Anti-ulcer activity of Cestrum nocturnum leaf in ethanol-and indomethacin-induced ulcer models

This study was planned to explore the antiulcer activity of the methanolic and n -hexane extracts of Cestrum nocturnum leaf against ethanol- and indomethacin-induced ulcer models in rats. The rats were administered orally vehicle in normal control group, methanolic and n -hexane extracts at doses 300, 500, 700 mg/kg, in treated groups and omeprazole 20 mg/kg, as standard drug. The gastric tissues/contents were examined to determine the ulcer index, antiulcer activity, gastric pH, gastric juice volume and acidity. Both extracts showed dose-dependent increase in antiulcer activity (%) in both ulcer models. Histopathology also supported these results. Gastric pH significantly increased while the gastric juice and acidity significantly decreased in the treatment groups of both ulcer models indicating the anti-secretory effect of extracts. It may be concluded that C. nocturnum protect gastric mucosa by decreasing gastric juice, acidity and increasing the gastric pH. Video Clip of Methodology : 6 min 58 sec: Full Screen Alternate


Introduction
In clinical practice peptic ulcer is the most common gastrointestinal disorder (Bafna and Balaraman, 2004).In such cases both synthetic and herbal medication are better and newer option for management and treatment of peptic ulcer disease (Dharmani et al., 2005).The patho-physiological causes of gastric ulcer were not completely defined but reasons behind ulcer are imbalance between the physiological and aggressive factors (pepsin and acid secretion) and gastric mucus membrane cytoprotective factors (bicarbonates and mucus secretion).Several endogenous factors of ulcer protection are also involved, which includes somatostatin, prostaglandins E2, sulfhydryl compounds and nitric oxide (de Sousa Araújo et al., 2008).Commercially available, antacids, H2 blockers, proton pump inhibitors are standard therapies for ulceration.But the use of these drugs is associated with number of side effects.WHO (1980) has suggested the research on natural products in those conditions where the use of synthetic drugs are not free from adverse effects.This recommendation creates a chance of discovering new therapeutic agents that are safe, less toxic and more efficacious in the treatment or prevention of ulcer from the community of medicinal plants.
Cestrum nocturnum belongs to solanaceae family with common name of "night blooming jasmine (Patil et al., 2011).It is perennial shrub and pollinated with the help of night flying insects and attracted by its fragrance (Sykorova et al., 2003).gastric ulcer.The present investigation was undertaken to provide scientific-based evidence to justify the folklore use as rational or irrational.Additionally the aim was to explore the cheap, easily available and having fewer side effects remedy for gastric ulcer.

Laboratory animals
Wistar albino rats between weights 150-200 g were purchased from University of Health Sciences-Lahore and accommodated at animal house of Faculty of Pharmaceutical Sciences Government College University Faisalabad, Pakistan.Rats were placed at the room temperature 22 ± 2°C and 12/12 period of light and dark with proper ventilation facility.The relative humidity 44 to 56% was maintained.Rats of different group were placed in different cages having wire bottom to avoid the coprophagy and to facilitate the accurate evaluation.Proceeding to the experimentation, for one week rats were acclimatized and given the standard diet and water ad libitum.The experiments were performed by the approval of Animal Ethics Committee of Government College University of Faisalabad, Pakistan.

Collection and identification of plant
Fresh leaves of C. nocturnum was collected from the botanical garden of University of Agriculture, Pakistan.The plant leaves were identified and authenticated by the taxonomist at the botany department of University of Agriculture, Pakistan.Leaves were separated, washed with water and dried under shade.Dried leaves were ground to fine powder.

Preparation of extract
C. nocturnum leaf extract was prepared by two methods: a) Maceration; b) Decoction.

Maceration
Leaves powder was soaked in methanol (1:4) for five days with vigorous shaking at regular time intervals.nhexane extract of leaves was prepared similarly.Extract was filtered with Whatman filter paper No. 1 and excess of solvent was removed with rotary evaporator.

Decoction
Freshly taken leaves of C. nocturnum (150 g) were boiled in one liter of water for the five minutes.Then decoction was allowed to cool for the 30 min and filtered through the Whatman filter paper No. 1.A fresh decoction was prepared after every three days.

Study design
Two ulcer models were used in this study which is as follows:

Ethanol-induced gastric ulcer model
The rats were separated into four groups, then group IV sub-divided into seven sub-groups, each of contain five animals and kept on fasting for the 24 hours with water ad libitum.Group I normal control received normal saline according to weight, Group II disease control, Group III standard control treaded with omeprazole (20 mg/kg, orally), Group IV (A, B, C ,D ,E, F and G) treated control administered methanolic and n-hexane extracts at doses 300, 500, 700 mg/kg, orally, decoction at dose (10 mL/kg, orally).After one hour dosing, ulcer was induced by administering ethanol (1 mL/kg, orally) to all groups except normal control.Animals were sacrificed by cervical dislocation, one hour after the administration of ethanol.Rats stomachs were removed, opened along the greater curvature, pinned on the soft board and stomach lesions were evaluated to determine the ulcer score.

Indomethacin-induce gastric ulcer model
The rats were separated into four group, then group IV sub-divided in seven sub-groups, each group contain five animals (n=5) and kept on fasting for 24 hours with water ad libitum before study.Group I normal control, Group II disease control, Group III standard control received omeprazole (20 mg/kg, orally), Group IV (A, B, C, D, E, F and G) treated with methanolic and nhexane extracts at doses 300, 500 and 700 mg/kg, orally respectively and decoction at dose (10 mL/kg, orally).After one hour dosing, ulcer was induced by administration of indomethacin (30 mg/kg, orally) to all animals except normal control animal.Animals were sacrificed by cervical dislocation, one hour after administration of indomethacin.Rat stomachs were removed, opened along the greater curvature, pinned on the soft board and stomach lesions were evaluated to determine the ulcer score.

Ulcer index calculation
The number of ulcers was counted by using the magnifying glass.Severity scores (Nwidu and Nwafor, 2009;Patidar, 2011)

Histopathological evaluation of gastric tissues
The wall of sacrificed rat stomach was fixed in 10% formalin and then paraffin blocks were made.Five micrometer thick section was cut from the paraffin block and stained with hematoxylin and eosin.Histopathological evaluations were carried out on the collected gastric tissue samples according to the method as reported elsewhere (Yu et al., 2015).

Statistical analysis
One-way ANOVA followed by Dunnett's posthoc by using GraphPad Prism version 5 was used for statistical analysis.Data were presented as mean ± SEM. p≤0.05 was taken as statistically significant value.

Results
Ulcer score, ulcer index and anti-ulcer activity (%) for ethanol-and indomethacin-induced ulcer models are given in Table I.In ethanol-induced ulcer models, there was dose-dependent increase in anti-ulcer activity in all treated groups.Methanol and n-hexane (700 mg/kg) showed 81% anti-ulcer activity which was higher than the standard value i.e. 75%.Decoction showed only 56% anti-ulcer activity.Indomethacininduced ulcer model also showed identical results.
Table II shows the effect of extracts on the gastric pH, acidity and juice.In both ethanol-and indomethacininduced ulcer models, both extracts at doses 300, 500, 700 mg/kg, orally, leaves decoction 10 mL/kg, orally and standard drug 20 mg/kg, orally significantly (p≤0.05)reduced the free acidity, total acidity and volume of gastric juice but significantly increased the pH of gastric volume in a dose-dependent manner when compared with the disease control group.

Histopathological evaluation
Histopathological assessment of the tissues of gastric mucosa of rats had also established the fact that Data represented as mean ± SEM; a p<0.05 when compared with disease control; ns = non-significant when compared with disease control Bangladesh J Pharmacol 2017; 12: 335-340 methanolic and n-hexane extracts of C. nocturnum leaves had shown significant anti-ulcer activity in a dose-dependent manner (Figure 1).

Discussion
Treatment groups in both ulcer models showed significant (p<0.05)anti-ulcer effect in methanol-and nhexane-treated groups which is comparable to the effect of standard drug while decoction showed less protecttion of mucosal and epithelial cells against corrosive action of ethanol and indomethacin in stomach.
Histopathological analysis of gastric mucosa of treated rats showed dose-dependent anti-ulcer effect of both extracts whereas decoction could not protect gastric mucosa to the level that extracts did.This might be due to chemical constituents that were extracted in methanol and n-hexane extracts.Banerjee and Firdous, (2015) studied Ipomoea staphylina hydroalcoholic extract anti-ulcer activity to find out new anti-ulcer drug with less adverse effects as compared to currently available ulcer treatment medicines.This plant extract showed significant decrease in free acicidity, total acidity, and ulcer index which was supported by histopathological analysis.Current results are in agreement with Banerjee and Firdous findings (Banerjee and Firdous;2015).Gul et al., (2015) explored anti-ulcer activity of Menthe longifolia and found decline in ulcer index (Gul et al., 2015).Dalbergia sissoo has been discovered to possess anti-ulcer activity by gastric mucosal protection (Gul et al., 2016).Our results are in accordance with the findings of studies conducted by Gul et al., 2015 are as follows:Ulcer score Ulcer index (UI) = 10 Anti-ulcer activity (%) calculation Bangladesh J Pharmacol 2017; 12: 335activity(%) /percentage protection is calculated by using following formula(Bhardwaj et al., 2012): Gastric volume, pH and acidity were determined by adoptingReignier et al., 2013, Delgado-Aros et al., 2002  and Segal et al., 1950 methods respectively.

Figure 1 :
Figure 1: Histological examination of gastric ulcers; A: Photomicrograph of rat stomach section from the control group showing the histological structure of the normal submucosa (SM), gastric gland (GG), serosa (S), muscularis externa (ME), outer longitudinal muscle (OLM); B: Standard group (omeprazole) is showing normal appearance of Gland cell and sub mucosal cell; C: Disease control (Ethanol induce ulcer) is indicating mucosal and sub mucosal ulceration along with loss epithelium cells in gastric mucosa; D: Disease control group (Indomethacin) is showing stomach of the ulcer control animal showing a severe effect on the submucosa with erosion, edema, moderate leucocyte infiltration and cellular debris; E: Methanolic extract 750 mg/kg + ethanol treated group is showing normal muscularis mucosa (MM), Submucosa (SM) and blood in mucosa cells; F: n-hexane 750 mg + ethanol treated group is indicating condensation of mucosa and clustering of RBC's in sub mucosal layer

Table II Effect of Cestrum nocturnum extracts on gastric juice pH, volume and acidity
and 2016.
a Data were represented as mean ± SEM; a p<0.05 when compared with disease control; ns = non-significant when compared with disease control