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ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 1  |  Page : 12-16

Macroscopic and microscopic assessment of gastroprotective effects of methanolic leaf extracts of Cissampelos owariensis (P. Beauv.) in rats using pyloric ligation method


Department of Anatomy, College of Health Sciences, Igbinedion University, Okada, Edo State, Nigeria

Date of Submission06-Jun-2019
Date of Decision09-May-2020
Date of Acceptance10-May-2020
Date of Web Publication20-May-2021

Correspondence Address:
Dr. Dayo Rotimi Omotoso
Department of Anatomy, College of Health Sciences, Igbinedion University, Okada, Edo State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_17_19

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  Abstract 


Background: Cissampelos owariensis is a tropical medicinal plant widely applied for diverse therapeutic uses such as treatment of circulatory, reproductive, and gastrointestinal conditions. In this study, the gastroprotective activity of methanolic leaf extracts of C. owariensis against prolonged exposure to acidic gastric acid was assessed in male Wistar rats. Materials and Methods: This study involved 25 male Wistar rats (180–200 g) divided into five groups (n = 5): Group A–E. Groups A and B were used as normal and test controls given distilled water, whereas Groups C–E were, respectively, administered with methanolic extracts of C. owariensis at dosage of 100 mg/kg, 300 mg/kg, and 500 mg/kg, respectively. The mode of administration was oral, and the study period was 21 days. Afterward, gastric mucosal injury was induced in Groups B–E animals through pyloric ligation method. Macroscopic and microscopic examinations of gastric tissues were done to ascertain the degrees of gastric mucosal protection or erosion using gross photographic and histological staining techniques. Results: The gross appearance of internal aspect of gastric tissues showed mildly eroded mucosal surface in treated Groups B–D, but intense erosion was observed in test control Group D. Similarly, for histological results, the treated Groups B–D showed moderate-to-mild mucosal surface erosion compared to intense erosion observed in test control Group E. Conclusion: Findings from this study indicated that prior treatment with methanolic extracts of C. owariensis resulted in gastric mucosal protection in experimental animals exposed to offensive factor such as gastric acid. This gastroprotective activity of methanolic extracts of C. owariensis may be associated with the antioxidant properties of the constituent phytochemical compounds.

Keywords: Cissampelos owariensis, gastroprotection, Wistar rats


How to cite this article:
Omotoso DR, Lawal OS, Olatomide OD, Okojie IG. Macroscopic and microscopic assessment of gastroprotective effects of methanolic leaf extracts of Cissampelos owariensis (P. Beauv.) in rats using pyloric ligation method. Niger J Exp Clin Biosci 2021;9:12-6

How to cite this URL:
Omotoso DR, Lawal OS, Olatomide OD, Okojie IG. Macroscopic and microscopic assessment of gastroprotective effects of methanolic leaf extracts of Cissampelos owariensis (P. Beauv.) in rats using pyloric ligation method. Niger J Exp Clin Biosci [serial online] 2021 [cited 2021 Jun 14];9:12-6. Available from: https://www.njecbonline.org/text.asp?2021/9/1/12/316524




  Introduction Top


Medicinal plants refer to plants that have parts such as seeds, leaves, barks, roots, stem, fruits, or the whole plant with constituent phytochemicals that can be applied for therapeutic purposes in order to ameliorate pains and cure diseases. These plants represent an important component of natural plant biodiversity, especially in many African countries where they constitute a vital source of health care for many individuals due to the relative accessibility, affordability, efficacy, and tolerability of medicinal plant products.[1],[2],[3] One of such medicinal plants is Cissampelos owariensis P. Beauvais ex DC. (also called lungwort) which belongs to the Menispermaceae family that comprises about 70 genera and 450 species. C. owariensis is a twiner plant found in tropical regions, especially in the wild of some sub-Saharan African countries.[4] Different parts of the plant (especially leaf and root) are used by traditional medical practitioners for various therapeutic applications. Extracts derived from C. owariensis have been applied in the treatment of metrorrhagia, wounds, snake bites, circulatory and reproductive diseases, amnesia, and psychosis;[5],[6] prevention of miscarriage and treatment of sterility;[7],[8],[9] and treatment of various gastrointestinal conditions such as dysentery, diarrhea, enteritis, and colic and intestinal worms.[10],[11] They also exhibited diverse biological activities such as insecticidal activity, antimicrobial and antiviral activity, and antibacterial and antifungal activity.[12],[13] Previous study had reported potent antioxidant activity of methanol extract of C. owariensis which was especially linked to the flavonoid component of its phytochemicals.[14] These phytochemical flavonoids are plant-derived phenols that exhibit free radical scavenging, anti-allergic, and anti-inflammatory activity and help to prevent diseases including gastric ulcers.[15],[16],[17] However, there is still a conspicuous shortfall in the availability of documented pharmacological studies on C. owariensis. Hence the need for this present study in which the gastric mucosal protective activity of methanolic leaf extracts of C. owariensis was investigated against erosive effect of acidic gastric juice after prolonged exposure.


  Materials and Methods Top


Plant material

Fresh whole C. owariensis plant was harvested from the Okada community, Ovia North-East Local Government Area, Edo State, Nigeria. The plant was verified at the Department of Biological Sciences, Igbinedion University, Okada, Edo State, Nigeria.

Method of extraction

The leaves of the plant were detached, dried, and pulverized into powdered form using mechanical grinder. 700 g of powdered leaves was infused in 5 l of methanol for 72 h. Thereafter, the preparation was filtered, the filtrate was evaporated using rotary evaporator (regulated at 40°C), and the residue obtained was cooled (at room temperature), weighed, and used as methanolic extracts for the study.

Experimental animals

This study involved 25 adult male Wistar rats weighing between 170 g and 200 g. The animals were divided into five groups – control Groups A and E and test Groups B–D. Each group comprises five animals (i.e., n = 5). Group A animals were given distilled water (5 ml/kg body weight), and they represented normal control animals that were not treated and not induced by pyloric ligation. Group B animals were given 100 mg/kg methanolic extracts of C. owariensis. Group C animals were given 300 mg/kg methanolic extracts of C. owariensis. Group D animals were given 500 mg/kg methanolic extracts of C. owariensis. Group E animals were given distilled water (5 ml/kg body weight), and they represented test control animals that were not treated but induced by pyloric ligation.

Period and mode of study

The treatment period of this study was 28 consecutive days, and all treatments were done orally using a flexible orogastric gavage.

Induction of gastric mucosal injury using pyloric ligation method

The animals were fasted for 24 h in separate cages but allow free access to water. Animals were anesthetized by intraperitoneal injection of ketamine/xylazine (50 mg/kg at 1:1). A small midline incision was made on the abdomen of animals to access pyloric part of the stomach. The pyloric end of the stomach was gently pulled up, ligated, and gently returned into the abdominal cavity, and the abdomen was closed. After an observatory period of 5 h, the animals were sacrificed and stomach tissues were harvested and prepared for macroscopic and microscopic examination.[18]

Ethical approval

This study was duly approved by the research and ethics committee of the university, and all procedures employed in this study conformed to standard guidelines for experimental animal handling.

Macroscopic examination

After the study period, the harvested stomach tissues of experimental animals were cut open along the greater curvature and the internal aspect of the stomach tissues was photographed, using a 20-megapixel digital camera, to document the gross appearance of the gastric mucosa and observable gastric mucosal erosion following the prolonged exposure to acidic gastric juice during pyloric ligation method.

Tissue processing

The stomach tissues of experimental animals were fixed in 10% neutral buffered formalin, dehydrated using ascending grades of alcohol (two changes each of 70% and 90% and absolute alcohol for 30 min each), cleared in xylene for 30 min, and embedded in molten paraffin and allowed to cool to form tissue blocks.

Sectioning

Blocks of tissue were cut into sections 5 μ thickness using rotary microtome and mounted on microscope slides made ready for histological staining.

Histological staining using hematoxylin and eosin staining technique

Tissue sections were dewaxed in xylene for 15 min, hydrated by treating with decreasing grades of alcohol (i.e., absolute alcohol, 90% alcohol, and 70% alcohol in succession) for 3 min each, stained in hematoxylin for 10 min, washed in running tap water for 3 min, and differentiated in 1% acid alcohol (prepared as 1% HCl in 70% alcohol) for 1 min. Then, sections were blued by washing in alkaline running tap water (Scott's tap water) for 10–20 min, rinsed in water, and stained in 1% aqueous eosin for 3 min. Sections were rinsed in water (to wash off excess stain), dehydrated using increasing grades of alcohol (i.e., 70%, 90%, and absolute alcohol in succession) for 2 min each, cleared in xylene for 2 min, and mounted in DPX. After staining, the tissue sections were allowed to dry and made ready for microscopic examination.


  Results Top


Macroscopic results

The gross appearance of the internal aspect of gastric tissues of experimental animals [Figure 1] showed normal gastric mucosa in the normal control Group A, varying degrees of mucosal surface protection in treated Groups B–D, and prominent mucosal surface erosion in test control Group E after exposure to the acidic gastric secretions during pyloric ligation method.
Figure 1: Macroscopic appearances of the internal aspect of gastric tissues of experimental animals (Groups A–E). Group A animals represented normal control animals, Group B, C, and D animals were given 100 mg/kg, 300 mg/kg, and 500 mg/kg methanolic extracts of Cissampelos owariensis, respectively, and Group E animals represented test control animals

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Microscopic results

This histological examination of the gastric tissues of experimental animals [Figure 2] showed normal histological architecture of the gastric mucosa of treated animals in normal control Group A, moderate mucosal surface erosion in treated Groups B and C, mild mucosal surface erosion in treated Group D, and intense mucosal surface erosion (as well as significant mucosal distortions) in test control Group E following exposure to acidic gastric secretions during pyloric ligation method.
Figure 2: Histological presentations of the gastric tissue of experimental animals (Groups A–E) (H and E, ×100). Group A animals represented normal control animals, Group B, C, and D animals were given 100 mg/kg, 300 mg/kg, and 500 mg/kg methanolic extracts of Cissampelos owariensis, respectively, and Group E animals represented test control animals. Arrows indicated mucosal surface erosion

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  Discussion Top


The gastric mucosa is constantly vulnerable due to exposure to different substances which may alter the structural integrity of the mucosa causing injury and ulceration, especially when gastric mucosal defensive factors are overwhelmed. These defensive factors (such as mucin, bicarbonate, nitric oxide, prostaglandins, and others) help to mitigate against erosive effect of offensive factors (such as gastric acid, pepsin, stress, Helicobacter pylori, and others), thereby preventing mucosal injury and ulcerations.[19],[20] Hence, a potent antiulcerogenic or gastroprotective agent or drug functions by counteracting aggressive factors or stimulating protective factors.[21] – In this study, the pretreatment of gastric mucosa of the experimental animals with methanolic extracts of C. owariensis conferred on it certain degrees of protection against the erosive effect of exposure to acidic gastric secretion during the pyloric ligation. According to the result of the macroscopic and microscopic examinations of gastric mucosa of experimental animals [Figure 1], the treated Groups B–D compared relatively with the mucosal morphology of the normal control Group A. The treated Groups B–D also showed very insignificant mucosal surface erosion when compared to the intensely eroded mucosal surface of test control Group E. The observed gastroprotective activity of methanolic extracts of C. owariensis in this study may be linked to its constituent bioactive phytochemical compounds. In general, the constituent phytochemical compounds, especially its high-value, low-volume secondary metabolites, confer on medicinal plants and or preparations the medicinal properties applied in ameliorating pains and treatment of diseases, hence, called active principles or bioactive components.[22],[23],[24] Previous studies have posited that flavonoids derived from medicinal plants exhibit a significant antioxidant effect and protect against toxic effects of reactive oxygen species due to their hydroxyl groups.[25],[26] These phytochemical flavonoids have been described to exhibit gastroprotective functions due to their ability to promote gastric mucus synthesis (by mucous cells), reduce gastric acid secretion (by parietal cells), and downregulate pepsinogen synthesis (by chief cells).[27],[28],[29] From the result of this study, the gastric mucosa of experimental animals exposed to methanolic leaf extracts of C. owariensis tends to exhibit some degrees of gastric mucosal protection against erosive effects of offensive factors (acidic gastric secretions). This is more prominent in higher dosage level of extracts (which invariably contain more flavonoids compounds). Hence, the gastroprotective activity of methanolic leaf extracts of C. owariensis (like other pharmacological activities of the plant extracts) can be described as a function of its constituent phytochemical compounds.

Recommendation

Based on the outcome of this study, methanolic leaf extracts of C. owariensis can be applied as a potent gastroprotective agent for therapeutic uses. However, further studies may be necessary to ascertain this using other extractive media (aqueous or other organic solvents) and other experimental models of induction of gastric mucosal injury or ulceration.


  Conclusion Top


This study affirmed the gastroprotective activity of methanolic leaf extracts of C. owariensis (P. Beauv.), and the extracts can be applied as a potent gastroprotective agent for therapeutic uses.

Acknowledgments

We acknowledge the contributions of Mr. Adebiyi, Central Animal House, Igbinedion University, Okada, and Mrs. Okoro, Histopathology Laboratory, University of Benin Teaching Hospital, Benin City, Edo State, Nigeria, toward the successful completion of this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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