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Study on antioxidant and antimicrobial activities of the Pistacia lentiscus and Glycyrrhiza glabra extracts on healing the gastric ulcer in rats.
Waheed A. Filimban
Department of pathology, Faculty of Medicine, Umm al Qura University, Makkah, Saudi Arabia.
Corresponding Author: Waheed A. Filimban, Department of pathology, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia, P.O. Box: 715. Email:
[email protected],
[email protected] Mobil: 00966566810828
Abstract: The present study aimed to evaluate the aqueous extract of Pistacia lentiscus leaves and Glycyrrhiza glabra roots as a possible treatment for aspirin induce ulcer in rats. A total thirty six adult male albino rats were used in this study. Rats were divided into 6 equal groups. Group (GP.1) was used as a negative control and fed on the control ration only. The GP. (2-6) oral treated with aspirin at a dose of 200 mg/ Kg bw, to induce gastric ulcer. GP.2 was left as a control +ve (ulcerated rats), whereas the GPs.3-4
were
administrated by aqueous extracts of Pistacia lentiscus a dose of groups200 and 400mg/kg. bw respectively. GPs. 5-6 were treated with aqueous extracts of Glycyrrhiza glabra a dose of groups 200 and 400mg/kg bw respectively. The treatment period was 7 consecutive days.
Results revealed that oral
administration of the aqueous extract of Pistacia lentiscus and Glycyrrhiza glabra showed decreases the length of gastric ulcer, significantly decreased the volume of gastric juice, and pH value, in addition to, increased curative ratio when compared to the GP.2 (control +ve). In addition to decreased lipid 1
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peroxidation and enhancement antioxidant system, reduced glutathione (GSH), Superoxide dismutase (SOD) and total antioxidant capacity (TAC). Oral administration of aqueous plant extracts decreased histopathological changes in the stomach layers and mucosa, especially with Glycyrrhiza glabra roots and Pistacia lentiscus with doses of 400mg/kg Bw. In conclusion, the aqueous extract of Pistacia lentiscus and Glycyrrhiza glabra have promise effect for healing gastric ulcer disease.
Key words:
Gastric ulcer - Pistacia lentiscus, Glycyrrhiza glabra,
Antioxidant, Antimicrobial, Histopathological.
1. Introduction: The peptic ulcer is an acute or chronic and recurrent disease, of the gastrointestinal diseases (Guyton, 2000). Gastric ulcer is among the most serious diseases in the world. The peptic ulcer disease affects about 10% of the population It is a major health problem. The development of gastric ulcer occurs with acid and the breakdown of mucosal defense (Goroll, and 2009). Peptic ulcer is the general term applied to a sore on the lining of the gastrointestinal tract (Bethesda 2010). It can be classified mainly into four types they are gastric, duodenal, esophageal and Meckel's Diverticulum ulcers (Goroll, and 2009). Gastric ulcer is a peptic ulcer that develops in the stomach. . Gastric ulcers are less common than duodenal ulcers and more patient’s age of both sexes. It occurs usually along the lesser curvature of the stomach and its size vary from millimeters to 2
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centimeters. However, gastric ulcers are more predisposed to develop into malignant disease (Alpers et al, 2002).
The main causes of peptic ulcer are infected with the bacterium called Helicobacter pylori (H. pylori) and the use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) such as aspirin (Goroll, and 2009). The causes of gastric ulcer disease include physical and psychogenic factors that involved an imbalance between the gastric acid and pepsin secretions and the degree of tissue resistance to the secretions. In ulcer, gastric acid formation, large amounts of hydrochloric acid and the strong gastric enzyme pepsin are secreted. These substances are essential for food digestion, but at the same time have the ability to erode the cell linings of the digestive system if secreted in excess amount (Mohamed, et al, 2011). Various topical irritants interfere with normal function of this tissue, including aspirin, alcohol, certain drugs, caffeine or bile acids that may come in contact with the mucosa (Mahan, and Stump, 2008)
The traditional medicine using herbal plants is prevalent among rural population in many developing countries. Medicinal plants contain many active constituents which are used for treatment of various diseased conditions. These constituents represent a natural source of drugs that have greater safety or lesser toxicity than chemicals when used for therapy (Awaad et al, 2013).
Glycyrrhiza glabra, is a plant which grows in Egypt and other countries of the world (Siracusa et al, 2011). Its roots are widely used as a cold beverage, in preparing some pharmaceutical preparations such as hematinic pills and to disguise the bitter taste 3
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of other remedies (Song, and Hu 2009). The extract of this plant is useful as a tonic, detoxifying, expectorant, anti-inflammatory, antimicrobial, antiatherogenic, antiallergic, antiviral agents against metabolic syndrome, obesity and immune system alterations, peptic ulcer, bronchitis, cough, arthritis, rheumatism, hypoglycemia and hypotension (Gupta et al., 2008, Tanaka et al., 2008, Visavadiya
et al., 2009;
Siracusa et al., 2011). Finally, Sancar et al., (2009) concluded, Glycyrrhiza glabra can be used for the treatment of aspirin-induced ulcers as an inexpensive alternative to chemotherapy. Pistacia species have a wide range of uses; in eczema treatment, paralysis, throat infections, renal stones, jaundice, asthma and antipyretic, pectoral, antiatherogenic effect, antioxidant, antihepatotoxic, antimicrobial, and antiproliferative activity in colon cancer cells (Angeliki et al, 2007; Balan et al, 2007; Dina et al, 2009). Moreover, Dellai., et al.(2013) investigated antiulcerogenic activities of Pistacia lentiscus leaves extracts in experimental gastric ulcer rats. The identified and quantified through the use of liquid chromatography and mass spectrometry of Glycyrrhiza glabra, and Pistacia lentiscus demonstrated the flavonoids compounds are the predominant constituent (Siracusa et al, 2011, Kaliora et al, 2004, Gardeli et al, 2008). Flavonoids possess both cytoprotective and antisecretory activities (Awaad et al, 2013). They exert a gastroprotective action in mammals by increasing endogenous prostaglandin levels, decreasing histamine secrestion, inhibiting Helicobacter pylori and scavenging oxygen derived free radicalsas well as the antioxidant property (Awaad et al, 2013; Olaleye and Farombi 2006). The aimed of the present study to investigate the effect of oral administration of aqueous extracts of Glycyrrhiza glabra roots and Pistacia lentiscus, on the healing of gastric ulcer in rats. 4
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2. Materials and methods: 2.1. Materials 2.1.1. Plants: Mastic gum (Pistacia lentiscus) and liquorice roots (Glycyrrhiza glabra L.) were purchased from the local market of KSA and classified in the Botany Department Faculty of Sciensces.
2.1.2. Aspirin: Aspegic (Mmiriya Pharmaceutical Industries, Cairo) prepared by dissolving one vial in 25ml distilled water. A volume of 1ml of this solution was orally given (at the level 200mg/kg body weight) for one day to induce acute gastric ulcer in male albino rats, according to the dose carried out by Agarwal et al, (2002). 2.1.3. Diet: The rats were fed on basal ration composed of wheat bran, soya bean powder 44%, fish meal, molasses, fibers 3.3%, sodium chloride, calcium carbonate, calcium phosphate, methionine and ash with net protein 22% and fats 4.7% (Dennis et al, 2009). The diet and water were provided ad libitum for all the experimental period. 2.1.4. Rats: Thirty six adult male albino rats (175+5g. bw.) of Sprague Dawley Strain were obtained from animal house of the faculty of medicine, Um Al Qura University. The rats were acclaimed for 10 days. Each rat was housed in an individual stainless steel cage under optimal controlled conditions in the Animal House of the Faculty of Medicine (Umm al
5
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Qura University). The experimental protocol was approved by institutional animal ethical committee. 2.2. Methods: 2.2.1. Preparation of aqueous extracts: The Mastic gum (Pistacia lentiscus) and liquorice roots (Glycyrrhiza glabra L.) were ground using a porcelain grinder to pass through a sieve-mesh pores of 1mm diameter. The extract of plants was prepared by mixing 1gm powdered with 100 ml deionized water. The mixture boiled for 10 minutes and filtered after cooling using filter paper to remove the particulate matter (0.2mm) then the filtrate was freely dried (Lyophilized) and reconstituted in 1.5 ml of deionized water (100 mg/kg bw) (Sofrata et al, 2007). 2.2.2. Experiment design The rats were divided into 6 equal groups (each is 6). The first group (GP.1) was fed on the basal diet only as a control negative (healthy rats). GP.2-5 were given oral aspirin at a dose of 200mg/kg B.Wt., for induction of acute gastric ulcer, according to the dose carried out by Agarwal et al, (2002). GP.2 was kept as positive (+ve) control. GP.3-4 were orally treated with Pistacia lentiscus extract at doses of 200 and 400 mg/kg Bw respectively. GP.5-6 were orally administrated with Glycyrrhiza glabra extract at doses of 200 and 400 mg/kg Bw respectively. All rats were fasted for 12-14hrs before the rats anesthetized and sacrificed. 2.2.3. Measurement the volume of gastric juice: Gastric juice was collected according to the methods of (Niida et al, 1991). The stomach and duodenum were exposed and a fistula made with a poly ethane tube inserted into the stomach from a small incision made in the duodenum and esophagus was clamped 6
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to prevent reflux and loss of the gastric mucosa. The gastric juice collected in graduated tubes and measured after centrifuging. 2.2.4. Measure the length of gastric ulcer and gastric juice: The stomachs were tied around both openings (cardiac & pyloric sphincters) and injected with distilled water (3ml). The stomachs opened longitudinally, and examined under dissecting microscope for ulcer after washing with saline. The length of gastric ulcer was measured and expressed as mean +SE for each group. The curative ratio was then calculated for each treated group, according to the method described by Akhtar and Ahmad (1995) using the following equation: Curative ratio (CR) = (LC - LT / LC) x 100. LC = length of ulcer in control positive group. LT = length of ulcer in treated group 2.2.5. Determination of pH: Determination of pH according to the method (A.O.A.C, 1995). 2.2.6. Humoral antibacterial studies 2.2.6.1. Bactericidal activity The bactericidal activity (BCA) of plasma determined by modifications (Kampen et a, 2005). One hundred microliters of plasma added in duplicate of 96 round bottom well microtiter plate and incubate for 2.5 h with 50 μL suspensions of 24h a live Staphylococcus aureus
culture (1 X107 CFU mL-1). Fifty μL diphenyltetrazolium
bromide solution (MTT; 2 mg mL-1) (Sigma) added to each well and incubated for 20 min at room temperature to allow the formation of the formation. Plates centrifuged for 10 min at 2000 g. The supernatant discard and the precipitate dissolved in 200 μL dimethyl sulfoxide (DMSO). The absorbance of the dissolved formazan read at 560 nm 7
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with a microtiter plate reader. The bactericidal activity calculated by subtracting the absorbance of samples from that of controls and reporting as absorbance units. 2.2.6.2 .Lysozyme activity Serum
lysozyme activity
measured, according to Ellis
modifications. A sample of 0.25 ml plasma
(1990) with some
mixed with 0.75 ml Micrococcus
lysodeikticus (Sigma Chemical Co) suspension (0.2 mg /ml in 0.05 M PBS, pH 6.2). The mixture reacts at 25 0C for 5 min, and then the OD measured at 1-min intervals for 5 min at 540 nm (5010, Photometer, BM Co. Germany). One unit of enzyme activity defined as the amount of enzyme causing a decrease in absorbance of 0.001 and the activity expressed as U/min/ml serum. 2.2.7. Antioxidant studies The antioxidant system, reduced glutathione (GSH), Superoxide dismutase (SOD) and total antioxidant capacity (TAC), in addition to lipid peroxidation, malnoaldhyde (MDH)
were determined by using ELISA commercial kits, (Kaymen Co. USA)
according to the enclosed pamphlet. 2.2.8. Histopathological study: Specimens from stomachs were collected from rats of all experimental groups at the end of the experimental period, fixed at 10% neutral buffered formalin (pH=7.0), dehydrated in ethyl alcohol and cleared in xylol and 4-6 microns thickness sections stained with heamtoxylin and eosin for examining (Bancroft and Gamble, 2008). 2.2.7. Statistical Analysis: Statistical analysis was achieved by using statistical package for the social science program version 20 (SPSS, 2008). Statistical analysis was performed by one-way 8
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analysis of variance (ANOVA) followed by least significant difference for multiple comparisons. The mean and standard errors were calculated for each variable. The significance of differences was accepted at p < 0.05 3- Results: The present study was designed to clear out the effect of aqueous extracts of Pistacia lentiscus and Glycyrrhiza glabra on healing acute gastric ulcer induced by aspirin in rats. 3.1. The length of gastric ulcer in rats. The effect of Pistacia lentiscus and Glycyrrhiza glabra at two doses on the length of gastric ulcer in rats were shown in table 1. The mean length of gastric ulcer in GP.2 (control +ve) was significantly higher (P
