Indian Journal of Research in Homeopathy

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 10  |  Issue : 2  |  Page : 40--46

Gastroprotective effects of aqueous extract of Hibiscus sabdariffa calyx on nonsteroidal anti-inflammatory drug-induced gastric ulcer in wistar rats


NK Sani1, C Onwuchekwa1, U Mohammed2, MB Abubakar3,  
1 Department of Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria
2 Department of Morbid Anatomy and Forensic Medicine, Faculty of Basic Clinical Sciences, College of Health Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria
3 Department of Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, Usmanu Danfodiyo University; Center for Advanced Medical Research and Training, Usmanu Danfodiyo University Sokoto, Nigeria

Correspondence Address:
Dr. N K Sani
Department of Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, Usmanu Danfodiyo University, PMB 2346, Sokoto
Nigeria

Abstract

Background: Gastric ulcer is the most common gastrointestinal disorder associated with high morbidity and significant mortality and has been described as the plaque of the 19th and 20th centuries. The most conventional antiulcer drugs are largely ineffective and associated with disease recurrence, thus identifying natural products with potential antiulcer properties is essential. Objective: The aim of this study was to investigate the potential protective effects of aqueous extract of Hibiscus sabdariffa calyx (AEHSC) against nonsteroidal anti-inflammatory drug (NSAID)-induced gastric ulcer. Materials and Methods: Thirty male rats weighing 170–230 g were randomized into five groups as: ulcerated control group (distilled water, n = 6), experimental extract groups 1, 2, and 3 (AEHSC 100 mg/kg, 200 mg/kg, and 400 mg/kg, respectively, n = 6 each), and positive control group (ranitidine 20 mg/kg, n = 6); ulcer was induced using oral indomethacin 40 mg/kg, 30 min after all interventions. Rats were sacrificed 5 h after ulcer induction. Blood samples were taken for serum hydroxyproline determination; gastric tissues were also visualized (ulcer index (UI) and percentage of ulcer protection), and later stained and viewed under a microscope. Results: There was a significant (P < 0.05) reduction in UI in treated rats compared with control. The percentage of ulcer protection was also significantly increased (P < 0.05). Histological slides also indicated increased epithelial mucus secretion in stomach tissues of rat treated with AEHSC. Conclusions: AEHSC may be potentially beneficial in protection against the development of NSAID-induced gastric ulcers.



How to cite this article:
Sani N K, Onwuchekwa C, Mohammed U, Abubakar M B. Gastroprotective effects of aqueous extract of Hibiscus sabdariffa calyx on nonsteroidal anti-inflammatory drug-induced gastric ulcer in wistar rats.Niger J Exp Clin Biosci 2022;10:40-46


How to cite this URL:
Sani N K, Onwuchekwa C, Mohammed U, Abubakar M B. Gastroprotective effects of aqueous extract of Hibiscus sabdariffa calyx on nonsteroidal anti-inflammatory drug-induced gastric ulcer in wistar rats. Niger J Exp Clin Biosci [serial online] 2022 [cited 2022 Dec 10 ];10:40-46
Available from: https://www.njecbonline.org/text.asp?2022/10/2/40/359778


Full Text



 Introduction



Peptic ulcer disease (PUD) is an acid-induced lesion of the gastrointestinal tract that usually occurs in the stomach, proximal duodenum, or lower esophagus, and is characterized by denuded mucosa with the fibrin-covered defect extending into the submucosa or muscularis propria.[1],[2] PUD is one of the most common gastrointestinal disorders with a worldwide annual incidence of 0.1%–0.3% and a prevalence of 5%–10%.[2] In the United States of America, PUD is responsible for an annual health-care burden of approximately $10 million,[3] whereas in Nigeria, the national direct cost implication is about 186 billion naira (≈$958 million).[4] The leading causes of PUD are nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori infection.[4],[5]

When PUD occurs in the stomach, it is referred to as a gastric ulcer, which has the highest prevalence among all gastrointestinal disorders and causes about 15 deaths in every 15, 000 complicated cases annually worldwide.[6] Under normal circumstances, the gastric mucosa is protected by factors such as gastric mucus, prostaglandins, and bicarbonates against ulceration by gastric acid, pepsin, bile salts, H. pylori infection, and NSAIDs. Gastric ulcer develops when aggressive factors overcome the protective mechanisms (Chai, 2011) and NSAIDs are among the leading causes.[5] NSAIDs remain one of the most commonly prescribed medications as more than 30 million people take them every day worldwide.[7],[8] More than 90% of all ulcers are caused by NSAIDs and nearly 25% of people taking NSAIDs to develop PUD.[9]

NSAIDs are a group of pharmacologic agents used for symptomatic relief of pain, fever, and inflammation.[10],[11] They bring about gastrointestinal injury through their local and systemic effects including cyclooxygenase enzyme inhibition in the metabolic pathway of arachidonic acid, leading to decreased synthesis of prostaglandins E2 and I2 which are particularly cytoprotective to the gastrointestinal mucosa and their deficiency could result into gastric mucosal injury.[10],[12]

Several antiulcer drugs have been associated with several adverse effects and disease recurrence, raising the need for the search for natural products with potential antiulcer properties.[13],[14] Hibiscus sabdariffa is one of the plant products that have been used in traditional medicine for the treatment of PUD.[15] It is a herbal shrub indigenous to tropical Africa but also cultivated in almost all warm countries.[16] Phytochemical analyses have revealed that HS contains biologically active substances such as flavonoids (mainly anthocyanins), tannins, phenols, glycosides, saponins, and alkaloids which have different curative properties (Nnamonu et al., Ines Da-Costa-Rocha, 2014, and Riaz and Chopra, 2018). Furthermore, HS was also reported to possess healing properties in ocular mucous membrane and cutaneous ulcers.[17],[18] Studies have shown that substances from plant sources such as polyphenols, tocopherols, alkaloids, and flavonoids, many of which are contained in HS, offer gastroprotection as well as healing potentials against PUD.[19] The aim of this study was to investigate the potential protective effects of aqueous extract of H. sabdariffa calyx (AEHSC) against NSAID-induced gastric ulcer.

 Materials and Methods



Experimental animals

A total of 30 male Wistar strain albino rats weighing 170–230 g were obtained from the animal house of a Nigerian university. The rats were maintained on a standard balanced rat feed (grower mash) and had free access to water. They were acclimatized to the animal room conditions for at least 2 weeks before the commencement of the study. These animals were kept in standard rat cages with commercial wood pellets (sawdust) as bedding, in well-ventilated animal rooms with natural light. The experimental protocol used in this study was approved by the research ethics committee of our institution.

Plant material

Dried HS calyces were obtained from a local market and were identified as HS Linn at the department of pharmacognosy and ethnopharmacy, faculty of pharmaceutical sciences of our university, with a sample kept at the herbarium.

Extraction of plant material

Impurities and other particulate matter were first removed from the calyces by handpicking. The calyces were then washed with tap water to remove dust and then dried at room temperature. The dried calyces were then grounded to a fine powder with the aid of a mortar and pestle. Using a 2000 ml flat bottom flask as a container, 135 g of the powder was mixed with 500 ml of distilled water and allowed to stay for 24 h, and the mixture was then stirred thoroughly and filtered using Whatman filter paper. The filtrate obtained was then dried using an oven dryer at 37°C. This yielded the AEHSC.[19],[20] The extract was later dissolved again in distilled water and administered orally to the animals according to their body weights.

Experimental design

This is an experimental study. The animals were fasted for 24 h before the commencement of the experiment but were given free access to drinking water.[21] A total of 30 rats were randomly divided into five groups (n = 6). All interventions were given as a suspension in 2 ml of distilled water and administered by oral gavage. The rats were grouped and treated as follows:[19],[20],[22],[23]

Ulcerated control group (UCG): Administered with single doses of distilled water 0.5 ml per ratPositive control group (PCG): Administered with single doses of ranitidine 20 mg/kg per ratExperimental extract group 1 (EEG-1): Administered with single doses of AEHSC 100 mg/kg per ratEEG 2: Administered with single doses of AEHSC 200 mg/kg per rat.EEG 3: Administered with single doses of AEHSC 400 mg/kg per rat.[19],[20],[22],[23]

Ulcer induction

Induction of ulcer was carried out 30 min after administration of all interventions, according to the method described by Azuumi et al.,[24] with slight modifications, using indomethacin 40 mg/kg orally to each rat.[13] Five hours after ulcer induction, the rats were sacrificed using chloroform inhalation.

Blood sampling and measurement of serum hydroxyproline

Immediately after sacrifice, blood samples were taken from all animals through cardiac puncture using 5 ml syringes and 23G needles. The samples were then transferred into plain blood collection tubes and allowed to coagulate, after which it was centrifuged at ×3000 g for 20 min.[25] The serum was then separated and kept at a temperature of −4°C for the determination of serum hydroxyproline.

Determination of serum hydroxyproline was carried out by the enzyme-linked immunosorbent assay method. Serum samples (50 μL each) from 30 rats were added to the 96-well antibody-coated plate and mixed with 50 μL of horseradish peroxidase conjugate antibody and incubated for 40 min at 37°C. It was then rinsed a number of times with diluted wash buffer and the substrate (3,3′,5,5′-tetramethylbenzidine, TMB); 90 μL was added to the plate and incubated for 20 min at 37°C in the dark. The reaction was ended by adding 50 μL stop solution to each well. The absorbance was measured at 450 nm within 5 min.

Measurement of ulcer index and percentage of ulcer protection

The rats were dissected, their stomachs removed and cut along the greater curvatures, and washed with normal saline to remove remnants of gastric contents.[19] The gastric tissues were then spread open over rectangular pieces of soft foam with their edges held with the aid of office pins. The stomachs were examined under a magnifying glass with ×5 for mucosal ulceration.[26] The total lengths of all ulcers in the gastric mucosa of each animal were designated as the ulcer index (UI) for that animal. The percentage of ulcer protection (gastroprotection percentage) was also calculated using the formula:

% gastroprotection/Inhibition = (UIC–UIT)/UIC × 100

Where UIC is the UI of the control group, and UIT is the UI in the test groups.[27]

Tissue fixation and histology

After visualization, the gastric tissues were fixed with 10% formaldehyde and kept in plastic containers. The tissues were then dehydrated using graded alcohol and xylene and then subsequently embedded in paraffin blocks. The tissue blocks were then sectioned at 5 μm widths, affixed onto glass slides, deparaffinized and stained with hematoxylin and eosin (H & E) and then finally examined under the microscope.[14],[28]

Statistical analyses

Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 26 (IBM company, Armonk, New York, USA). Differences among groups were determined by a one-way analysis of variance followed by Tukey's multiple comparison tests with P < 0.05 considered statistically significant. Results are expressed as mean ± S. E. M.

 Results



Effect of aqueous extract of Hibiscus sabdariffa calyx on the gross appearance of gastric mucosa

The gross appearance of representative gastric tissues from all rat groups is shown in [Figure 1]. Rats that received only distilled water (UCG) had extensive ulcerations. However, there was a dose-related reduction in gastric ulceration in rat groups pretreated with AEHSC in comparison to the UCG. Among the extract-administered groups, gastric ulceration was least observed in the group that received 400 mg/kg AEHSC, followed by the group pretreated with 200 mg AEHSC, and the highest ulceration was seen in the group that received 100 mg/kg AEHSC. The rat group pretreated with ranitidine 20 mg/kg (PCG) showed an almost ulcer-free mucosa (Photo Plate 1E) in comparison to the UCG.{Figure 1}

Effect of aqueous extract of Hibiscus sabdariffa calyx on the ulcer index

[Figure 2] shows the UI in all experimental groups of rats. A dose-dependent reduction in the UI with an increasing dose of extract was also observed. The reduction was statistically significant in the EEG-2 and EEG-3 and PCG when compared with the UCG. Similarly, the UI in EEG-3 and PCG were significantly low when compared with EEG-1.{Figure 2}

Effect of aqueous extract of Hibiscus sabdariffa calyx on percentage of ulcer protection

[Table 1] shows the percentage of ulcer protection in the interventional groups of rats. There was a dose-dependent increase in the ulcer protection percentage in groups that received AEHSC. However, the highest percentage of ulcer protection was seen in the PCG which received ranitidine 20 mg/kg.{Table 1}

Serum hydroxyproline levels

[Figure 3] shows the serum hydroxyproline levels of the experimental groups. High serum hydroxyproline levels were seen in the PCG and EEG-3. However, there was a statistically significant reduction in serum hydroxyproline levels in the UCG, EEG-1, and EEG-2 in comparison with PCG.{Figure 3}

Histologic appearance of gastric tissues

Histologic appearance of representative gastric tissues from groups of rats showed that pretreatment with AEHSC appeared to cause a dose-dependent reduction in ulceration as well as an increase in epithelial mucus secretion. As shown in [Figure 4], there was mucosal ulceration in groups that received distilled water or low-to-moderate doses of AEHSC, i.e. UCG, EEG-1, and EEG-2, but no ulceration was observed in PCG and EEG-3 (groups that received ranitidine and high doses of AEHSC, respectively). In Photo Plate 2A, which represents the UCG, mucosal discontinuity was observed as indicated by the yellow line. In Photo Plate 2B, which represents the PCG, an intact ulcer-free mucosa was observed. In [Figure 4]c which represents EEG-1, mucosal ulceration was also observed and spans the length of the yellow line. In [Figure 4]d, which is representative of EEG-2, mucosal ulceration was observed as indicated by the black arrow. There was also an increased mucus secretion in adjacent epithelial cells (red arrow). Finally, in Photo [Figure 4]e, which represents EEG-3, an intact ulcer-free mucosa with increased epithelial mucus secretion was observed.{Figure 4}

 Discussion



Effect of AEHSC on the ulcer index

It was observed that AEHSC was associated with a significant reduction in the UI of treated rats compared to the control. This implies that AEHSC might have offered significant protection against the development of NSAID-induced gastric ulcer in the treated rats. This is in agreement with previous findings[29] in which a significant reduction in UI was seen as a result of the use of hydro-ethanolic extract of HS on indomethacin-induced gastric ulcer. Eddy et al.,[30] demonstrated that ethanolic extract of HS calyx has the ability to physically adhere to a mild steel surface, so the AEHSC used in the present study might have also adhered to the gastric epithelial surface and served as a physical barrier protecting the mucosa from the ulcerative effect of the NSAID, just the same way as gastric wall mucus protects the mucosa from the acidic gastric content.[10] NSAIDs are known to cause damage to the gastric mucosal cells through different mechanisms including the release of free radicals which cause injury to the gastric mucosa that results in ulcer.[31] Interestingly, it has been established that extracts of both leaves and flowers of HS such as polyphenols, tocopherols, alkaloids, and flavonoids are known to possess high antioxidant properties.[32] Therefore, the AEHSC used in the present study might have protected the gastric mucosa from a free radical injury that might have been caused by the NSAID indomethacin. It has also been shown by Mohamed et al.,[33] that aqueous calyx extract of HS prevents oxidative stress as it lowers MDA levels in red blood cell membranes of diabetic rats, compared with control. Yusof et al.,[34] also reported a significant reduction in MDA and advanced oxidation protein product as well as increased levels of reduced glutathione in diabetic rats treated with polyphenol-rich HS extract. Therefore, the presence of antioxidants such as polyphenols, flavonoids, and other phytochemicals in the AEHSC could have prevented oxidative damage to the mucosal membrane due to free radicals released by NSAIDs and therefore preventing the mucosa from ulceration.

Effect of aqueous extract of Hibiscus sabdariffa calyx on the percentage of ulcer protection and gastric tissue histology

In the present study, it was also found that AEHSC provided a dose-dependent protection in ulcer percentage. At high doses, AEHSC provided almost the same ulcer protection as the conventional antiulcer drug used. This agrees with the findings of Nyam et al.,[35] in a study using seed oil and seed extracts of both H. sabdariffa and Hibiscus cannabinus on different ulcer models. Furthermore, as demonstrated by Alqasoumi, Al-dosari[28] that ethanolic extract of HS reduces gastric acid secretion and increases the production of gastric wall mucus; AEHSC in the present study might have prevented the development of NSAID-induced gastric ulcer by the similar mechanisms because increased mucus secretion was observed in histologic slides of gastric tissues of rats treated with AEHSC compared with control. It has long been established that gastric acid secretion is a strong predisposing factor to the development of gastric ulcer and gastric wall mucus serves as a vital protective factor that shields the gastric mucosa from acid injury.[10]

Perhaps AEHSC also prevented local damage to the gastric mucosal cells caused by NSAIDs by preventing their direct cytotoxic action on epithelial cells as demonstrated by Lee et al.[36] on the beneficial effects of HS polyphenol extract on streptozotocin-induced diabetic nephropathy.

Serum hydroxyproline levels

Hydroxyproline (trans-4-hydroxy-l-proline) is a derivative of posttranslational hydroxylation of the nonessential amino acid proline and a major component of collagen that serves as a key stabilizing factor of the collagen triple helix. Hydroxyproline can be used as an indicator of the collagen content of a given tissue (https://pubchem.ncbi.nlm.nih.gov). It has been demonstrated that gastric ulceration is associated with a decrease in gastric collagen (and therefore hydroxyproline) content as a result of an increase in collagenase enzyme activity and this can be monitored in the serum and reflects the induction of gastric ulcer regardless of cause.[37] Decreases in hydroxyproline levels in both serum and stomach extracts of rats induced with gastric ulcer using NSAIDs have also been reported Takeuchi et al.,[38] In the present study, serum hydroxyproline was found to be significantly decreased in rats that had significant gastric ulceration. This is in agreement with previous reports of low serum hydroxyproline levels in ulcerated control rats compared with pretreated groups.[39] It was also observed in the present study that serum hydroxyproline levels remained high in rat groups that did not develop significant gastric ulceration and this agrees with previous findings of Takeuchi et al.,[40] and may be a result of the prevention of gastric ulceration and breakdown of gastric mucosal collagen by ranitidine and AEHSC in pretreated rats.

 Conclusions



The present study indicates that AEHSC may be beneficial in protecting against NSAID-induced gastric ulcer as it may promote gastric mucus secretion as well as protect from local mucosal injury from NSAIDs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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