Research Articles

2016  |  Vol: 1(1)  |  Issue:

Anti-ulcerogenic effect of ethanolic extract of Morus alba fruits in chemically induced ulcer

M. Sajjad Khan¹*, Pushpendra Kumar Jain²

¹Monash University, Melbourne, 3052 Australia

²Vidyawati Pharmacy College, Jhansi, U.P., India

*Corresponding Author

M Sajjad Khan

Monash University, Melbourne, 3052 Australia

Abstract

Objective: Aim of present study was study antiulcer activity of ethanol extract of Morus alba fruits in rats. Materials and methods: Phytochemical screening of ethanol extract was performed to detect the presence of chemical constituents. Ethanol extract of M. alba fruits was selected for study with 200 mg/kg and 300 mg/kg bw doses on ethanol induced gastric ulcer. Effect of extract was observed by calculating ulcer index based on lesion index and pH. Results: Pytochemical screening of ethanol extract reveals the presence of flavonoids, tannins, carbohydrates, amino acid and proteins. Ethanol extract with 300 mg/kg dose produced significant inhibition on the ulcer lesion index in rats. Ethanol extract with 300 mg/kg dose significantly (p<0.01) change the gastric volume, ulcer index and pH. Conclusion: The results of the study indicated that significant inhibition of gastric lesions was found. The results of macroscopic observation also supported the antiulcer effect with antioxidant mechanism of flavonoids present in fruits extract.

Keywords: Morus alba, antiulcer, flavonoids, antioxidant, omeprazole

Introduction

Peptic ulcer disease is a chronic, multifactorial disease characterized by benign lesions of gastric or duodenal mucosa. Around 20% of global population is affected by ulcer during their lifetime (Levenstein, 2000). Peptic ulcer is a common gastrointestinal disorder bearing the several side effects (arrythmias, gynaecomastia and haematopoeitic changes) of modern medicine (Akhtar et al., 1992).  Traditional medicines possessing lesser side effects could be selected for better alternative treatment of peptic ulcer. Various studies have reported that alterations in antioxidant status following ulceration, indicating that free radicals to be associated with the pylorus ligation-induced (Rastogi et al., 1998) and ethanol-induced (Pihan et al., 1987; Mizui et al., 1987) ulcer. Some drugs with multiple mechanisms of protective action, including antioxidant properties, may be used to minimizing tissue injury (Barry, 1991).

M. alba (family: Moraceae) is commonly known as Mulberry, a large, deciduous trees native to India and subtropical regions of Asia, Africa, and the Americas. Mulberry fruits are delicious, fleshy, which are low in calories and contain health promoting phytonutrient compounds like polyphenols, minerals and vitamins that are essential for optimum health. It has long been used in diabetes, and as a general tonic to enhance health in traditional medicine. Mulberry extract has been reported to have potent antioxidant activity (Zhang et al., 2008; Yang et al., 2010), hypolipidemic effect (Liu et al., 2009; Chen et al., 2005), macrophage activating effect (Kim et al., 2013), and neuroprotective activity. Many of these bioactivities were linked to the presence of phenolics in mulberry fruit. Mulberry fruit contains high amounts of flavonoids, including anthocyanins that are responsible for its color, and other phenolics. Anthocyanins isolated from mulberry fruits has been reported to exhibit potent neuroprotective and antitumor activity (Huang et al., 2008; Jeong et al., 2010). In present study, Morus alba fruits were selected as promising alternatives used traditionally as medicine for wound healing activity.

Materials and Methods

Phytochemical studies

M. alba fruits were purchased from local market and identified before extraction of plant materials. The plant material was powdered and extracted with petroleum ether for defatting. The defatted materials were extracted with ethyl alcohol using a soxhlet apparatus at 70-80 ⁰C up to complete extraction (Lodhi et al., 2010). The ethanol extract was concentrated and dried under reduced pressure and yield was calculated. Extract was investigated for qualitative chemical test for different chemical constituents.

Ethanol induced ulcer

Wistar albino rats (180-200g) of either sex were selected for antiulcer activity. They were housed and divided into six groups containing each group of six animals in polypropylene cages, under standard laboratory conditions of temperature (25±2^oC). The animals were allowed to free access standard food (Brooke Bond-Lipton, India) and water. The animals were acclimatized for minimum 7 days before experiment. All experimental protocol was approved by the Institutional Animals Ethics Committee as per CPCSEA guidelines. The control group was given only vehicle (sodium carboxymethyl cellulose, 0.5 % p.o.), test group II and III were given ethanol extract with 200 and 300 mg/kg, p.o., respectively. Standard group IV was given standard Omeprazole (20 mg/kg, p.o.).

The ulcer was induced using 1 ml of 80 % ethyl alcohol administered orally to each animal. After 1 hr the rats were anesthetized and die by cervical dislocation. The macroscopic observation of stomach was recorded for ulcer scoring after removal of stomach. Whole stomachs were gently rinsed with water to remove the gastric contents and blood clots. Stomach was examined for lesions in the four stomach portion for indexed according to severity (Jhariya et al., 2015; Jain and Bhandarkar, 2016). Mean ulcer score for each animal was expressed as ulcer index.

Gasric contents from each stomach were collected and volume was measured. The pH of the gastric secretion was recorded and determines total acidity of the gastric juice by titration with 0.01 N NaOH and phenolphthalein as indicator. The total acidity is expressed as mEq/l using the following formula:

Total/free acidity = n×0.01×40×1000

Determination of antioxidants levels

The tissues were collected from stomachs were tested for antioxidants assay. Reduced glutathione (GSH) level was determined by the method of Moron (Moron et al., 1979). Superoxide dismutase (SOD) was assayed (Misra and Fridovich, 1972) based on the inhibition of epinephrine autoxidation by the enzyme. Catalase was estimated following the breakdown of hydrogen peroxide (Beers and Sizer, 1952). Skin homogenates were immediately precipitated with 0.1 ml of 25% TCA and removed after centrifugation. Free-SH groups were assayed in a total 3 ml volume by the addition of 2 ml of 0.6 mM DTNB and 0.9 ml 0.2 mM sodium phosphate buffer (pH 8.0) to 0.1 ml of the supernatant and the absorbance was read at 412 nm using UV spectrophotometer.

Statistical Analysis

Pharmacological data were represented as the mean ±S.D. for six rats. All data were statistically evaluated using the Tukey test. Values of P <0.05 were considered to be statistically significant.

Results and discussion

Phytochemical studies

Phytochemical screening reveals the presence of flavonoids, amino acids, proteins, carbohydrates and glycosides in ethanol extract. Practical yield of ethanol extract found 5.3 w/w%. The ethanol extract was reported safe dose up to a dose of 2000 mg/kg body weight. The extract dose of 200 and 300 mg/kg p.o. was selected for the gastroprotective study.

Ethanol induced ulcer

Administration of ethanol in higher doses resulted in the production of gastric mucosal damage. The ulcer index of control group was found 26.21±2.45. Ethanol extract at 300 mg/kg showed 12.62± 1.02 inhibition of the ulcer index which were significantly reduced. The reduction in ulcer index by alcoholic extracts at 200 mg/kg was 39.18%. Standard Omeprazole, showed anti-ulcer effect with 68.44% ulcer index inhibition (Table 1). Total gastric acidity of control group of animals was found 524.4±42.38. Ethanol extract at 300 mg/kg was showed significant decrease in total acidity 398.2±30.71 mEq/l, compared to control group (Table 2).

Table 1. Effect of ethanol extract of M. alba fruits on ulcer index in ethanol induced model

Treatment groups	Doses	Ulcer Index	% Inhibition
Control	5 ml/kg	26.21±2.45	-
Omeprazole	20mg/kg	8.27±0.52*	68.44± 3.28
EEMA	200 mg/kg	15.94± 1.37	39.18± 2.17
EEMA	300 mg/kg	12.62± 1.02*	51.85± 3.15

MAEE: Ethanol extract of M. alba; Values are mean ± SD; N = 6 in each group; ^*P < 0.01, when experimental groups compared with control 

Table 2. Effect of ethanol extract of M. alba fruits on ulcer parameters in ethanol induced model in rats

Experimental groups	Doses	Gastric volume (ml)	pH	Total acidity   (mEq/l)
Control	5 ml / kg	6.52±0.48	1.8±0.74	524.4±42.38
Omeprazole	20 mg/kg	3.74±0.35*	3.8±0.45*	253.8±21.53*
EEMA	200 mg/kg	4.35±0.54	3.2±0.35	328.6±28.62
EEMA	300mg/kg	4.86±0.65*	4.2±0.15*	398.2±30.71*

MAEE: Ethanol extract of M. alba; Values are mean ± SD; N = 6 in each group; ^*P < 0.01, when experimental groups compared with control 

Table 3. Effect of ethanol extract of M. alba fruits on antioxidants level in stomach tissues

Experimental groups	Doses	Antioxidants
		SOD	GPX	CAT
Control	5 ml / kg	11.87±0.57	18.62±0.82	10.92±0.29
Omeprazole	20 mg/kg	25.31±0.47*	31.28±1.62 *	22.30±1.05*
EEMA	200 mg/kg	17.29±0.29	19.62±0.85	15.62±0.56
EEMA	300mg/kg	22.60±0.95*	26.72±1.65*	19.67±1.76*

MAEE: Ethanol extract of M. alba; Values are mean ± SD; N = 6 in each group; ^*P < 0.01, when experimental groups compared with control 

The macroscopic observation of stomach showed that control group of animals found necrosis and hemorrhage in gastric lesions predominant over vast surface area. It showed perforations with complete mucosal destruction. The animals treated with dose of 300 mg/kg ethanol extract and standard drug were not found any necrosis and hemorrhage that reflects the protective effect of extract and drug.

The anti-ulcer activity was tested against gastric lesions induced by ethanol, the experimental models related to lesion pathogenesis with production of reactive species. Ethanol extract of M. alba found to increase the pH and decrease the gastric acid content and total acidity of gastric fluid. Alcohol rapidly penetrates the gastric mucosa which can cause cell membrane damage and leading to increased intra cellular membrane permeability to sodium and water. The enormous intracellular gathering of calcium represents a major step in the induction of gastric mucosal injury. This may support to cell death and exfoliation of epithelium surface (Raju, 2009). The antioxidant effects of M. alba fruits extract was already reported that can support for gastroprotective effect of M. alba. Some oxygen free radicals and causing agents have been implicated in the pathogenesis of ethanol-induced gastric ulcers (Shardul and Gangadhar, 2010).

Free radicals are generated during numerous normal metabolic processes and also required for normal functioning of living organism. A balance is necessary for better good health but imbalance produces several disorders in human body. Different antioxidant enzymes like superoxide dismutase (SOD), gluthathione peroxidase (GPX) and catalase (CAT) are present in different tissues and also control their accumulation (Fridovich, 1986). Any imbalance in the activity of these enzymes normally leads to faulty disposal of free radicals. These ROS are responsible for oxidation of tissues leading to lipid peroxidation and tissue damage. So, the antioxidants seemed to have protective role in gastric ulcers (Ito et al., 1996). The possible mechanism involves the antioxidant properties of flavonoids present in ethanol extract of M. alba, which produces protective effect against lesions induced by 50 % ethanol. This was probably by reducing the levels of lipoperoxides and increasing the activity of the antioxidant enzyme glutathione peroxidase. In conclusion the ethanol extract of M. alba protect the mucosa layer of stomach may be due to its flavonoid constituents. Thus, this finding confirms the traditional use of M. alba in the treatment of internal wounds as well as ulcer.

Conflict of interest

The authors report no conflicts of interest.

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