraction of MP was administered to groups C, D, and E at graded doses for 21 days, whereas group F rats received omeprazole at 1.8 mg/kg. On days 14, 21, and 28, five rats from each group were sacrificed. The pH value of the gastric contents was determined, gastric tissues were processed using paraffin wax embedding method; sections were stained with hematoxylin and eosin and Alcian blue/PAS. Data were analyzed using descriptive and inferential statistics.
Results: Histomorphological and histomorphometric studies showed a restorative effect of flavonoid fraction of MP in all the treated groups of rats as there was a significant (P = 0.0001) (P = 0.0001) increase in the total mucosal and glandular mucosal layers when compared with the test control. There was a progressive and dose-dependent improvement in staining for mucus-secreting cells in the treated groups as well as a significant (P = 0.0001) increase in a number of cells in the treatment groups when compared with the control.
Conclusion: MP attenuated the deleterious effects of aspirin on the stomach by strengthening mucosa defensive factors.

Keywords: Gastric corpus, gastric ulcer, mucus-secreting cells, Musa paradisiaca


How to cite this article:
Alese MO, Adewole SO, Alese OO. Microanatomical evaluation of flavonoid-rich fraction of Musa paradisiaca in Aspirin-induced gastric lesion. J Exp Clin Anat 2017;16:60-70

How to cite this URL:
Alese MO, Adewole SO, Alese OO. Microanatomical evaluation of flavonoid-rich fraction of Musa paradisiaca in Aspirin-induced gastric lesion. J Exp Clin Anat [serial online] 2017 [cited 2018 Sep 26];16:60-70. Available from: http://www.jecajournal.org/text.asp?2017/16/1/60/212643




  Introduction Top


The stomach can resist endogenous aggressive factors such as hydrochloric acid, pepsin, refluxed bile, leukotrienes, and reactive oxygen species (ROS); however, the degree of resistance depends on cytoprotective and physiologic factors, including mucus-bicarbonate barrier, surface active phospholipids, prostaglandins (PGs), mucosal blood flow, cell renewal and migration, nonenzymatic and enzymatic antioxidants and some growth factors (Bhattacharjee, et al. 2002). Gastric lesions may occur when there is a breakdown in protective mechanisms due to injurious factors.

Gastric ulcer results in a discontinuity in the gastric mucosa penetrating through the muscularis mucosa (Bandyopadhyay, et al. 2001). It has a worldwide prevalence of about 5% with the potential for significant morbidity (such as bleeding and perforation) and mortality. The pathogenesis of ulcers includes diverse factors such as a stressful lifestyle, alcohol consumption, use of steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs) and drugs which stimulate gastric acid and pepsin secretion, Helicobacter pylori infections, trauma, shock, smoking, low socioeconomic status, and family history (Bhattacharjee, et al. 2002). Traditional theories on the pathogenesis of peptic ulcers focus on acid hypersecretion and NSAIDs which disrupt the normal mucosal defense and repair, making the mucosa more susceptible to the attack of acid (Blaser 2005). NSAIDs are a major cause of gastric ulceration as they result in a “chemical gastritis” characterized by mucosal hyperplasia and edema with the infiltration of little inflammatory cells (Higham, et al. 2002). Aspirin is a NSAID routinely used for the treatment of rheumatoid arthritis and related diseases as well as the prevention of cardiovascular thrombotic diseases (Zhongzhi, et al. 2011). It causes inhibition in the gastric mucosal protective factors and at the same time increases the aggressive factors (mainly acid and pepsin) to which the mucosa of the stomach is exposed (Beers and Berkow 2006). These predisposes to gastrointestinal effects of variable severities ranging from mild dyspepsia to severe fatal gastric bleeding.

Aspirin often causes acute gastric mucosal damage which is apparently related to the dose administered. This dose-response effect, evident in both endoscopic and micro-bleeding studies done after acute or short-term aspirin administration, is also associated with the risk of developing chronic gastric ulcer (Graham and Smith 1986). In previous studies, oral administration of aspirin has been used as a model for gastric lesions in the animal as it induces the formation of the reactive oxygen metabolites (McAllindon,et al. 1996; El-Far, et al. 2012). This may contribute to mucosal injury and cause a dose-dependent reduction in mucosal PG E2 and I2 biosynthesis accompanied by an increase in the mean area of gastric ulcerations (Obi, et al. 2000). This effect has been attributed to the fact that aspirin irreversibly inactivates the PG synthetase system which mediates synthesis of PG in the mucosa (Obi, et al. 2000). The pathogenesis of NSAIDs-induced gastric ulceration includes the block of cyclooxygenase activity that leads to lowered mucus and bicarbonate secretion, decreased mucosal blood flow, neutrophil infiltration, alteration of microvascular structures, and increase of acid and pepsinogen secretion. In addition, increased production of ROS, increased lipid peroxidation, and neutrophil infiltration has been demonstrated to play a role in the pathogenesis of NSAIDs induced ulcers, including aspirin-induced ulcer (Jainu, et al. 2006).

The therapy for the treatment of gastric ulcer involves the control of gastric acid secretion and reinforcement of gastric mucosal production using antacids, H2-receptor blockers, proton pump inhibitors, anticholinergics, and cytoprotective agents (Rao, et al. 2004). A major drawback of these therapies is the challenge posed by high recurrence rate and side effects of most of the drugs. These include diarrhea, itching, dizziness, arrhythmia, impotence, gynecomastia, and hematopoietic changes (Akhtar, et al. 1992; Bandyopadhyay, et al. 2002).

In developing countries, there is an increase in the use of alternative therapies and natural products, especially those derived from plants. Furthermore, in the past few decades, medicinal plants are among the novel sources of drugs and have shown promising results in the treatment of various diseases including gastric ulcer. The role of a mucosal factor in gastric ulcers has been well elucidated as it is well established that gastric ulcer can both be prevented and cured by enhancing the defensive mechanisms of gastric mucosa. The antiulcerogenic activity of many natural products of plants and herbs in the management of gastric ulcer is due to an increase in mucosal defensive factors rather than a decrease in the offensive factors (Akomas, et al. 2014).

Musa paradisiaca (MP) is a tropical plant which is a staple source of nutrition but has to be cooked to be edible. It is called plantain or vegetable banana and grows to about 9 m high; with a robust tree-like pseudostem, a crown of large elongated oval deep-green leaves (up to 365 cm in length and 61 cm in width), with a prominent midrib. The plant produces a dark red and fleshy single inflorescence-like drooping spike with large bracts which are ovate, 15–20 cm long and concave, opening in succession. The fruits are oblong, fleshy, 5–7 cm long in wild form and longer in the cultivated varieties. Different parts of the plant are used in traditional folk medicine for numerous purposes including antidiarrheal, antiulcerogenic, antimicrobial, hypoglycemic, hypocholesterolemic, antihypertensive, and diuretic. It is also known to be used as a cardiac depressant, bronchodilator, expectorant, oral contraceptive, abortifacient, and wound healing herb (Sharma, et al. 2002; Imam, et al. 2011). The extract of unripe MP has been suggested to have high antioxidant activity due to the presence of phytochemicals which are potent antioxidants with free radical scavenging activities (Eleazu, et al. 2011). MP has also been found effective in the management of gastric ulcer (Goel and Sairam 2002).

Flavonoids are phytochemicals present in most plants used in the treatment of gastric ulcers; they could be influenced by other organic and inorganic compounds such as coumarins, alkaloids, terpenoids, tannins, phenolic acids, and antioxidant micronutrients (Czinner, et al. 2001). Phytochemical analysis of MP showed the presence of flavonoids and related compounds (Lewis, et al. 1999; Lewis, et al. 2001).

The histomorphological alterations that occur in the gastric mucosa following MP treatment of experimentally-induced gastric damage have not yet been elucidated, hence this study.


  Materials and Methods Top


Ethical Approval for this study was obtained from the Health Research Ethics Committee (HREC), Institute of Public Health, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, IPHOAU/12/187. All animals were handled in accordance with the Guidelines for Animal research as detailed in the NIH Guidelines for the Care and Use of Laboratory Animals (National Institute of Health 2011).

Plant material

Mature, unripe fruits of MP Linn (Musaceae) were purchased from a farmland in Ado– Ekiti, identified by a taxonomist at the Department of Plant Science and Forestry, Ekiti State University, Ado Ekiti, Nigeria and a voucher specimen (UHAE 2014/84) was deposited at Herbarium of the Department.

Preparation of extract

The fruits were washed, peeled; the pulp cut into pieces, air-dried and pulverized using an electric grinder. The powdered sample was extracted three times with 70% methanol (20% w/v) with continuous stirring using an Orbital shaker at room temperature for 48 h each. The mixture was filtered and the filtrate concentrated at 40°C in a vacuum rotary evaporator under reduced pressure to obtain a dark brown extract. To obtain the flavonoid-rich fraction, the crude extract was successively partitioned by solvent-solvent extraction using n-hexane, dichloromethane, ethyl acetate, and n-butanol in successive order as described by (Owoyele, et al. 2008). The resulting flavonoid fraction was concentrated, freeze-dried in a vacuum freeze drier and stored in a desiccator until needed.

Test for flavonoids

The flavonoid fractions were subjected to phytochemical analysis using Shibata's reaction according to (Markham 1982); then observed for red coloration of flavonoids and further confirmed with Ferric chloride test; and observed for the bluish green coloration of flavonoids (Trease and Evans 1989).

Animal care and management

Ninety healthy male Wistar rats weighing between 120 and 150 g were obtained from the Animal House of the College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria used for this study.

The rats were fed on standard rat pellets procured at once from Ladokun feeds, Ibadan to avoid change in the ration of the animals in the course of the research work. They were given tap water ad libitum and acclimatized for a week. They were housed in plastic cages in the Animal Holding of the Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife, Nigeria under standard laboratory conditions of natural light/dark cycle at room temperature and humidity.

Chemicals and equipment

Reference standard acetyl-salicylic acid (ASA) was purchased from Sigma Chemicals, USA while omeprazole (OMZ) was obtained from the Department of Pharmacy, Ekiti State University Teaching Hospital, Ado-Ekiti. Other chemicals, reagents and solvents used for extraction, tissue processing and histochemical studies were procured from ADFOLAK (Nig) Ltd., Ibadan, Nigeria. Equipment in the Department of Anatomy and Cell Biology; and the Drug Research and Production Unit, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria were used for the study.

Experimental design, induction of gastric lesion and treatment

At the commencement of the experiment, stool samples of each animal were obtained and tested for the presence of H. pylori antigen using the H. pylori Stool Antigen Test Kit (Biotech, China). The rats were randomly assigned into 6 groups (A, B, C, D, E, and F) of 15 rats each. Before the induction of gastric lesions, rats in the experimental group were starved overnight to ensure complete gastric emptying and a steady state gastric acid secretion but allowed free access to water. Gastric lesions were induced in Groups B, C, D, and E rats by a single oral administration of 400 mg/kg reference standard aspirin (Sigma Chemicals, Co., USA) dissolved in 0.5 ml of distilled water. Preliminary investigations have revealed that gastric lesions were induced at this dose in Wistar rats. Group A rats received an equivalent volume of distilled water used in dissolving aspirin. After 24 h, flavonoid fraction of MP dissolved in distilled water was administered by gavage to the rats in groups C, D, and E at graded doses of 100, 200, and 400 mg/kg, respectively, at a particular time (10.00–11.00 h) for 21 days. Group F rats received Omeprazole (Divine Essential Formulations, Lagos, Nigeria) dissolved in distilled water at 1.8 mg/kg by gavage for 21 days. The rats in group A received equivalent volumes of distilled water used in dissolving the extract and drug for the same period.

Sacrifice of animals

On the 14th and 21st days of treatment, five rats from each group were sacrificed under ketamine anaesthesia at a dose of 75 mg/kg, whereas the remaining were left untreated for another 7 days before sacrifice. Each of the animals was quickly dissected through a mid-line incision in the anterior abdominal wall.

Determination of pH of stomach content

The stomach of each sacrificed rat was excised and opened along the greater curvature; the content was drained and completely recovered by washing with 10 ml of normal saline. The gastric content and washing for each animal were combined and centrifuged at 3500 rpm for 10 min and the pH value of the supernatant was measured using a digital pH meter (Alvarez-Suarez, et al. 2011).

Tissue processing

Samples of the gastric corpus from each group of animals were fixed in 10% neutral buffered formalin and processed according to the general technique of tissue processing and microtomy as described by (Bancroft and Gamble 2008). Sections of 4-5 μm thickness were produced on a Rotary microtome and stained with hematoxylin and eosin (H and E) for a demonstration of general gastric architecture, whereas Alcian blue and PAS stains were employed for demonstration of mucus secreting cells.

Photomicrography and image analysis

OMAX × 40–×2000 Digital Light microscope was used to examine the stained sections and digital photomicrographs taken at various magnifications. Image Analysis and Processing for Java (Image J) software (National Institute of Health, USA) was used to analyze and quantify photomicrographs. Motic Image plus software was used to identify and quantify the number of mucus-secreting cells stained by Alcian blue and PAS.

H and E slides of the stomach were used for histomorphometric studies. Photomicrographs of the stained sections were analyzed with the ScopeImage 9.0 Professional imaging software. The thickness of four areas that were localized at equal distances from one other on each tissue was measured (in μm) for the following parameters:

  1. Mucosa layer: from the gastric epithelial lining to the muscularis mucosa
  2. Subglandular mucosal layer: from the lamina propria to the muscularis mucosa
  3. Glandular mucosa layer: calculated by subtracting the values of the measurements of subglandular mucosa from the mucosa layer (De Conto, et al. 2010).


Furthermore, the percentage of mucosa that is glandular was calculated as;



Determination of ulcer index

Microscopic ulcer index was obtained separately by two pathologists, and a mean index was calculated according to the method of Pandit, et al. 2000.

Normal tissue = 0; Local damage to gastric pits cells = 1; Local damage to gastric glands = 2; Deep damage to gastric glands = 3.

Microscopic ulcer index = (number of lesion 1) + (number of lesion 2) ×2+ (number of lesion 3) ×3.

Statistical analysis

GraphPad Prism version 5.00 for Windows (GraphPad Software, USA) was the statistical software used for analysis. The results were expressed as a mean ± standard error of the mean two-way ANOVA was used for comparative analysis of the data between the groups of rats, followed by Bonferroni tests for multiple comparisons. Statistical significance was set at P < 0.05.


  Results Top


Phytochemical analysis of the ethyl acetate fraction of MP for the presence of flavonoid was positive as there was a coloration of red for the Shibata's reaction and bluish green for the Ferric chloride test, respectively.

Results of stool analysis for H. pylori in all the rats were negative as a pink line appeared in the control line, and there was no line in the test region.

pH of gastric contents of control and treated rats

Two-way ANOVA of the pH of gastric content showed that there was a significant difference between control and treated groups at week 1, 2, and 3 (P = 0.0001). Aspirin increased gastric acidity as there was a significant reduction (P 0.0001) in the pH of gastric content in the aspirin-only group when compared with both the control and treated groups [Figure 1]. A significant increase (P = 0.0089) in the pH of the gastric contents in the treated groups was observed when compared with the control on the 14th day of the experiment; however on the 21st day of the experiment, a significant increase was observed in only the ASA + MP400 group of rats when compared with the control. On the 28th day of the experiment (withdrawal period), there was no significant difference when all the treated groups were compared with the control (P = 0.1). Furthermore, there was no significant difference (P = 0.23) in the pH of gastric content among the treated groups throughout the experimental period except for the ASA + MP400 group which showed a significant increase (P< 0.05) when compared with ASA + MP100 on day 14 and ASA + OMZ on days 14 and 21.
Figure 1: Weekly pH value of gastric contents of control and treated rats. *P < 0.05., α and β – significant difference compared to control and acetyl.salicylic acid only, respectively

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Ulcer index of the corpus of control and treated rats

There was a significant difference in the ulcer index in the corpus of control and treated rats throughout the experimental period (P = 0.0001). As shown in [Table 1], in the gastric corpus, administration of aspirin resulted in a significant increase in ulcer index (P = 0.0015) in both the ASA only and the treated groups when compared with the control. This indicates that aspirin induced ulceration and hemorrhagic lesions in the corpus. On the 14th and 21st days of the experiment, there was a significant increase (P 0.0001) in ulcer index in all the treated groups when compared with the control, but on the 28th day of the experiment, apart from the ASA + MP100 group of rats, there was no significant difference (P 0.12) in the ulcer index between the control, all the treated groups of rats (ASA + MP200 and ASA + MP400 and ASA + OMZ). This indicates the efficacy of both MP (200 mg/kg and 400 mg/kg) and OMZ in the reduction of aspirin-induced gastric lesions over a period. Furthermore, throughout the experimental period, a significant increase (P = 0.0001) was observed in the ulcer index of the ASA only group of rats when compared with all the treated groups (ASA + MP100, ASA + MP200, ASA + MP400 and ASA + OMZ). On the 14th day of the experiment, there was no significant difference (P 0.0012) in ulcer index among the treated groups of rats (ASA + MP100, ASA + MP200, ASA + MP400 and ASA + OMZ). At the end of the treatment period, a significant increase (P< 0.05) was however observed when ASA + MP100 was compared with ASA + MP200, ASA + MP400 and ASA + OMZ. This shows the efficacy of ASP + MP200, ASA + MP400, ASA + OMZ over ASA + MP100 in reducing the ulcer index.
Table 1: Ulcer Index of the corpus of control and treated rats

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Haematoxylin and eosin staining results

On day 14 of the experimental period, as shown in [Figure 2], the corpus of the control group presented with a well-organized histological outline of the gastric architecture. A continuous epithelial lining was also observed. There was discontinuation of the epithelial lining in the aspirin-only group as evidenced by the ulcer craters. In the treated groups, a gradual restoration of the epithelial lining was observed. The ASA + MP200 and ASA + MP400 groups presented with a proper alignment of the connective tissue fibers similar to what is observable in control. However, some pockets of ulcerations were still observed in the ASA + MP100 and ASA + OMZ groups.
Figure 2: Photomicrograph of the gastric corpus of control (a), acetyl-salicylic acid Only (b), MP treated (c-e) and omeprazole treated (f) Groups on Day 14. H and E, ×100. Mucosa (m), Muscularis mucosa (arrows), Submucosa, Muscularis propia (MP), Ulcer crater (dashed arrow)

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On day 21 of the experimental period, the control group displayed a well-structured histoarchitecture with well-arranged gastric layers. Severe ulcerations characterized by epithelial distortions were observed in the ASA only group. Evidence of distortion of the connective tissue fibers within the mucosa was also observed. The photomicrographs in the treated groups displayed a normal histoarchitecture as compared with the control. No ulcerations were observed, and the gastric layers were in proper alignment [Figure 3].
Figure 3: Photomicrograph of the gastric corpus of control (a), acetyl-salicylic acid only (b), MP treated (c-e) and omeprazole Treated (f) Groups on Day 21. H and E, ×100. Mucosa (m), Muscularis mucosa (arrows), Submucosa, Muscularis propia (MP), Ulcer crater (dashed arrow)

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On day 28 of the experimental period, a well-organized histoarchitecture was observed in the gastric layers of the control and treated groups. The ASA only group, however, showed areas of ulceration with irregularly arranged connective tissue fibers in the submucosa [Figure 4].
Figure 4: Photomicrograph of the gastric corpus of control (a), acetyl-salicylic acid only (b), MP treated (c-e) and omeprazole treated (f) Groups on Day 28. H and E, ×100. Mucosa (m), Muscularis mucosa (arrows), Submucosa, Muscularis propia (MP), Ulcer crater (dashed arrow)

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Histomorphometry of the corpus

[Table 2], [Table 3], [Table 4] present the results of histomorphometric measurement of the corpus. There was a significant difference between the mucosal layers and percentage glandular mucosal layers in week 1, 2, and 3 (P = 0.0001) (P = 0.0011), respectively. Throughout the experimental period, aspirin caused a significant reduction (P 0.001) in total and glandular mucosal layers in all the groups of rats that received it when compared with control. This effect was attenuated in the treated groups of rats (ASA + MP100, ASA + MP200, ASA + MP400 and ASA + OMZ) as a significant increase (P 0.001) was observed in the total and glandular mucosal layers when compared with the ASA only group during the treatment period. There were no significant differences among the treated groups when compared with one another. Furthermore, during the treatment period, the percentage glandular mucosa layer showed comparable results among the treatment groups, but there were significant differences when the groups were compared with both the control and the aspirin only groups. This indicates the efficacy of the treatments in both MP and OMZ groups.
Table 2: Histomorphometric measurements on Day 14

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Table 3: Histomorphometric measurements on Day 21

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Table 4: Histomorphometric measurements of the corpus on Day 28

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Histomorphology of the corpus as revealed by Alcian blue/PAS staining

On day 14 of the experimental period, as shown in [Figure 5], the corpus of the control Group (A), ASA + MP200 and ASA + MP400 and ASA + OMZ show normal luminal surface mucus secreting cells and numerous mucous neck cells in the mucosa (arrows). However, a reduction in the staining of the cells is observed in the ASA only (B) and the ASA + MP100 groups.
Figure 5: Photomicrographs of the gastric corpus of control (a), acetyl-salicylic acid only (b), MP treated (c-e) and omeprazole treated (f) Groups on day 14. Alcian Blue/PAS × 400. Arrows show mucus secreting cells (signet rings) in the gastric mucosa

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[Figure 6] presents the corpus on day 21. All the treated groups displayed a positive staining for mucus secreting cells. A reduction in staining was however observed in the ASA only group.
Figure 6: Photomicrographs of the gastric corpus of control (a), acetyl-salicylic acid only (b), MP treated (c-e) and omeprazole Treated (f) groups on day 21. Alcian Blue/PAS, ×400. Arrows show mucus secreting cells (signet rings) in the gastric mucosa

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On day 28, the treatment groups ASA + MP100, ASA + MP200, ASA + MP400 and ASA + OMZ groups show positive staining of mucus secreting cells when compared with control [Figure 7]. The ASA only group however displayed a weak staining of the cells when compared with the control.
Figure 7: Photomicrographs of the gastric corpus of control (a), acetyl-salicylic acid Only (b), MP treated (c-e) and omeprazole Treated (f) groups on day 28. Alcian Blue/PAS, ×400. Arrows show mucus secreting cells (signet rings) in the gastric mucosa

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Motic image plus count of the mucus-secreting cells stained by Alcian blue/PAS

Using Motic Image plus software to count the number of mucus-secreting cells stained by Alcian blue/PAS, results from this study showed that in the gastric corpus, there was a significant difference at week 1, 2 and 3 (P = 0.0001). The presence of gastric lesions caused a reduction in the number of mucus-secreting cells as there was a significant decrease in the ASA only group when compared with the treated groups on days 21 and 28 of the experimental period (P = 0.001). However, there was no significant difference between the ASA + MP100, ASA + MP200, and ASA + OMZ groups of rats. This indicates that restoration of the mucus secreting cells was progressive. Comparison among the treated groups showed that there was no significant difference (P = 0.1) in the number of secreting cells throughout the experimental period except for that observed between ASA + MP100 and ASA + OMZ on day 14 and ASA + MP400 and ASA + OMZ on day 28. When the control was compared with the treated groups, throughout the experimental period, there was a significant difference (P 0.0001) except in the ASA + MP400 group on day 14 which showed no significant difference when compared with the control. This indicates a better efficacy of the extract in increasing the number of mucus-secreting cells at a dose of 400 mg/kg [Figure 8].
Figure 8: Weekly Motic Image plus count of mucus secreting cells stained by Alcian blue/PAS in the corpus of control and treated rats. *P < 0.05., α and β – significant difference compared to control and acetyl.salicylic acid only, respectively

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  Discussion Top


Although the incidence of gastric ulcers is not as high as duodenal ulcers, it has higher mortality rate due to its prevalence in older patients and predisposition to malignancy in poorly managed cases. (Langman 1988; Chai 2011). In spite of the usefulness of NSAIDs such as aspirin in therapeutic medicine especially in the geriatric population, the major deterrent to their use are serious side-effects, including damage of gastrointestinal mucosa, aggravation of stress ulcerations, and exacerbation of preexisting gastric ulcerations (Konturek, et al. 1990; Mizuno, et al. 1997; Schmassmann and Stettler 1997). These could be due to their topical irritating effect, activation of neutrophils, ROS, fall in the microcirculation and the reduction in mucosal generation of PGE2 which is essential for homeostatic functions including maintenance of the mucosal integrity and mucosal blood flow. Hence, there is a decrease in cell function, and mucosal cell damage occurs.

Administration of aspirin has been proven to result in enhancement of apoptosis rate and the development of histopathological characteristics and functional alterations which result in gastric mucosa injuries (Popovic, et al. 2009; Scheiman, et al. 2010). A good number of drugs developed to minimize the adverse effects in the digestive tract have been found to be related to the decrease in the concentration of PGs; and these lipids are vital to the process of the mucosa tunic integrity maintenance, and gastric ulcer healing (Berenguer, et al. 2002; Brunton, et al. 2007). These contribute to recurrence rates and a total lack in the efficacy of treatment regimen for gastric ulcers, hence the need for more efficacious therapy (Jones and Sherman 1999).

In this study, aspirin caused a significant reduction in pH of rat's stomach when compared with both the control and treated groups. This correlates with work of (Olaleye, et al. 2013). The level of acidity of gastric secretions is demonstrated by the pH and low value indicates decreased hydrogen ion concentration in gastric juice. This has been linked to the pathogenesis of gastric lesions in experimental animals. Aspirin-induced mucosal damage occurs due to interference with PG synthesis, increase in acid secretion, decrease in mucin activity and back diffusion of H + ions (Jaikumar, et al. 2010; Rao, et al. 2000). Results from this study show the efficacy of the treatments with various doses of MP and OMZ in increasing the pH on the 14th day of treatment. However, at the end of the experimental period, MP at a dose of 400 mg/kg had a more significant effect than MP at a dose of 100 mg/kg and OMZ in elevating the gastric pH. Comparable results were, however, obtained for MP at 200 mg/kg and 400 mg/kg. It has been reported that flavonoids inhibited acid production in isolated parietal cells in response to histamine stimulation (Beil, et al. 1995). The results of this study are consistent with this report.

It has been established that aspirin causes gastric lesions when administered to fasted Wistar rats (Popovic, et al. 2009; Scheiman, et al. 2010). The present study shows that there was ulceration in the stomach of the rats treated with aspirin. In the corpus, the aspirin-only group was seen to present with severe ulceration. At the end of the treatment period, there was evidence of restoration in the gastric tissues of treated groups of rats when compared with the control. The effectiveness of MP at doses of 200 mg/kg and 400 mg/kg in gastric ulcer healing was however demonstrated as pockets of ulceration were still observed in the gastric tissues of rats treated with OMZ and MP at 100 mg/kg; and these were not present in the groups that received higher doses of the extract. Furthermore, during the withdrawal of treatment, the attenuating effect of MP on gastric ulcers was still observed as there was an improvement in the gastric morphology in the treatment groups. Similar findings were made by (Best, et al. 1984) who reported that the anti-ulcerogenic activity of MP against aspirin-induced gastric ulcers in rats was due to its ability to stimulate the growth of gastric mucosa. Furthermore, Ghosal 1985 reported that ethanolic extract of MP increased the accumulation of (PGE and PGI2) in the human gastric mucosal incubates. Prostaglandins are important in the maintenance of mucosal integrity and blood flow. The effect of extracts on the regeneration of glandular epithelium was also observed as results of the histomorphometric study show that throughout the experimental period, there was a significant increase in the glandular mucosal layer in the treatment groups when compared with the aspirin-only group. This is in agreement with Goel, et al. 1986 who reported that the dried powder of MP displayed anti-ulcerogenic activity by strengthening of mucosal resistance. Mukhopadhyay, et al. 1987 also found out that apart from this, it also increased cell proliferation as observed from an increase in DNA and [3H]-thymidine uptake by the mucosal cells and increase in mucosal thickness. This property was also reported to be essential in the healing of ulcers. The findings from this study are similar to those obtained by Gonzalez and Di Stasi 2002 who concluded that flavonoids are active compounds against gastric lesions. This study and others, MP have been confirmed to contain flavonoids which are implicated in cellular regeneration and cytoprotection (Lewis, et al. 1999; Kumar, et al. 2013).

MP has been reported to promote ulcer healing by its various effects on gastric mucosal defensive factors including enhanced cell proliferation and reducing free radicals levels (Kumar, et al. 2012).

In this study, there was a significant increase in ulcer index in the Aspirin only group when compared with the control. At the end of the experimental period, MP was effective in reducing ulcer index in a dose-dependent manner as there was no significant difference in the ulcer index in the MP200 and MP400 groups and the control. This is in conformity with Carvalho, et al., 2011 who reported the decrease in gastric lesion following treatment with leaves of Brassica oleracea in aspirin-induced gastric ulcer in rats. Similar effects were also observed in OMZ treated groups. There was a reduction in effectiveness of MP100 as there was a significant difference in ulcer index between MP100 and the control.

Mucins are glycoproteins of high molecular weight which are synthesized, stored and secreted by the epithelial mucosal cells of several organs including the gastrointestinal tract. According to Brunton, et al., 2007 a decrease in the synthesis of PGs is one the factors involved in the pathogenesis of the gastric ulcer; this results in a reduction in mucus secretion and direct damage to the mucin layer or mucin synthesis resulting in apoptosis of the gastric cells (Hoshino, et al. 2002). NSAIDs possess free carboxyl group that form a strong electrostatic bond with positively charged head of Zwitterionic phospholipids of mucus layer; the consequent increase in solubility of the phospholipids neutralises its surface activity and topically act on tissues to disrupt the hydrophobic protective lining of the mucus gel layer resulting in ulcerations (Al-Harbi, et al. 1995). A decrease in gastric mucus is associated with the significant production of free radicals; this results in increased lipid peroxidation which in turn causes damage to both cell and its membranes and the accumulation of neutrophils (Reshma, et al. 2000). Findings from this study indicate that aspirin results in a decrease in the number of mucus-secreting cells. In addition, MP and OMZ attenuated these effects as there were increased staining intensity and a number of mucus-secreting cells. However, a dose-dependent effect was observed as MP200 and MP400 significantly increased the number of mucus-secreting cells as against MP100 and OMZ. Since all cell membranes have a lipid bilayer; the importance of mucus secretion in the maintenance of mucosal integrity cannot be over-emphasized. Mucus secreting cells secrete mucin which creates a protective barrier as well as lubrication for the underlying epithelium. Furthermore, mucins act as free radical scavengers, due to their ability to bind to lipids (Prathima and Kumar 2012). Previous reports have shown that in gastric ulcer, the protective effects of MP was due to increase in the defensive factors including an increase in mucin secretion, mucosal glycoproteins, life span, and cell proliferation rather than affecting the offensive acid-pepsin secretion (Goel, et al. 1986; Kumar, et al. 2013). This is in agreement with our work.


  Conclusion Top


Results from this study have shown that oral administration of flavonoid fraction of mature and unripe fruit extract of MP at a dose of 200 mg/kg and 400 mg/kg was effective in restoring the damaging effects of aspirin on the gastric mucosa. Furthermore, there was no recurrence of lesions after discontinuation of treatment.

There is a need for more information on the effects of the extract at higher concentrations, produce the active ingredient (s) or its synthetic analog in higher quantities and test the extract under other routes of administration and pharmaceutical formulations.[56]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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