Antiulcerogenic activities of Agelanthus dodoneifolius leaf against acetyl salicylate and ethanol induced ulceration in rats

Antiulcer and antioxidant activities of crude methanol (800 and 1600 mg/kg bw), n-hexane and ethyl acetate fractions (800 mg/kg bw) of Agelanthus dodoneifolius were investigated against acetylsalicylate and ethanol induced ulcer in rats. The methanol fraction had the highest ulcer protective effect (73.33 ± 7.40%), better than the cimetidine (60.00±8.54 %) while the ethyl acetate fraction had the least (26.66 ± 6.66 %) in ethanol model, however, in acetyl salicylate model, crude extract exhibited the highest (54.16 ± 4.16 %) and the lowest (33.33 ± 4.16 %) protective effect at 1600 and 800 mg/kgbw respectively. The methanol faction preserved the integrity of the epithelial mucosal better than the other fractions. Treatment with crude and fractions of A. dodenofolius significantly (p<0.05) increased the activities of SOD and Catalase and decreases the MDA concentration when compared with ulcerative untreated rats in both ulcer models. In conclusion, A. dodoneifolius could serve as a sources of new plant based antiulcer agent.


Introduction
Ulcer disease is a condition that affects the skin around the lining of human body either externally or around its internal viscera: typically characterized by the presence of an open sore in the lining of epithelial cells or deep lesion in the body [1]. Ulcer disease is seen as one of the world's most common illnesses, which is a global health problem affecting the youths [2]. The major aggressive factors that cause ulcer are pepsin, gastric acid, non-steroidal anti-inflammation drugs (NSAID), bile salts, abnormal motility, alcohol and a bacterial infection known as Helicobacter pylori. Although, there are ample factors that could be protective in function which safeguard the stomach from ulcer formation and these include among others; bicarbonate production, mucus secretion, gastro-protective prostaglandin synthesis and normal tissue microcirculation [3]. The proposed strategies put in place for the prevention of peptic ulcer disease are those of attenuating gastric acid production and increasing gastric mucosal protection [4]. Evaluation of therapeutic potential of medicinal plant against gastric ulcer is attested by employing the use of ethanol to induce gastric injury [5]. Ethanol elicit its effects by causing intense lesions in the sub-mucosa by penetrating through the membrane [6], elevating Reactive Oxygen Species (ROS) formation, and reduces the mucus membrane formation [7] which can cause death of cells around the gastric mucosa. Ethanol inhibits endogenous prostaglandins formation and also suppresses cyclooxygenase enzyme activitiues [8].
Herbal medicine has witnessed widespread attention globally in recent years because of their use in the field of medicine for treating many diseases. Loranthaceae also known as is a parasitic plant growing on a large number of tree species such as Parkia biglobosa, Tamarindus indica, acacia, kola, citrus, orange and many other trees as a host plants. Presence of tannins, flavonoids, alkaloids and saponins were revealed in the methanol and chloroform extracts of the plant [9] Incidence of ulcer was discovered in about 87.4 million people worldwide in 2015 and about 10% of people develop peptic ulcer at some point in their life time. Peptic ulcer also accounted for the deaths of 267,500 people in 2015 alone [10]. It is known to be the most predominant of the gastrointestinal diseases with a global prevalence of around 4% of the population [11].
Liver toxicity also known as hepatotoxicity is a common disease resulting into serious debilities such as serious metabolic disorders to even death. Hepatotoxicity in often due to increase free radical. Free radicals produced by toxicant metabolism initiate what is termed toxicant cascade [12]. The free radical scavengers known to be endogenous in the body includes among others; catalase, super oxide dismutase, reduced glutathione, vitamin C and vitamin E, are present in the body. Cell necrosis is initiated by inhibited free radicals thus leading to hepatotoxicity [13]. Synthetic drugs have little to offer for alleviation of these disorders. Natural occurring antioxidants have better antioxidant activity from their natural source and this is why they are preferred to synthetic antioxidants [14]. The use of NSAIDs is crucial for the treatment of inflammatory diseases, it however presented some adverse effects such as gastric/ duodenal ulceration and renal failure which pose a serious threat in their medical use for treatment [15] The objectives of the present research were to evaluate the antiulcer activities of the methanol extract of the leaves of Agelanthus dodoneifolius using various animal models. Therefore, the present study was undertaken for the purpose of verifying its use by the locals in the treatment of ulcer.

Material and methods
The present work was designed to determine the anti-ulcer activities of the medicinal plant Agelanthus dodoneifolius. This was performed at the Department of Biochemistry, Federal University of Technology, Minna, Nigeria. In order to ascertain the anti-ulcer activities activities of Agelanthus dodoneifolius, the following steps were involved.

Animals
Wistar rats of about 180 ± 2 g weights were obtained, and are in the range of 6 weeks old from Animal House of the Federal University of Technology Minna were used for the experiments. The animals were conveniently housed under standard environmental conditions Temperature (27 ± 2) °C; 70% relative humidity; 12h daylight/night cycle) and had free access to commercial feed pellets and water.

Fractionation of the extract
Solubilization of the extract was done in methanol: water in 80:20 and the resulting solution was subjected to successive extractions in separating funnel with the solvents: n-hexane, ethyl-acetate (AcOEt) and methanol (MeOH). Three fractions were obtained from the extracts.

Antiulcer and Cytoprotective Studies
Three (3) animals for each group: Oral administration of drugs or saline was achieved at 9:00am daily for 8 days. Treatment was conducted for 8 days, the rats to be experimented were kept fasting for 36 hours in separate cages with raise wide mesh wire bottom to ensure complete emptying of the stomach and to prevent coprophagia (eating their faeces) before subjecting them to ulcerogen, and allowed water ad-libitum Same design was used for ethanol model using ethanol.

Ethanol (EtOH) and aspirin (ASP) ulcer induction
The Ethanol induced ulcer was carried in rats by administering Ethanol as described by Hollander et al. [16] and Aspirin Induced ulcer was carried out as described by Rajkapoor et al. [17].

Induced ulcers
Aspirin was administered orally on the day of experiment at about 10 AM with the help of an orogastric tube in the form of an aqueous water suspension (200 mg/kg, per oral) and animals were sacrificed after 4 hours of administration. The stomach was incised along with the greater curvature and examined for ulcers as described earlier.

Biochemical parameters
A 50 dilution of the stomach homogenate was prepared and the activities of superoxide dismutase and catalase were evaluated as described by Ekanem & Yusuf, [18]. The levels of Malonaldehyde as an index of lipid peroxidation was estimated as described previously [19] while method described by [20] was used for total proteins estimation. Histological examination was conducted following the procedure of [21].

Statistical analysis
Data obtained in this study were analysed using the statistical software SPSS 16.0, 2016. Numerical data were presented as Mean ± SD. The significance of the mean difference between two independent groups was determined using Student's t-test and one-way analysis of variance (ANOVA) while multiple comparisons were used when comparing more than two groups. A p-value<0.05 was considered significant.

Results and discussion
The methanol fraction had the highest ulcer protective effect (73.33 ± 7.40%), better than the cimetidine (60.00±8.54 %) while the ethyl acetate fraction had the least (26.66±6.66 %) in ethanol model, however, in acetylsalicylate model, crude extract exhibited the highest (54.16±4.16 %) and the lowest (33.33±4.16 %) protective effect at 1600 and 800 mg/kg bw respectively. The methanol faction preserved the integrity of the epithelial mucosal better than the other fractions. Treatment with crude and fractions of A. dodenofolius significantly (p<0.05) increased the activities of SOD and Catalase and decreases the MDA concentration when compared with ulcerative untreated rats in both ulcer models.
Methanol crude extracts and fractions of A. dodoneifolius significantly (p<0.05) decrease the Malonaldehyde (MDA) content, compare to the control group of both models. An increase in MDA content is correlated to an increase in tissue damage. The control group of both the acetylsalicylate model and the ethanol model were elevated compared to the treated groups which had significantly (p<0.05) reduced MDA content. The MDA is the functional product of lipid peroxidation and it is used to determine lipid peroxidation levels [22]. Increasing lipid peroxidation production is an important cause of NSAID's damaging effects on stomach tissue [22]. Earlier studies have shown that gastric damage caused an increased MDA level [23] The result indicates marked increase in the catalase level when administered with methanol crude extract and fractions for the two models (ethanol and acetylsalicylate) as compared to the control. Catalase involvement in gastric injury caused by ethanol is seen when catalase functions in the conversion of hydrogen peroxide to water. Catalase activity is indicative of oxidative stress and significantly (p<0.05) increased upon induction by the ethanol and acetylsalicylate models [24]. These findings were in accordance with the main experimental studies which showed that catalase activity in stomach tissue increased gastric damage [25] In intact rat stomach tissue superoxide dismutase (SOD) activity was significantly (p<0.05) lower than that of the control rats in the ethanol model and there were no significant (p<0.05) difference in SOD content of the acetylsalicylate model. SOD activity is least in damage tissues and it is highest in healthy tissues [26] The results revealed that the mean ulcer score of the two models (acetylsalicylate and ethanol) in the untreated groups were 8.00 ± 0.52 and 5.00 ± 0.57 respectively. The mean ulcer scores indicated the severity of the ulcer induction and the acetylsalicylate has more ulceration as compared with the ethanol model. . The higher mean ulcer score of the acetylsalicylate indicated higher epithelial damage than the ethanol model during ulceration and this findings can be compared to the earlier studies [27]. The methanol fraction had the highest ulcer protective effect (73.33 ± 7.40%), better than the cimetidine (60.00±8.54 %) while the ethyl acetate fraction had the least (26.66±6.66 %) in ethanol model, however, in acetylsalicylate model, crude extract exhibited the highest (54.16±4.16 %) and the lowest (33.33±4.16 %) protective effect at 1600 and 800 mg/kg bw respectively.
The histological findings revealed that the methanol faction preserved the integrity of the epithelial mucosal better than the other fractions. The result may not be unconnected with the earlier report of the acetylsalicylate damaging more epithelium, hence low level of protection as compared to the ethanol model in this study. The negative control without treatment shows discontinuity in mucosal epithelium in acetylsalicylate model of rat ulceration. A standard drug (cimetidine) of 50g/kg bw showed no ulcer formation and a similar result was gotten from the treated group when a crude extract of A. dodoneifolius with a dose of 1600mg/kg bw was administered.

Conclusion
The study showed that the crude extract and fractions (methanol, n-haxane and ethylacetate) of Agelanthus dodoneifolius have significant anti-ulcer activity with high safety margin. The crude methanolic extract of the leaves and its various fractions possess significant phytochemicals and antioxidant activities demonstrates potent anti-ulcer activities on the Wister rats. The evidence obtained supported the use of the plant for treatment of ailment as traditional herb by the natives, particularly Africans. Therefore, the crude extract and fractions of Agelanthus dodoneifolius are potential sources of new plant based antiulcer agent.

Statement of ethical approval
The principles governing the use of laboratory animals as laid out by the Federal University of Technology, Minna Committee on Ethics for Medical and Scientific Research and also existing internationally accepted principles for laboratory animal use and care as contained in the Canadian Council on Animal Care Guidelines and Protocol Review were duly observed.