1. Introduction

Peptic ulcer disease (PUD) is a problem of the gastrointestinal tract characterized by a break in the normal gastric mucosa integrity secondary to gastric acid and pepsin secretion[1]. It represents a serious and growing health problem in the whole world[2]. It has a worldwide prevalence of about 40% in the developed countries and 80% in the developing countries [3]. It is the most prevalent gastrointestinal disorder ever known, accounting for an estimated 15 mortalities out of every 15,000 complications yearly in the world. The disease develops as a result of altered balance between offensive and defensive factors. The major offensive factors are gastric acid secretion, pepsin secretion, non-steroidal anti-inflammatory drugs (NSAIDs), Helicobacter pylori (H. pylori) infection and bile salts. Defensive factors include the mucosal barrier, mucus secretion, prostaglandin, bicarbonate, blood flow, cellular regeneration, epidermal growth factors and motility[4, 5]. Stomach colonization by the bacterium H. pylori and indiscriminate use of NSAIDs are the two most prominent causes of PUD [6]. The most common symptoms of PUD include episodic epigastric pain or gnawing.  During the last two decades, the use of plants for the prevention, treatment and/or management of PUD has been advocated. This is due to several reasons, namely, orthodox drugs provoke many adverse effects, perceived effectiveness, affordability, ease of accessibility and safety of medicinal plants. Moreover, a large percentage of the world’s population does not have access to conventional pharmacological treatment [7]. Lannea acida is a small deciduous shrub or tree belonging to the family Anacardiaceae [8]. It is a valuable multi-purpose tree widely used by local people. The bark is used internally in the treatment of stomach troubles, beriberi, schistosomiasis and haemorrhoids (Burkill, 2004). The powdered root, mixed with salt, is made into a tampon for application to the scrotum in treating orchitis [9]. The kernel is purgative, applied externally, it is used to treat eye-troubles. The root bark is considered good for treating skin-infections. The leaves and bark have been reported to be useful in the treatment and management of gout, rheumatism, wounds, swelling and burns [9]. Lannea acida (A. Rich) is noted for being effective in the treatment of different ulcer cases among the Hausa and Fulani in Sokoto State, North Western, Nigeria [10]. To the best of our knowledge, a scientific investigation on the antiulcerogenic effect of this plant based on its folkloric use has not been evaluated. Therefore, the present study is designed to evaluate the antiulcerogenic effect of stem bark extract and solvent fractions of L. acida on ethanol induced mucosal injury in albino rats.

2.      Materials and Experimental Methods

2.1    Chemicals and Reagents

Solvents for extraction and partitioning of the plant materials and all other chemicals were of analytical grades.

2.2    Experimental Animals

Albino rats weighing between 150-180g of both sexes were used for the study. The animals were obtained from the animal house of Department of Pharmacology and Toxicology, Usmanu Danfodiyo University, Sokoto. The rats were housed in clean and disinfected plastic cages and were allowed to acclimatize for fourteen (14) days. The rats were fed with a standard rat chow and allowed to drink water ad libitum. All experiments were carried out in accordance with the WHO guidelines for the use of experimental animals [11].

2.3    Plant collection and identification

The fresh stem bark of Lannea acida was collected from Zuru town in Kebbi State, Nigeria. The plant was authenticated by a taxonomist from Botany Unit in Biological Science Department, Usmanu Danfodiyo University Sokoto where a voucher specimen has been deposited.

2.4    Preparation of Plant Extracts

The preparation of extract was carried out according to the method of Hassan et al. [12]. Briefly, the stem bark of the plant collected was cleaned, air dried at room temperature and then cut into small pieces. Two hundred grams (200g) of it was macerated using 2L of 95% methanol for 48hrs and was filtered with Whatman sized 1 filter paper. The filtrate was concentrated in a rotary evaporator at 45ºC. The dried extract was kept in a dried clean air tight container until used.

2.5    Fractionation of the extract

This was carried out according to the method of Hassan et al. [12]. Exactly about fifty grams (50g) of the crude methanol stem bark residue was subjected to fractionation by solubilisation in water in a separatory funnel and sequential partition with hexane (3×200 mL), ethyl acetate (3×200 mL) and saturated butanol (3×200 mL). Each fraction was evaporated to dryness and subjected to antiulcerogenic activity.

2.6    Antiulcer Studies

2.6.1 Gastric Ulcer Induction by Ethanol

The induction of gastric mucosal damage was carried out by the method of Almasaudi et al. [13]. Albino rats of either sex were divided into six (6) groups of five (5) animals each as follows: Group E1 (Control): rats in this group received distilled water only, Group E2 (Cimetidine): rats in this group received cimetidine at a dose of 100mg/kg for seven days before the induction of ulcer, Group E3 (Positive Control): rats in this group received ethanol at a dose of 1ml/200g on the seventh day only, Group E4, E5 and E6: rats in this group received the plant extract at a dose of 100, 200 and 300mg/kg respectively for 7 days before the induction of ulcer.

Following 24 hours of food deprivation on the seventh day, the standard drug and test substances were administered. One hour after the pretreatment, the animals were administered ethanol at a dose of 1ml/200g body weight by intragastric gavage.  Sixty minutes later the rats were sacrificed. Immediately after sacrificing the animals, the stomachs were removed, cut along the greater curvature and the mucosa was washed with 0.9% saline solution to clean away the blood. This was followed by macroscopic examination of the stomach for the detection of any hemorrhagic lesions on the glandular mucosa. 

2.6.2 Measurement of Ulcer Score

The ulcer score was determined by scoring of severity of mucosal lesions as reported by Almasaudi et al. [13]. This was done as follows: no ulcer (0), small ulcer (1-2 mm) (1), medium ulcer (3-4 mm) (2), large ulcer (5-6 mm) (4) and huge ulcer (>6 mm) (8).

2.6.3 Measurement of Ulcer Index

Measurement of ulcer index was carried out as reported by Ugwah et al. [14] and Wasagu and Shehu [15]. The average of the length in mm of all lesions for each stomach was measured to determine the mean ulcer index (UI).

Where; UN = Average of number of ulcer per animal, US = Average of severity score

UP = Percentage of animal with ulcer

2.6.4 Measurement of Percentage Inhibition

The percentage inhibition was calculated using the method of Mahmood et al. [16].

2.6.5 Histological Evaluation of Gastric Lesions

For histopathological examination, the tissues were fixed in 10% formalin solution. Then, the formalin-fixed stomach specimens were embedded in paraffin wax and serially sectioned (3–5 𝜇m) and further stained with hematoxylin and eosin. The stained tissues were observed for pathological changes using light microscopy [13].

2.7    Antiulcer Activity of Solvent fractions

The solvent fractions were tested for antiulcer activity using the ethanol model described earlier. Albino rats of either sex were divided into seven (7) groups of five (5) animals each as follows: SF I: rats in this group received distilled water only, SF II: rats in this group received cimetidine at a dose of 100mg/kg for seven days before the induction of ulcer, SF III: rats in this group received ethanol at a dose of 1ml/200g on the seventh day only, SF IV-VII: rats in this group received hexane, ethylacetate, butanol and last remaining aqueous fraction at a dose of 300mg/kg for 7 days before the induction of ulcer. Induction of ulcer and measurement of ulcer score, ulcer index, percentage inhibition and histological examination of gastric tissues were carried out as described above.

2.8    Mechanism of Antiulcer Activity of the Most Potent Solvent Fraction (MPSF)

A frozen portion of the stomachs was thawed. Thawed tissues were homogenized in 50Mm potassium phosphate, pH 7.5 and 1Mm EDTA.

Antioxidant Activity of Gastric Homogenate

Catalase activity,  reduced glutathione, malondialdehyde (MDA),   superoxide dismutase activity, Vitamin A, Vitamin C and Vitamin E were estimated by the method which is described in previous papers [17-24].

2.9    Evaluation of the Involvement of K_ATP Channels

To study the possible involvement of K_ATP channel in the antiulcer activity of the MPSF; a separate experiment was conducted. Albino rats of either sex were divided into six (6) groups designated control, ethanol, ethanol and EtyAc, ethanol and glibenclamide, Ethanol+EtyAc+Glibenclamide, and Ethanol+Drug. The treatment groups were pretreated with the EtyAc fraction. Thirty minutes after, the animals were administered with glibenclamide a blocker of K_ATP channels (6 mg/kg). After another 30 mins, all animals received absolute ethanol (1ml/200g) for the ulcer induction. Sixty minutes after the administration of ethanol, the rats were sacrificed, and their stomachs removed for examination, as previously described.

2.10  Data Analysis

All data was reported as means ± standard error of mean (SEM). The values were analyzed using Statistical Package for Social Sciences (SPSS) windows program version 20. Statistical significance of difference between means was carried out using one-way analysis of variance (ANOVA). p

3.      Results

3.1    Anti-Ulcerogenic Studies of the Crude Extract

Oral administration of absolute ethanol produced mucosal lesions in the albino rats (Table 1). The gastric mucosal lesions produced in the ulcer control group; marked by elevated ulcer index were very extensive. Pretreatment with 100, 200 and 300 mg/kg of the methanol solvent extact, demonstrated significant reductions in ulcer index (p<0.05) in a dose dependent manner when compared to the ethanol ulcer control group. The standard drug; cimetidine (100mg/kg) showed less mucosal injury with significant (p<0.05) reductions in ulcer index. The reductions in ulcer index produced by the extract at 200 and 300mg/kg was comparable with that of the standard drug. Although, the extract at 100mg/kg showed significant reductions in ulcer index, the values were far less when compared with the reference drug.

This result was confirmed after histological examination as shown in Fig. 1A-F. In the ethanol ulcer control group, there were severe disruptions, erosion and vacoulation of the stomach glands with infiltration of inflammatory cells (Fig. 1B).

Table 1: Effect of Different Doses of Methanol Stem bark Extract of Lannea acida and Cimetidine on Ethanol-Induced Ulcer in Rats.

Values are expressed as Mean± SEM (n=4).

^*p**pLannea acida.

The rats pretreated with 100, 200 and 300 mg/kg showed markedly better reductions in gastric lesion evident by moderate sloughing of epithelian infiltration and vacoulation, severe fibrous connective tissue formation suggestive of healing process and normal glandular stomach (Fig. 1D-F). The drug control group demonstrated significantly better mucosal protection with less mucosal damage and massive fibrous connective tissue formation (Fig. 1C).

3.2    Anti-Ulcerogenic Activity of Solvent Fractions

The solvent fractions (Hexane, Ethylacetate, Saturated Butanol and Last Remaining Aqueous Fraction) significantly mitigated the development of gastric lesions and significantly (p<0.05) decreased ulcer index when compared to the ethanol ulcer control group (Table 2). EtyAc fraction was the most potent solvent; accomplishing similar efficacy to the standard drug. Least reduction in ulcer index was observed in the hexane fraction treated group. Rats administered with ethanol showed marked increase in ulcer index when compared to the normal control group.

 Fig. 1- NORMAL Control. Normal Glandular Stomach (palte A), Ethanol Ulcer Control showing severe disruption, erosion and vacoulation of the stomach glands with infiltration of inflammatory cells (palte B), Cimetidine Control showing Massive Fibrous Connective Tissue Formation with cellular infiltration and vascular congestion (palte C), 100mg/kg of MSELA. Showing congestion, moderate sloughing of epithelium and vacoulation (H&E stain, 20x magnification) (palte D), 200mg/kg of MSELA with Massive fibrous connective tissue formation, infiltration of inflammatory exudattes and congested blood vessels (palte E), 300mg/kg of MSELA showing Normal glandular stomach (palte F).

Fig. 2- NORMAL Control. Normal Glandular Stomach (palte A), Ethanol Ulcer Control showing severe disruption and erosion of the gastric pits (palte B), Cimetidine Control showing normal glandular stomach (palte C), HEX Treated group showing normal glandular stomach with severe vascular congestion in the submucosal surface (palte D), EtyAc Treated group showing near normal glandular stomach (palte E), BuOH Treated group showing normal glandular stomach with mild vascular congestion (palte F), LRAF Treated group showing connective tissue formation suggesting healing process (palte G),

Table 2: Effect of Different Solvent Fractions of MSELA and Cimetidine on Ethanol-Induced Ulcer in Rats.

Values are expressed as Mean± SEM (n=4).

 ^*p

 ^**p

 HEX: Hexane Fraction, EtyAc: Ethylacetate Fraction, BuOH: Saturated Butanol Fraction and LRAF: Last Remaining Aqueous Fraction

The findings of histopathological analysis of the solvent fractions is summarised in Fig. 2A-G. The mucosa layer of the stomach of the control rats showed normal histology with intact epithelial lining and gastric pits (Fig. 2A). The rats treated with ethanol showed severe disruption and erosion of the gastric pits (Fig. 2B). The rats treated with cimetidine showed no ulceration evident by normal glandular stomach (Fig 2C). Near normal stomach mucosa to normal glandular stomach with massive formation of connective tissue suggestive of healing were observed in the rats treated with HEX, EtyAc, BuOH and LRAF (Fig 2D-G).

3.3    Mechanism of Action

3.3.1. In-vivo Antioxidant Activity

In an attempt to investigate the possible mechanism involved in the antiulcerogenic activity, the level of antioxidant enzymes; GSH, SOD, CAT, and MDA; an index of lipid peroxidation were evaluated. The results showed that rats administered with ethanol had marked significant (p<0.05) reductions in the level of GSH, activities of SOD, CAT, and an increased MDA compared to normal. Pretreatment with the EtyAc fraction significantly increased the level of GSH, SOD and CAT activities. It also significantly decreased the level of MDA when compared to the ethanol induced ulcer control group (Tables 3). Similarly, rats pretreated with the standard drug had significantly (p<0.05) increased level of GSH, SOD and CAT activities and decreased levels of MDA. The increased level of GSH, SOD and CAT activities and decrease in the level of MDA observed in the EtyAc pretreated groups were comparable with that of the normal and the standard drug control group.

3.4    Investigation of the Involvement of K_ATP Channels in the Antiulcerogenic Activity of the EtyAc fraction

The result of involvement of K_ATP channels in the antiulcerogenic activity of the EtyAc fraction is shown in Fig. 3. Ethanol induced marked ulcerative lesions as observed earlier.

Table 3: Effect of the EtyAc Fraction of Lannea acida on in-vivo antioxidants in Ethanol Induced Ulcer

Values are expressed as Mean± SEM (n=4). Mean having different Superscriptin a column are significantly different (p<0.05) (One-Way ANOVA followed by Dunnett’s Multiple Comparison Test Using SPSS Version 20).  EtyAc: Ethylacetate Fraction.