GC-MS ANALYSIS AND ANTIULCER ACTIVITY OF ETHANOL EXTRACT OF TUBERS OF MOMORDICA TUBEROSA COGN. (CUCURBITACEAE) IN RATS
Pramod Kumar*1, Devala Rao G2, Lakshmayya3, Ramachandra Setty S4
1 Department of Pharmacognosy, V.L. College of Pharmacy, Raichur-584103 2 KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada- 520010 3 GRD Institute of Management and Technology (Pharmacy), Dehradun-248001 4 Govt. College of Pharmacy, Bengaluru- 560027, India
Keywords: Anti-oxidant, Aspirin, Ethanol, Free acidity, Pyloric ligation, Total acidity.
Abstract

70% ethanol extract of tubers of Momordica tuberosa (TMT) showed the presence of saponins and triterpenoids. GC-MS analysis of the extract showed presence of 42 compounds with Isopulegol, a monoterpene and a steroid, Androstane as main constituents. Many fatty acid derivatives like myristic acid, margaric acid, oleic acid etc. were also present. The extract was then tested for its antiulcer activity using three different experimental models of ulcers in rats viz., aspirin, ethanol and pyloric ligation induced ulcers in rats. The LD50 in rat was 200mg/kg. One fifth and one tenth of maximum dose i.e.40 and 20mg/kg were used to assess the antiulcer activity. In pylorus ligation model, parameters like ulcer index, volume of gastric juice, free acidity, total acidity and pH were measured. The extract showed antiulcer activity in a dose dependant manner. The 40mg/kg dose reduced ulcer to an extent of 95% in aspirin model and 82% in pyloric ligation method. The results suggest the anti-ulcer activity of tubers of Momordica tuberosa, probably due to its antioxidant nature.

Article Information

Identifiers and Pagination:
Year:2011
Volume:3
First Page:359
Last Page:269
Publisher Id:JAppPharm (2011 ). 3. 259-269
Article History:
Received:May 21, 2011
Accepted:September 11, 2011
Collection year:2011
First Published:October 10, 2011

INTRODUCTION
Precise etiology of ulcer is not clear and a satisfactory regimen remains elusive. Ulcers are believed to be imbalance in offensive factors like acid and pepsin and defensive factors like mucin secretion, tissue glutathione (GSH), cell proliferation, prostaglandins (Goel & Bhattacharya,1991 and Itoth & Guth, 1985). Peptic ulcer being one of the most rampant gastrointestinal disorders continues to occupy the key position in concern of both, clinical practitioners and researchers. As a consequence, many synthetic drugs are explored offering newer and better options for treatment of peptic ulcer. However, these drugs differ from H2 receptor antagonists, proton pump inhibitors or cytoprotective agents such as sucralfate. Unfortunately, most of these drugs confer simpler to severe side effects like arrhythmias, gynaecomastia, enterochromaffin like cell (ECL) hyperplasia and hematopoietic changes (Akhtar et al., 1992). Thus, there is an urgent need to search an indigenous drug with fewer side effects to have a better and safer alternative for the treatment of peptic ulcer. In this context, extensive studies and research has been undertaken which mainly focuses on search of antiulcer agents of plant origin. The participation of reactive oxygen species in the etiology and pathologies of human diseases, such as neurodegenerative disorders, inflammation, viral infections, autoimmune pathologies and digestive system disorders such as gastrointestinal inflammation and gastric ulcer has been reported earlier (Repetto & Llesuy, 2002). Several studies have shown alterations in the anti-oxidant status following ulceration, implying that free radicals may be associated with ethanol induced gastric mucosal damage in rats (Pihan et al., 1987 & Mizui et al., 1987). Drugs with multiple mechanisms of protective action, including antioxidant properties, may be beneficial in minimizing tissue injury in human disease (Barry, 1991). The plant Momordica tuberosa Cogn. (Cucurbitaceae) growing abundantly in and around Raichur is traditionally used as abortifacient (Kirtikar & Basu, 1991). Fruits of the plant possess hypoglycemic activity (Kameshwar Rao et al., 2003).The tubers have anti implantation activity (Koneri et al., 2006). Fruits contain citric acid and maleic acid (Parvati & Kumar, 2002) and are antioxidant and hepatoprotective (Swamy & Jayaveera, 2007). We have earlier reported the in vitro and in vivo antioxidant and hepatoprotective property of the tubers in rats in CCl4 induced toxicity (Kumar et al., 2008). Since the fruits of M.tuberosa are reported to contain Vitamin C (Parvati & Kumar, 2002), a known antioxidant, it was hypothesized that the tubers may also contain antioxidant principles and may possess antiulcer property. Hence, they were selected for phytochemical screening and evaluation for antiulcer properties.


MATERIALS AND METHODS
Plant material
The tubers of M.tuberosa were collected from the suburban fields of Raichur during January and were identified and authenticated by Prof. Srivatsa, Retired Professor of Botany, L.V.D. College, Raichur. A herbarium specimen (VLCP-02/05) was deposited in the Department of Pharmacognosy, V.L. College of Pharmacy, Raichur.
Preparation of extract
The coarse powder of shade-dried tubers of M. tuberosa was extracted successively with petroleum ether, chloroform, ethanol and water (Kokate, 1996). Similarly, 70% ethanol extract (TMT) was also prepared after defatting of the drug. The obtained extracts were dried under reduced pressure by using Rota-flash evaporator.
Preliminary phytochemical screening
All extracts obtained were screened for the presence of phytoconstituents by using the qualitative tests (Kokate, 1996 & Khandelwal, 2005).
GC-MS analysis of 70 % ethanol extract of tubers
The GC-MS analysis of 70% ethanol extract was carried out in a GC-MS Model GC-MS-QP2010 Plus. Make: SHIMADZU gas chromatograph fitted with ZB-624 30 m X 1.4 mm ID 0.25µm film thickness or equivalent column. Carrier gas was helium with a flow rate of 2.5 mL/min; column temperature initially was at 120°C for 2 min. Then rose to 250°C at the rate of 10°C per minute maintained at 250°C for 20 minutes; injector temperature was 220°C, detector temperature 260°C, Volume injected was 1µL with liquid injector of 70% ethanol extract in ethanol (1g in 5 mL ethanol); The Mass spectra operating parameters were, ionization potential, 70 eV; ion source temperature; 250°C, Solvent delay 3.0 min , Program Run time: 31 minutes and scan range 30-350 amu, EV voltage 3000 volts.
The identification of components present in the 70% ethanol extract was based on direct comparison of the retention times and mass spectral data by computer matching with the commercial library Wiley 229.
Animals
Albino rats (150 – 200 g) and mice (20 – 25 g) of both sex were obtained from Sri. Venkateshwara Enterprises, Bangalore and housed in plastic animal cages in groups of 6 - 8 animals with 12:12 h of light: dark cycle under standard husbandry conditions. The animals were fed with standard rodent diet and provided water ad libitum. The animals were used for the study after one week of acclimatization. The approval of Institutional animal ethical committee was obtained prior to the experiments.
Ethanol induced ulcer
The ulcers were induced by ethanol (Luis et al., 2002). Five groups of six animals each were fasted for 18h and water was given ad-libitum. The first group animals served as negative control and received saline. The animals of second group served as positive control and received ethanol 100% (1mL/200g) for inducing gastric ulcers. The animals of third group orally received standard drug, lansaprazole (8 mg/kg). Animals of group four and five received the TMT at 20 mg/kg and 40 mg/kg dose po, respectively. All the animals in group three, four and five received ethanol 100% (1mL/200g) after 60min of respective treatments. The animals were sacrificed by cervical dislocation after one hour of ethanol administration and stomach was incised along the greater curvature and examined for ulcers. The number of ulcers per stomach was noted by using simple microscope and score was given (Kulkarni, 2002) viz. 0 for normal coloured stomach, 0.5 for red colouration, 1 for spot ulcer, 1.5 for hemorrhagic streaks, 2 for ulcer > 3 but < 5mm and 3 for ulcer > 5mm.Mean ulcer score for each animal is expressed as ulcer index. The percentage protection was calculated.
Pylorus-ligated rats’ method
In the pylorus ligation method (Shay, et al., 1945), albino rats were divided into five groups of six animals each and fasted for 18h. Care was taken to avoid coprophagy. The treatment schedule and grouping was similar to ethanol model. The pyloric ligation was performed under light ether anesthesia 60 min after respective treatments. The animals were not given water during the post operative period. After 4h stomachs were dissected out, contents were collected into tubes for estimation of volume, acid content and pH. The stomachs were cut open along the greater curvature and ulcer index and percentage of protection were determined as mentioned in ethanol model.
Estimation of Acid content and pH
The gastric juice was collected 4h after pylorus ligation; volume was measured and centrifuged for 5 min at 2000 rpm. The pH of the gastric juice was recorded. Then the contents were subjected to analysis for free and total acidity. Free acidity was determined by titrating with 0.01mol/L NaOH, using Topfer’s reagent as indicator until solution turns to yellowish orange and noted the volume of NaOH (this volume represents free acidity) then titration was continued with phenolphthalein as indicator until red tinge as end point. This volume corresponds to total acidity.
Aspirin induced ulcer method
In the aspirin-induced ulcers (Banerjee et al., 2005), five groups of albino rats (150–175 g), with each group consisting of six animals were used. The first group served as a control group and received only saline for 8 days, the second group served as positive control administered with Aspirin (200mg/kg), the third group served as the standard, received Lansaprazole (8 mg/kg), fourth and fifth group received TMT 20 and 40 mg/kg. The groups III to V received lansaprazole, TMT 20 and 40 mg/kg for 8 days orally respectively. After 8 days of treatment animals were fasted for 24 h and ulcer was produced by aspirin 1 % (200 mg/kg, in CMC suspension) po. Animals were sacrificed after 4 h of treatment under over dose of anaesthesia with ether and stomach was dissected out. The stomach was cut open along the greater curvature and ulcer index and percentage of protection were determined as mentioned in other models.
Statistical Analysis
Data was shown as the mean ± SEM and analysed by one way ANNOVA followed by students’’ test. The level of significance for all experiments was P<0.05*, 0.01** and 0.001***.


RESULTS AND DISCUSSION
The preliminary phytochemical investigations showed the presence of sterols in the pet ether extract, saponins, cardiac glycosides, triterpenoids and bitters in the ethanol extract and carbohydrates and constituents of ethanol extract in aqueous extract. The phytoconstituents present in the 70% ethanol extract were similar to that of ethanol and aqueous extracts. The presence of saponins was confirmed by the persistent foam test and haemolysis test. GC-MS analysis of the extract showed presence of fragments like cyclopentane acetic acid, myristic acid, margaric acid, a monoterpene isopulegol, thymin and arachidic acid. A steroid of androstane derivative was present up to 2.88% and a phenol derivative, 2-methoxy-4-vinylphenol was present at 1.13%. A complete analysis report is given in table 1.
Pretreatment with 70% ethanol extract of tubers of M.tuberosa cogn. protected the animals from alcohol, aspirin and pylorus ligation induced gastric ulcerations. Gastro protection offered by TMT at 40 mg/kg dose was almost equivalent to lansaprazole 8mg/kg in aspirin model. The percentage of reduction in ulcer index and percentage of protection at different doses is tabulated in table 3 and reduction in both total and free acidity together with pH changes in table 2.
NSAIDS are known to induce peptic ulcer by not only denaturing mucous glycoprotein but also by free radical formation (Shirwaikar et al., 2006). Similarly, ethanol is also known to produce free radicals and induce peptic ulcer (Soll, 1990). Pyloric ligation induced ulcers are due to increased presence of acid and pepsin in the stomach (Pillai et al., 1982). Also, high turnover rate of mucin forms a protective barrier to epithelial digestion. The action can also be explained in light of the hydroxyl radical scavenging action of mucin (Grisham et al., 1987).
Although in most of the cases, the etiology of ulcer is unknown, it is generally accepted that it results from an imbalance between aggressive factors and the maintenance of the mucosal integrity through the endogenous defense mechanism (Goel & Bhattacharya,1991 and Itoth & Guth, 1985).To regain the balance, different therapeutic agents including herbal preparations are used to inhibit the gastric acid secretion or to boost the mucosal defense mechanisms by increasing mucus production. Reactive oxygen species are involved in the pathogenesis of ethanol-induced gastric mucosal injury in vivo (Akhtar et al., 1992). Lipid peroxidation is a free radical mediated process, which has been implicated in the variety of disease states. It involves the formation and propagation of free radicals, the uptake of oxygen and rearrangement of double bonds in unsaturated lipids, which eventually results in destruction of membrane lipids. Biological membranes are often rich in unsaturated fatty acids and bathed in oxygen-rich metal containing fluid. Therefore membrane lipids are susceptible to proxidative attack (Cheesman, 1993). We have earlier reported (Kumar et al., 2008) a significant decrease in lipid per oxidation by 70% ethanol extract of M. tuberosa in the CCl4 model of hepatotoxicity. In the present study, the ethanol extract of tubers of M.tuberosa has shown significant protection against ulcers in dose dependant manner in all the experimental models of ulcers. In addition, gastric volume and total acidity were reduced significantly but, to our surprise, without much change in pH. Research is on to ascertain the reasons for the same.
Though we have not studied the active principles responsible for its antiulcer property, it is likely that antioxidant principles, saponins and triterpenoids present in the ethanol extract of M.tuberosa may be involved in this action. Some saponins produced in legumes, namely, group B soya saponins, contain an antioxidant moiety attached at C23 (Yoshiki et al., 1998).This unique sugar residue 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), allows saponins to scavenge superoxides by forming hydro peroxide intermediates, thus preventing bio-molecular damage by free radicals (Yoshiki & Okubo, 1995). Hence, the antiulcer activity of TMT could be attributed to the antioxidant potential of saponins and triterpenoids present in it. There is a claim (Kirtikar & Basu, 1991) and report (Koneri et al., 2006) that the plant is an abortifacient. Therefore, its use as antiulcer agent in pregnancy may be contraindicated.
Acknowledgements: Authors take this opportunity to express their gratitude to the Principal and management of V.L.College of Pharmacy, Raichur, India and S.C.S. College of Pharmacy, Harapanahalli, India for providing necessary research facilities.
Place of work (Institution): Pharmacology Research Laboratory, V.L.College of Pharmacy, RAICHUR, and S.C.S College of Pharmacy, HARAPANAHALLI, INDIA.

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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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