Tropical Journal of Natural Product Research

Phytochemical evaluation The screening for phytoconstituents of the plant leaf extract was carried out according to the methods of Harbone 12 and Trease and Evans. 13 Quantitative analysis was carried out as described by Harbone 12 and Soni and Sosa. 14


Introduction
Peptic ulcer disease (PUD) has been referred to as the collapse of the stomach and/or duodenum epithelial mucosal barrier, characterized by inflammation and ulcer formation. 1 Helicobacter pylori infection, use of non-steroidal anti-inflammatory drugs (NSAIDs), lifestyle factors such as diet, smoking, and alcohol use are some of the causes of peptic ulcer disease. 2 NSAID-induced ulcer is regarded as the second major cause of ulcer after H. pylori. NSAIDs such as aspirin, indomethacin and ibuprofen are known to inhibit prostaglandin synthesis through cyclooxygenase pathway. 3 Several plants have been reported to be effective in the treatment of peptic ulcer. Some of these plants include Hibiscus asper, 4 Eremurus spectabilis, 5 Cnidoscolus aconitifolius. 6 Serafino et al. 7 reported that many plant extracts are as effective as the synthetic drugs with no or insignificant adverse effect. Acalypha torta is one of the several useful plants in folk medicine. According to Ezekwesili and Nwodo. 8 Acalypha torta leaves have been useful in the treatment of high blood pressure in Nigeria folklore medicine. Irobi and Banso 9 reported the antimicrobial activity of Acalypha torta leaves against Staphylococcus aureus, Serratia marcesens, Yersinia spp, Bacillus spp, Trichophyton rubrum, and T. mentagrophytes while Ezekwesili and Nwodo 8 opined that the ethanolic leaf extract of the plant has demonstrated antidiarrhoeal, antithrombotic and immunosuppressive activities.
Acalypha torta is one of the plants used in the treatment of various diseases in traditional medicine. The plant is known to possess antidiarrhoeal, immunomodulatory and antimicrobial activities. Despite the ethnomedicinal uses of this plant, there is a paucity of studies on its gastroprotective activity. However, this study was designed to investigate the gastro-protective properties of Acalypha torta leaf extract. The gastro-protective effect of Acalypha torta leaf methanol extract was evaluated using HCl/ethanol ulcer-induction model. A total of twenty-four (24) animals were randomly grouped into six (6) treatment groups labelled groups 1-6. Groups 1 and 2 received 1% carboxymethyl cellulose (CMC). Group 3 received 30 mg/kg b.w lansoprazole while groups 4-6 received 200, 400 and 600 mg/kg b.w extract, respectively. The present findings revealed that methanol extract of Acalypha torta leaf exerted gastro-protective activity, as shown by its consistent and significant dose-dependent increase in mean ulcer index (MUI) inhibition. Histological investigation of gastric lesions showed that the plant stimulates the scarring cicatrizing process. The gastro protection observed could be attributed to the phytoconstituents of the plant extract which exerted gastro-protective and anti-ulcerogenic effects, possibly by increasing antioxidant enzymes thereby reducing oxidative stress. The gastro-protective properties of the extract could also be due to the ability of the extract to ISSN 2616-0692 (Electronic) 300 © 2020 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License Animals Male Wistar rats of weight range 150 -180 g, used for this study were obtained from the Department of Zoology and Environmental Biology, University of Nigeria, Nsukka.

Experimental design
After acclimatization, the animals were randomly distributed into six (6) groups having four (4) animals each. The extract and standard drug were administered for one week. The route of administration of the extract and standard drug was oral using oral intubation tube. The rats were handled according to the guidelines of the National Institute of Health on the case and use of laboratory animals (NIH, 1985). The research was approved by the Ethical Committee of Faculty of Biological Sciences, University of Nigeria Nsukka. Group 1 served as the normal Control (1% CMC only) while Group 2 was the untreated group. Group 3 received 30 mg/kg b.w. lansoprazole while Groups 4, 5 and 6 received the graded doses of the extract (200, 400 and 600 mg/kg b.w, respectively).

Induction of ulcer using HCl/Ethanol model
After 24 hours of fasting, the graded doses of the extract of Acalypha torta leaf (200, 400 and 600 mg/kg b.w.) and 30 mg/kg b.w. lansoprazole were administered. One hour later, the ulcer induction was done according to a modified method of LiraMota et al. 15 using 0.8 mL of 5% 0.3 M HCl/60% ethanol in groups 2-6. The rats were monitored for one hour and sacrificed, and the stomachs removed and opened along the greater curvature to remove gastric content. The gastric contents were weighed using a graduated cylinder while the pH was determined by the aid of a digital pH meter (HI 9021). The ulcers were viewed and counted with the aid of a magnifying hand lens (×10) and the ulcerative lesion index was calculated based on the following keys: Mildly ulcered = 1, moderately ulcered = 2, severely ulcered = 3. The ulcer preventive/inhibition index was calculated using the formula prescribed by Onwukwe et al. 16 Ulcer protective index (%) =

Determination of gastric parameters
Gastric juice volume was determined according to the method of Kiranmai et al. 17 The collected gastric content was centrifuged at 3000 rpm for 10 min, then separated and the volume measured using a graduated cylinder.

Histological studies
The stomachs of the sacrificed rats were excised and immersed in 10% formalin solution. The fixed specimens were trimmed, washed and dehydrated in ascending grades of alcohol. The Specimens were further cleared in xylol, embedded in paraffin, sectioned at 4-6 microns thickness and stained with heamtoxylin and eosin for examination as described by Drury et al. 18

Statistical analysis
The results obtained from the study were statistically analysed using One-way ANOVA, and the Duncan multiple test range was used to compare means. The results were expressed as mean ± SD. The analysis was carried out using the IBM SPSS statistical package version 20.

Results and Discussion
Phytochemical evaluation of ethanol extract of Acalypha torta leaf The phytochemical profiling of the extract indicated a relative abundance of steroids, flavonoids, alkaloids, saponins, cardiac glycosides and tannins as shown in Table 1. This agrees with the report of Irobi and Banso 9 on the phytochemical composition of the leaf of Acalypha torta where steroids, tannins, saponins and cardiac glycosides were detected. However, alkaloids and flavonoids were absent in the reports of Irobi and Banso. 9 According to Sumbul et al., 19 flavonoids act in the gastrointestinal tract exerting anti-spasmodic, anti-secretory, anti-diarrheal, anti-ulcer and antioxidant activities. In addition, flavonoids offer protection against a variety of ulcerogenic agents through several mechanisms of action which include free radical scavenging and antioxidant properties, increased mucus secretion, anti-secretory action, and inhibition of Helicobacter pylori proliferation in the gastric mucosa. 19 Also, de Jesus et al. 20 reported that tannins act as ulcer preventive agents resulting from their protein precipitating effects. The astringent action of tannins has also been reported to precipitate microproteins on the ulcer site, thus forming an impervious layer over the lining, which inhibits ulceration. 19

Effect of ethanol extract of Acalypha torta leaf on ulcer parameters
The results of the effect of Acalypha torta leaf extract on gastric juice volume, pH, ulcer index and ulcer protective index are shown in Table  2. Administration of HCl/Ethanol to experimental rats produced a significant increase (p < 0.05) in gastric juice volume as observed in the group treated with HCl/Ethanol only (2.83 ± 0.06 mL), when compared to the normal control (1.17 ± 0.13 mL). Pre-treatment with graded doses of the extract exerted a significant (p < 0.05) dosedependent decrease in gastric volume from 1.77 ± 0.04 to 1.35 ± 0.03 mL as the doses increased from 200 to 600 mg/kg b.w. of extract when compared to the ulcer group. Also, there was an improvement in the pH of the intestinal content of the groups that received the graded doses of the extract compared to the untreated group. The results of ulcer index revealed a significant (p < 0.05) decrease when the groups that received graded doses of the extract and standard drug (lansoprazole) were compared to the untreated group. There was also a significant (p < 0.05) increase in the ulcer protective index of normal control and groups that received the graded doses of the extract and standard drug were compared to the untreated group. Scanlon and Sanders 21 reported that the increase in gastric volume of treatment groups compared to the normal control could be due to the HCl used in ulcer induction. According to Ezeasiliji et al., 22 ethanol induces ulceration by releasing superoxide anions and hydroperoxyl free radicals which leads to an increased lipid peroxidation of lipids in the membranes of stomach mucosa as a result of ethanol metabolism. The result of this study is consistent with the findings of LiraMota et al., 15 who reported that oral administration of HCl/ethanol causes necrotizing lesions in the gastric mucosa. The lesions which are described by the ulcer index are caused by loss of mucous layer and increase of acid secretion, hence an increase in gastric volume leading to ulcer occurrence. 24,25 The gastro-protective effect of the extract could be attributed to its phytoconstituents such as flavonoids, which have anti-secretory effect, 19 inhibiting secretion of HCl by the parietal cells of the gastric mucosa and hence reducing the volume of gastric content and increasing the pH of the gastric content. The standard drug, lansoprazole is a proton-pump inhibitor and acts by inhibiting gastric acid secretion by blocking the H + /K + -adenosine triphosphate enzyme system (the proton pump) of the gastric parietal cells. 25 It is effective in alleviating ulcer, but in this study, the graded doses of the extract indicated a better ulcer protective effect as shown in Table 2.

Histological examination of gastric tissues
There was no mucosal damage in the normal control as shown in Figure 1A. The histology revealed an intact gastric wall (muscularis mucosa) (green colour), submucosa (red colour) and serosa (black colour) with no lesions to the gastric mucosa observed. The histological examination of the gastric tissues of the animals indicated severe and extensive mucosal damage in the animals that were induced but not treated ( Figure 1B) while there was a progressive ameliorative effect in the groups that received 200 and 400 mg/kg b.w extract as shown in Figures 2A-B and 3A. The animals that received 600 mg/kg b.w extract exerted the most ulcer protective effect characterized by an intact muscularis mucosa, granulated tissue, hyperplasia of epithelial cells (black arrow) and large aggregates of lymphocytes and fibrous scar (blue arrow) (H&E ×100) as shown in Figure 3B.

Conclusion
The present findings have shown that Acalypha torta leaf ethanol extract possesses gastro-protective activity, as shown by its significant and dose-dependent increase in ulcer protective index against gastric mucosa damage induced by HCl/ethanol induction. The antiulcerogenic effects of the extract could be attributed to its phytoconstituents.

Conflict of interest
The authors declare no conflicting interest Values are mean ± SD, (n = 4). Values in the same column having different superscripts differ significantly (p < 0.05).