"Evaluation of antiulcer activity of bark extract of albizzia "
1 Received by Belinda Received on 2012-7-16 2 ID No. B541 Revised on 2012-7-27 3 Pages 13 4 评价溪谷合欢树皮提取物的抗溃疡性 5 Evaluation of Antiulcer Activity of Bark Extract of Albizzia lebbeck Linn. 6 Neelam Balekar*, Dinesh Kumar Jain, Hitesh Jawanjal. 7 College of Pharmacy IPS Academy, Rajendra Nagar, A.B. Road, Indore – 452012 8 (M.P.), INDIA 9 10 11 12 13 *Corresponding author: Dr. Neelam Balekar, 14 Tel.: +919893405071; fax: +07314041627. 15 E-mail address: firstname.lastname@example.org (Neelam Balekar). 16 Postal Address: Department of Pharmacology, College of Pharmacy, IPS Academy, 17 Hukhmakedhi, Rajendra Nagar, A.B. Road, Indore – 452012 (M.P.), 18 INDIA 19 20 21 22 23 1 1 2 3 4 5 Abstract 6 Objective: To determine the antiulcer properties of the ethanolic extract from the stem 7 bark of Albizzia lebbeck Linn. in experimental rats. 8 Methods: Antiulcer potential of ethanolic stem bark extract of A. lebbeck were 9 determined using ethanol induced, aspirin induced, cold stress restraint and pylorus 10 ligated ulcer in rats. 11 Results: The ethanol extract (100, 250, 500,and 1000 mg/kg, p.o.) significantly 12 suppressed the peptic ulcer induced by ethanol. Ethanolic extract (500 mg/kg) showed 13 promising activity. Hence, this dose was selected for the further evaluation of antiulcer 14 studies. The ethanolic extract (500 mg/kg) showed significant (P< 0.05) reduction in 15 gastric volume, free acidity, total acidity, ulcer index, protein and pepsin content and 16 increase in mucus content as compared to control. 17 Conclusions: The present study indicates that A. lebbeck bark extract have potential 18 antiulcer activity in all models which might be due to its antisecretory activity and 19 increased resistance to necrotizing agents, providing a direct, protective effect on the 20 gastric mucosa. 21 Keywords: Albizzia lebbeck, Gastric ulcer, cytoprotection, antisecretory. 22 23 2 1 1. Introduction 2 Gastric ulcer, most common disorder of gastrointestinal tract has multifunctional causes 3 in its pathophysiology. The disorder affects large population in all countries. It is now 4 assumed that these drugs ultimately balance the aggressive factors (acid, pepsin, H. 5 pylori, bile salts) and defensive factors (mucin secretion, cellular mucus, bicarbonate 6 secretion and mucosal blood flow). Peptic ulcer is a lesion of gastric or duodenal 7 mucosa occurring at site where the mucosal epithelium is exposed to aggressive 8 factors. Herbal medicine is fast emerging as an alternative treatment to available 9 synthetic drugs for treatment of ulcer possibly due to lower costs, availability, fewer 10 adverse effects and perceived effectiveness. Albizzia lebbeck Linn. (family- 11 Mimosaceae) is a tropical evergreen tree widely distributed in India, South Africa and 12 Australia. Its stem bark has been used for a long time in Southeast Asia as a traditional 13 medicine for toothache, diseases of gum, skin infection, ulcer, piles, dysentery and 14 diarrhea[3,4]. A. lebbeck contains tannin, flavonoids, steroids, and triterpenoids. The 15 pharmacological activities previously reported are antibacterial, antiinflammatory, 16 antimicrobial, and antioxidant. 17 The aim of the present study was to investigate the antiulcer activitiy of the bark of 18 A. lebbeck commonly employed by traditional healers. Parameters like ethanol induced, 19 aspirin induced, cold restraint stress and pylorus ligated ulcer model in rat were utilized 20 to clarify its traditional use in a scientific investigation. 21 22 2. Materials and Methods 23 2.1 Plant material and chemicals 3 1 The fresh stem bark of A. lebbeck Linn. (Mimosaceae) was collected in October, 2009 2 from A.B. Road, Indore (M.P.). The plant was authenticated and voucher specimen 3 (ALHIT1) was deposited at the herbarium of the Botanical survey of India, Pune. 4 Aspirin was obtained as gift sample from Cyno Pharma, Indore, India and omeprazole 5 and ranitidine were obtained from Alpa Laboratories Indore, India. All the other 6 chemicals and reagent used were of analytical grade, obtained from Kasliwal Brothers, 7 Indore, India. 8 9 2.2 Extraction 10 The stem bark (500 g) of A. lebbeck was shade dried, coarsely powdered and defatted 11 by maceration with petroleum ether for 48 h. The defatted marc was subjected to soxhlet 12 extraction with 95% ethanol (2.5 L) at (60-80 ºC). The solvent was filtered through 13 Whatman No. 1 filter paper and evaporated to dryness under vacuum at 40 ºC using 14 rotary evaporator. The ethanolic extract was stored in tightly closed container and kept 15 in refrigerator. 16 17 2.3 Preliminary phytochemical screening 18 The extract was qualitatively analyzed for the presence or absence of phytoconstituents 19 like glycosides, flavonoids, saponins, alkaloids, carbohydrates, sterols, proteins using 20 standard methods. The quantitative determination of total flavonoid content was done 21 by the method of Chang et al., 2002. 22 4 1 2.4 Animals 2 The Wistar rats of either sex weighing 120-200 g were used. Animals were housed 3 under standard condition of temperature (25 ± 2 ºC), 12 h/12 h light/dark cycle and fed 4 with standard pellet diet and water ad libitum. The animals were allowed to acclimatize 5 for one week before the experiments. All experimental protocols were approved by the 6 IAEC under the supervision of CPCSEA. 7 8 2.5 Ethanol induced ulcer 9 Wistar rats were divided into six groups, each group containing five animals. Group I 10 was control (0.5% Sodium carboxymethyl cellulose (SCMC)), group II served as 11 standard (Omeprazole 20 mg/kg p.o.), group III-VI served as ethanolic extract ALEE 12 (100, 250, 500, 1000 mg/kg) were administered to rats 1 h before ethanol (5 ml/kg) 13 treatment. After 1 h, the animals were sacrificed by cervical dislocation and the gastric 14 lesions were counted. 15 16 2.6 Aspirin induced ulcer 17 Wisar rats were divided into three groups. Group I was control (0.5% SCMC), group II 18 served as standard (ranitidine 50 mg/kg), group III was ALEE (500 mg/kg). Each group 19 was containing five animals. All treatments were administered 30 min before aspirin. 20 Aspirin (1000 mg/kg) was administered orally to rats. The animals were sacrificed by 21 cervical dislocation 6 h later and the stomach was removed to calculate the ulcerative 22 index. 23 24 2.7 Cold stress restraint ulcer 5 1 Wistar rats were divided into four groups. Group I was positive control (restraint with 2 cold stress), Group II served as negative control (restraint without cold stress), 3 Group III served as standard (omeprazole 20 mg/kg). Group IV was ALEE (500 4 mg/kg). Each group was containing five animals. All treatments were administered 1 h 5 before stress in restraint cages that were kept at 2 ºC in chamber for 4 h treatment. After 6 4 h, the animals were sacrificed using cervical dislocation and the gastric lesions were 7 counted as earlier and mucus content was estimated. 8 9 2.8 Pylorus ligated ulcer 10 Wisar rats were divided into three groups. Group I was control (0.5 % SCMC), group 11 II served as standard (omeprazole 20 mg/kg), group III was ALEE (500 mg/kg). Each 12 group was containing five animals. All treatments were administered to animals 45 13 mins before pylorus ligation. Pylorus ligation was done by ligating the pyloric end of 14 stomach of rats of respective groups under ether anaesthesia. Ligation was done 15 without causing any damage to the blood supply of the stomach. Animals were allowed 16 to recover and stabilize in individual cages and were deprived of water during 17 postoperative period. After 6 h of surgery, rats were sacrificed by overdose of ether and 18 ulcer scoring was done. Gastric juice was collected and mucus content, protein, pepsin 19 content, free and total acidity, pH were determined. 20 21 2.9 Statistical analysis 22 Data are expressed as a mean ± S.D. Statistical evaluation was carried out using one- 23 way (ANOVA followed by Tukey’s test) using “Graphpad Instat” version 3.00 for 6 1 Windows 95, Graphpad software, San Diego California USA. The values of P < 0.05 2 were considered to be statistically significant. 3 4 Results 5 3.1 Phytochemical analysis 6 The percentage yield of the ethanolic extract was found to be 12.36%. The ethanolic 7 extract showed the presence of flavonoid, tannin, saponin, protein. The total flavonoids 8 content in ethanolic was found to be 12.15 mg quercetin equivalents/g. 9 10 3.2 Ethanol induced ulcer 11 Oral administration of the ethanolic extract of the stem bark of Albizzia lebbeck (100, 12 250, 500, and 1000 mg/kg, p.o.) dose dependently reduced the ulcer index induced by 13 ethanol in rats as compared to control (P < 0.01). The mean ulcer index was found to be 14 control, (22.12 ± 0.74), omeprazole 20 mg/kg (2.1 ± 0.74) and ALEE 500 and 1000 15 mg/kg, (3.3 ± 0.84, 3.1 ± 0.89), respectively. At the dose of 100, 250, 500 and 1000 16 mg/kg, ulcer index was inhibited by 37.16, 69.71, 85.08 and 85.99% respectively. The 17 reference drug, omeprazole (20 mg/kg), also produced a significant protective effect 18 towards the ethanol induced ulcer by 90.51% inhibition (Fig 1). The reduction in ulcer 19 index produced by ALEE 500 and 1000 mg/kg was found to be comparable to 20 omeprazole. 21 22 3.3 Aspirin induced ulcer 23 The ethanolic extract of A. lebbeck stem bark (500 mg/kg, p.o.) and ranitidine 24 (50 mg/kg, p.o) significantly exert protective effect on aspirin induced ulcer in rats. The 7 1 ulcer protective effect exhibited by A. lebbeck and standard ranitidine was found to be 2 82.43 % and 89.86% respectively (Fig 2). The reduction in ulcer index produced by 3 ALEE 500 mg/kg (2.6 ± 0.65) was found to be comparable to ranitidine (1.5 ± 0.5). 4 5 3.4 Cold restraint stress ulcer 6 The ethanolic extract of A. lebbeck stem bark (500 mg/kg, p.o.) and omeprazole 7 (20 mg/kg, p.o) significantly exert protective effect on cold restraint stress ulcer in rats. 8 The mean ulcer produced by ALEE 500 mg/kg was significantly reduced as compared 9 to positive control (P<0.05). The ulcer protective effect exhibited by A. lebbeck and 10 standard omeprazole was found to be 78.46% and 85.93 % respectively (Table 1). 11 12 3.5 Pylorus ligated ulcer 13 In pyloric ligation induced ulcer model, oral administration of ethanolic extract 14 (500 mg/kg) showed significant reduction in ulcer index, gastric volume, free acidity, 15 total acidity as compared to the control group (P<0.05). It showed protection index of 16 77.89 % at the dose of 500 mg/kg in comparison to control whereas omeprazole as 17 reference standard drug showed reduction of ulcer by 85.26 % (Table 2). 18 19 3. Discussion 20 The antiulcer activity of the bark of A. lebbeck was evaluated by employing ethanol, 21 aspirin, cold restraint stress and pylorus ligation ulcer models. These models represent 22 some of the most common causes of gastric ulcer in humans. Ulcers caused by 23 ethanol are due to superficial damage to mucosal cells and damage by NSAIDs are 24 due to a decrease in PG synthesis, and ulcers due to stress are due to both physiological 8 1 and psychological factors and those by pyloric ligation are due to increased 2 accumulation of gastric acid and pepsin leading to auto digestion of the gastric 3 mucosa. 4 Antiinflammatory drugs like aspirin administered in toxic doses (1000 mg/kg), produce 5 visible gastric ulcers in animals. Aspirin is a potent inhibitor of prostaglandin 6 biosynthesis. Prostaglandins are known to play an important role in maintaining 7 mucosal integrity. An increase in certain endogenous prostaglandins can enhance 8 gastric mucosal resistance to ulcerogenic agents. The mechanisms involved in 9 prostaglandin action are multiple, including stimulation of mucus and bicarbonate 10 output, gastric mucosal blood flow that may be due to the presence of flavonoid 11 in A. lebbeck extract. 12 Ethanol induced gastric ulcer was employed to study the cytoprotective effect of the 13 extracts. Ethanol challenge induces gastric injury due to production of oxygen free 14 radicals leading to increased lipid peroxidation, which causes damage to cell and cell 15 membrane presenting as red streaks of sores. Ethanol induced gastric lesion 16 formation may be due to stasis in gastric blood flow which contributes to the 17 development of the haemorrhage and necrotic aspects of tissue injury. A. lebbeck 18 extracts has significantly decrease the ulcer index, may be due to the presence of 19 flavonoid which may showed antisecretory activity. Omeprazole were employed in this 20 study for the latter’s cytoprotective but not anti-secretory effect and its effectiveness 21 against experimentally induced ethanol ulcers. 22 On the other hand, pyloric ligation induced ulcers develop as a result of accumulation 23 of the gastric acid and distention of the stomach which in turn weakens the mucosal 24 defenses. It has been proposed that in pyloric ligation, the digestive effect of 9 1 accumulated gastric juice and interference of gastric blood circulation are responsible 2 for induction of ulceration. 3 In the cold-restraint stress model, gastric ulcer formation was mainly due to gastric 4 hypermotility, which could lead to mucosal over friction. Hence, the gastric mucus 5 layer is extremely important and the mucus is generally believed to contribute to a 6 cytoprotective action. In the stomach, prostaglandin synthase is the major pathway, 7 since leukotriene synthesis is approximately half of prostaglandin synthesis. Stress 8 induces an increase in leukotriene synthesis in the stomach which, compared to PGE2, 9 indicates that arachidonate oxidation changed toward leukotriene production. The 10 ethanolic extract of A. lebbeck (500 mg/kg, p.o.) showed significant reduction in cold 11 restraint stress in rats 12 The possible mechanism of gastric mucosal protection by ALEE may be partially due 13 to the reinforcement of resistance of the mucosal barrier by a protective coating. The 14 ethanolic extract of A. lebbeck in concentration dependent manner exhibited marked 15 gastroprotection in ethanol and aspirin induced ulcer model. The present investigation 16 thus, establishes the ulcer protective and healing effects of ALEE in different gastric 17 ulcer models and activity seemed to be due to its effect both on offensive and defensive 18 mucosal factors. 19 20 Conflict of interest statement 21 We declare that we have no conflict of interest. 22 23 24 Acknowledgements 10 1 The authors would like to acknowledge College of Pharmacy, IPS Academy, Indore, 2 India for providing the necessary facilities to carry out the study. 3 4 Research grant information; 5 SOURCE: This work was supported by IPSA Research Devlopment Scheme 6 GRANT NO- IPSA/RDS/2009-10/02 7 8 References: 9  Surana SJ, Tatiya AU, Jain AS, Ushir YV. Antiulcer activity of Eranthemum 10 roseum (Vahl) R. Br on Ethanol induced ulcer in Albino rats. Int J Pharmacol Biol 11 Sci 2007; 1: 65-69. 12  Govindarajan R, Vijaykumar M, Singh M, Rao CV, Shirwaikar A, Rawat AK, et al. 13 Antiulcer and antimicrobial activity of Anogeissus latifolia. J Ethnopharmacol 14 2006; 106: 57-61. 15  Singh PK, Kumar V, Tiwari RK, Sharma A, Rao CV, Singh RH. Medico 16 Ethnobotany of ‘Chatara’ Block of District Sonebhadra, Uttar Pradesh, India. Adv 17 Bio Res 2011; 4 (1): 65-80. 18  Bobby NMD, Wesely EG, Johnson M. High performance thin layer 19 chromatography profile studies on the alkaloids of Albizia lebbeck. Asian Pac J 20 Trop Biomed 2012: S1-S6. 21  Kumar, A., Saluja, A.K., Shah, U.D. and Mayavanshi, A.V. 2007. Pharmacological 22 potential of Albizzia lebbeck: A Review. Pharmacog Rev 2007; 1: 171-74. 23  Harborne, J.B. Phytochemical methods–a guide to modern techniques of plant 24 analysis. 2nd ed. New York: Chapman and Hall; 1984, p. 54-64. 11 1  Trease GE, Evans WC. Pharmacognosy. WB Saunders, London 2008. p. 554-55. 2  Malarajan, P, Gopalakrishnam G, Narasimhan S, Vehi KJ, Karimani S. Antiulcer 3 activity of crude alcoholic extract of Toona ciliate Roemer (heart wood). J 4 Ethnopharmacol 2007; 110: 348-351. 5  Bodhankar SL, Jain BB, Ahire BP, Dande RB, Shitole PP. The effect of 6 rabeprazole and its isomer on aspirin and histamine induced ulcer in rats. Indian J 7 Pharmacol 2006; 36: 357-358. 8  Dandagi PM, Patil MB, Mastiholimath VS, Gadad AP, Kulkarni AR, Jalalpure, 9 SS. Antiulcer activity of extracts of Cyperous rotundus rhizome in Pylorus 10 ligated Rat model. Adv Pharmacol Toxicol 2007; 8: 117-122. 11  Patil P, Prakash T, Shivakumar H, Pal S. Anti-ulcer and anti-secretory properties 12 of the Butea monosperma (Lam) Bark extract with relation to antioxidant studies. 13 Iranian J Pharmacol Ther 2009; 8: 1-6. 14  Pillai SI, Kandaswamy M, Subramanian S. Antiulcerogenic and ulcer healing 15 effects of Indian propolis in experimental rat ulcer models. J ApiProd ApiMed Sci 16 2010; 2 (1): 21-8. 17  John W. Prostaglandins, NSAIDs, and Gastric Mucosal Protection: Why Doesn’t 18 the Stomach Digest Itself. Physiol Rev 2008; 88: 1547–65. 19  Krishnaveni M, Anbu J, Nithya S, Anjana A, Prema S. Gastric antisecretory, 20 antiulcer and cytoprotective, properties of Tamira parpam (Bio-Copper) from 21 Earthworm. Int J Pharm Bio Sci 2012; 3(2): 250-59. 22  Singh AP, Shukla V, Khare P. Effects of Plumeria obtuse linn. In peptic ulcer 23 induced by pylorus ligation & indomethacin. J Pharm Sci Innov 2012; 3 (2): 26- 24 32. 12 1  Kamada T, Sato Y, Shiotani A, Haruma K. Treatment and prevention of 2 histamine-2 receptor antagonist for elderly gastric ulcer. Nihon Rinsho. 2010; 68 3 (11): 2064-70. 4  AlRashdi AS, Salama SM., Alkiyumi SS, Abdulla MA, Hadi A, Hamid A, et al., 5 Mechanisms of gastroprotective effects of ethanolic leaf extract of Jasminum 6 sambac against HCl/Ethanol-induced gastric mucosal injury in rats. Evidence- 7 Based Comp Alt Med 2012; 12: 1-15. 8  Mughrabi FF, Hashim H, Ameen M, Khaledi H, Ali HM. Cytoprotective effect of 9 Benzyl N'-(indol-3-ylmethylidene)-hydrazinecarbodithioate against ethanol- 10 induced gastric mucosal injury in rats. Afr J Pure App Chem 2011; 5 (3): 34-42. 11  Nair V, Arjuman A, Gopalakrishna HN, Dorababu P, Mirshad PV, Bhargavan D, 12 Chatterji D. Evaluation of the anti-ulcer activity of NR-ANX-C (a polyherbal 13 formulation) in aspirin & pyloric ligature induced gastric ulcers in albino rats. 14 Indian J Med Res 2010; 132: 218-23. 15  Devi MR, Subramanian NS, Anbazhagan S, Telrandhe UB. Anti gastric ulcer 16 activity of Ficus nervosa bark in wistar albino rats. J Chem Pharm Res 2012; 17 4(2): 1288-95. 18  Penissi AB, Giordano OS, Guzmán JA, Rudolph MI, Piezzi RS. Chemical and 19 Pharmacological Properties of Dehydroleucodine, A Lactone isolated from 20 Artemisia douglasiana Besser. Mol Med Chem 2006; 10: 1-11 21  Hegazy HG. Ameliorative effects of ginger and α-lipoic acid on oxidative stress 22 and inflammation in senile female rats. Afr J Pharm Pharmacol. 2011; 5(8): 23 1096-1105. 13