Ulcer-protective effect of Avicennia officinalis L., A common mangrove plant

N. Aparna et al., Pharmaceutical and Biological Evaluations 2014; vol. 1. www.onlinepbe.com ISSN 2394-0859 Ulcer-protective effect of Avicennia officinalis L., A common mangrove plant N. Aparna 1, Saumya Ranjan Pani 1, Suman Kumar Mekap 1, Nabin Kumar Dhal 2, Sabuj Sahoo *1, Sagar Kumar Mishra 1 1 University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Odisha-751 004. 2 Institute of Minerals and Materials Technology, Bhubaneswar, Odisha 751014. *For correspondence Dr. Sabuj Sahoo University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Odisha-751 004. Tel: - E-mail: sabujbiotech@rediffmail.com Received: 15 July 2014 Accepted: 5 August 2014 ABSTRACT Objective: Avicennia officinalis L. belonging to family Acanthaceae is a common mangrove plant known as Bani (Odiya) was investigated to evaluate the antiulcer activity of methanol leaf extracts (200 and 400 mg/kg b.w.) by two different models viz. Ethanol-Hydrochloric acid induced and Modified Pyloric ligation in Wistar albino rats. Method: The antiulcer potential of the extracts was compared with the standard drug Omeprazole (20 mg/kg b.w., p.o.). The percentage of ulcer inhibition and ulcer index was calculated and other biochemical parameters like free acidity, total acidity and ph were determined. Result: The methanolic extract at both the doses exhibited significant (p<0.01) antiulcer activity by inhibiting the ulcer and decreasing the ulcer score in both the ulcer models. Keywords: Antiulcer activity, Ethanol-acid induced model, Modified pyloric ligation model, Avicennia officinalis L. Introduction Peptic ulcer is worldwide problem and its prevalence is quite alarming in India. The exact cause of peptic ulcer is not known, the disease results in chronic suffering, loss of working hours and occasional fatality. Smoking, alcoholism, and spices add to the severity of the disease that often precipitate serious complication of ulcer [1]. Over the past few decades, there has been surge in research activity aimed towards the development of effective and safe antiulcer drugs both synthetically and from natural resources. The extracts and compounds from medicinal plants and other natural products have become the widely acceptable source of therapeutic changes for the treatment of peptic ulcers. However, mechanism underlying the gastro protection has not been well understood [2]. Avicennia officinalis L. belonging to family Acanthaceae, a common mangrove plant known as Bani in Odiya (White mangrove in English) is found in almost all the coastal states of India, Bangladesh, Srilanka, Pharmaceutical and Biological Evaluations 1 Research Article

Myanmar, Indo-China, Thailand and Malaysia [3]. The bark of A. officinalis acts as an astringent and is used in small pox [4] and the roots are aphrodisiac [5]. Unripe seeds are poulticed onto abscesses, boils, and smallpox sores. The present study has been carried out to investigate the anti-ulcer activity of the methanolic extract of A. officinalis L. on the basis that its aqueous leaf extract was determined to have anti ulcer activity in NSAID induced ulcer model [6]. Materials and Methods Collection and extraction of plant material The plant was collected from Astharanga, Odisha in the month of November and the identification was done by referring The Flora of Orissa Volume-III [3] and consulting herbarium (RRL-B) and was submitted with voucher specimen number 12672. The leaves were dried in shade at room temperature. The dried leaves were powdered by using grinder to coarse powder, packed into Soxhlet extractor [7] and extracted with methanol for 48 h. The excess of solvent was removed using Rotary vacuum evaporator (Heidolph Laborota 4000 Efficient). The obtained crude extract was stored in airtight container in refrigerator below 10 0 C for further studies. The prepared extracts were subjected to several preliminary qualitative chemical analyses to identify the various phytoconstituents present in them. Experimental design Wistar albino rats weighing 140-200g of either sex were used for the study. The experimental design was approved by Institutional Animal Ethical Committee (IAEC) and animals were maintained under standard conditions for an acclimatization period of 15 days before performing the experiment. All rats were housed individually in metabolic cages and temperature of 22±2 C. The condition in the animal house was approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (Regd. No. 90/c/05/CPCSEA). The methanolic leaf extract of A. officinalis was suspended in 0.5% DMSO as per the dose requirement and used in our various screening studies. The acute oral toxicity study [8] was done according to OECD guideline at dose range 1000 to 5000 mg/kg. The selected animals were fasted, and grouped as appended herewith. Ethanol- hydrochloric acid induced ulcer model [9] Group I: Toxic control (25 ml/kg of 0.3M HCl in 60% ethanol). Group II: Standard group (Omeprazole (20 mg/kg, p.o.) and 25 ml/kg of 0.3M HCl in 60% ethanol). Group III: Test group treated with methanol extract 200 mg/kg. Group IV: Test group treated with methanol extract 400mg/kg. The extract was administered orally as in the above case by suspending in 0.5% DMSO. Thirty minutes after oral administration of methanolic extract, ulcer was induced with oral administration of ethanol-acid [9] (25 ml/kg of 0.3M HCl in 60% ethanol). One hour later, the animals were sacrificed, stomachs removed and cut along the great curvature. The abdomen of each animal was opened and the stomach was isolated after suturing the lower esophageal end. The open stomach was rinsed in a stream of water and the lesions on the gastric mucosa observed and scored as described. The gastric contents collected were titrated against 0.01N NaOH solution using Topfer s reagent till the solution turns to canary yellow color. Pharmaceutical and Biological Evaluations 2

The volume of sodium hydroxide required corresponds to free acidity. The solution was further titrated till the solution regained pink color. The volume of sodium hydroxide required corresponded to the total acidity. The gastric ulcers were counted and the ulcer index was determined [10]. Modified pyloric ligation The selected animals were divided into four groups, each containing six animals. Group I categorized as Toxic control [Aspirin (200 mg/kg, p.o.) + Pyloric ligation (PL)], Group II animals were treated with Standard (Omeprazole (20 mg/kg, p.o.) and Aspirin + PL), Group III and Group IV animals were administered methanol extract 200 and 400 mg/kg, p.o. + PL, respectively. Groups II IV received the assigned drug treatment for the respective 10 days daily. From days 8 to 10, animals of Groups I-IV received aspirin orally as an aqueous suspension at the dose of 200 mg/kg, 2 h after the administration of the drugs [11]. Animals in all groups were fasted for 18 h after the assigned treatment, anesthetized by giving a combination of Ketamine and Midazolam (50 + 4 mg/kg body weight respectively) and the pylorus was ligated [12] i.e. the abdomen was opened by a small midline incision below the xiphoid process, pyloric portion of stomach was slightly lifted out and ligated providing traction to the pylorus without causing any damage to the blood supply. The rats were sacrificed after 4 h. The stomach was removed, opened along greater curvature; the mucosal layer was washed with 1ml distilled water. The gastric contents collected, the free and total acidity was assayed. The gastric ulcers were counted and the ulcer index was determined as described earlier. The gastric juice was collected, centrifuged and the volume of the supernatant was expressed as ml/100 gm b. w. Free acidity and total acidity were determined by titrating with 0.01 N NaOH using Topfer's reagent and phenolphthalein as indicators [13]. The free and total acids were expressed as meq/l. The total acid output was determined and expressed as meq/l. Total acidity was calculated by using the formula Acidity = meq/1/100g Histopathological examination For histopathological examination, the stomach was washed thoroughly with saline, dehydrated in gradual ethanol (50 100 %), cleared in xylene and embedded in paraffin. Sections (4 5 μm) were prepared and then stained with hematoxylin and eosin (H E) dye for photo microscopic observation (magnification 100 x). The sum of the length (mm) of all lesions for each stomach was used as the ulcer index (UI). In each rat, the macroscopic injury of each ulcer was scored by an independent observer according to a scale ranging from 0 to 4 as follows [14]: (0) no macroscopic changes, (1) Mucosal erythema only, (2) Mild mucosal edema, slight bleeding or small erosions, (3) Moderate edema, bleeding ulcers or erosions, and (4) Severe ulceration, erosions, edema and tissue necrosis. Statistical analysis Results were indicated in terms of mean ± SEM. Statistical significance of data were assessed by analysis of variance (oneway ANOVA), followed by comparison between different groups using Dunett's Pharmaceutical and Biological Evaluations 3

multiple comparison test. The significance level was considered at the level of p<0.05. Results and Discussion The phytochemical tests of the methanolic leaf extract of A. officinalis L. revealed that it contains all the necessary secondary metabolites such as alkaloids, glycosides, steroids, triterpenoids, tannins, saponins, carbohydrate, proteins and flavonoids. No mortality of animals was observed upto 24 h at the maximum dose range of 5000 mg/kg and hence two different doses 200 and 400 mg/kg b.w. were taken for the study. This study revealed a significant antiulcer effect of the methanol leaf extract in experimental model of gastric lesion induced by Ethanol-HCl acid. The ulcer formation was inhibited up to 50.80 % and 76.10 % at the doses of 200 and 400 mg/kg body weight as shown in figs. 1 and 2 respectively. The total acidity value in meq/l of the methanol leaf extract at doses 200 and 400 mg/kg as shown in Table 1 registered 30.50 and 25.48, where as omeprazole (fig. 3) and control groups registered 18.21 and 87.93 meq/l respectively. The quantity of free acidity for both the doses were recorded as 30.50, 25.48 meq/l and that of the control and standard groups were 55.68 and 11.25 meq/l respectively. The ulcer index score for the two different doses of the extract were found to be 1.585 and 0.769 whereas for the control and standard groups were 3.219 and 0.30 respectively. In Modified Pyloric ligation model the findings of the studies revealed that the methanol leaf extract of A. officinalis posses significant anti-ulcer activity in terms of total acidity, free acidity and ulcer index when compared to solvent control (0.5 % DMSO), which is depicted in Table 2. The value of total acidity in meq/l of the methanol leaf extract at doses 200 and 400 mg/kg registered 58.76 and 33.16 respectively where as omeprazole and control groups registered 29.41 and 102.65 meq/l respectively. The quantity of free acidity for both the doses were recorded as 34.25, 25.48 meq/l and that of the control and standard groups were 62.35 and 20.75 meq/l respectively. The ulcer index score for the two different doses of the extract were found to be 1.083 and 0.583 whereas for the control and standard groups were 2.91 and 0.166 respectively. On the whole the percentage of ulcer inhibition for the methanol leaf extracts in different doses and standard was found to be 62.85, 80.00 and 94.30 respectively. This can be clarified by referring the figs. 4, 5 and 6. A peptic ulcer is an erosion or sore in the wall of the gastrointestinal tract. The mucous membrane lining the digestive tract erodes and causes a gradual breakdown of tissue. This breakdown causes a gnawing or burning pain in the upper middle part of the belly (abdomen). Although most peptic ulcers are small, they can cause a considerable amount of discomfort. Peptic ulcers occur when the acid and enzyme overcome the defense mechanisms of the gastrointestinal tract and cause erosion in the mucosal wall [15]. The Ethanol-HCl acid causes more severe gastric mucosal ulceration. The ulcers are caused either by a direct effect of the ethanol acid solution on the gastric epithelium, or are modulated indirectly by the release of vasoactive products from mast cells [16], resulting in the release of mediators such as histamine [17]. Ethanol induced gastric mucosal lesions, predominant in the glandular Pharmaceutical and Biological Evaluations 4

part of the stomach, are caused by the direct toxic action of ethanol, reduction of the secretion of bicarbonate and depletion of gastric wall mucus. Table 1: Biochemical Estimation of Methanol Extract of A. officinalis Leaves Treated in Ethanol-HCl Induced Rat Ulcer Model: Group I II III IV Body Weight (gm) 165.16 ± 10.527 154.5 ± 9.773 150.33 ± 9.969 161.16 ± 11.603 Acid Content (ml) 10.25 ± 1.199 4.42 ± 1.018 ** 6.99 ± 0.886 ns 6.22 ± 0.926 * ph 2.942 ± 0.341 5.018 ± 0.527 ** 3.420 ± 0.331 ns 4.272 ± 0.463 ns Free Acidity (m Eqv/L/100 gm) 55.68 ± 3.379 11.25 ± 2.225 ** 30.50 ± 8.004 * 25.48 ± 6.976 ** Total Acidity (m Eqv/L/100 gm) 87.93 ± 7.481 18.21 ± 1.693 ** 40.08 ± 7.911 ** 31.25 ± 7.028 ** Ulcer Index 3.219 ± 0.686 0.30 ± 0.144 ** 1.585 ± 0.487 * 0.769 ± 0.282 ** Values are given in mean ± SEM. *p<0.05, **p<0.01, for n=6. Group I: Toxic control (25 ml/kg of 0.3M HCl in 60% ethanol). Group II: Standard group (Omeprazole (20 mg/kg, p.o.)) and (25ml/kg of 0.3M HCl in 60% ethanol). Group III: Treated with methanol extract 200 mg/kg, p.o + (25ml/kg of 0.3M HCl in 60% ethanol). Group IV: Treated with methanol extract 400mg/kg, p.o + (25ml/kg of 0.3M HCl in 60% ethanol). ns: non-significant, NA: not applicable % of Ulcer Inhibition 0 (NA) 90.70 % 50.80 % 76.10 % Table 2: Biochemical Estimation of Methanol Extract of A. officinalis Leaves Treated in Modified Pylorus Ligated (Shay) Rat Ulcer Model. Group I II III IV Body Weight (gm) 164 ± 9.384 170.5 ± 6.386 162.83 ±9.414 156.66 ± 10.849 Acid Content (ml) 6.15 ± 0.624 3.32 ± 0.238 * 4.95 ± 0.966 ns 4.27 ± 0.976 ns ph 2.046 ± 0.173 4.106 ± 0.740 * 3.012 ± 0.587 ns 3.937 ± 0.397 * Pharmaceutical and Biological Evaluations 5 Free Acidity (m Eqv/L/100 gm) 62.35 ± 7.754 20.75 ± 4.165 ** 34.25 ± 8.755 * 25.48 ± 6.976 ** Total Acidity (m Eqv/L/100 gm) 102.65 ± 13.220 29.41 ± 6.946 ** 58.76 ± 8.563 ** 33.16 ± 6.453 ** Values are given in mean ± SEM. *p<0.05, **p<0.01. For n=6. Group I: Toxic control (Aspirin (200 mg/kg, p.o.))+ Pyloric ligation (PL). Group II: Standard group (Omeprazole (20 mg/kg, p.o.)) and Aspirin dose + PL). Group III: Treated with methanol extract 200 mg/kg, p.o. + Aspirin dose + PL. Group IV: Treated with methanol extract 400 mg/kg, p.o. + Aspirin dose + PL. ns: non-significant, NA: not applicable Ulcer Index 2.91 ± 0.396 0.166 ± 0.105 ** 1.083 ± 0.374 ** 0.583 ± 0.327 ** % of Ulcer Inhibition 0 (NA) 94.30 % 62.85 % 80.00 %

Fig. 1: Ulcer inhibition by the test drug at 200mg/kg (Ethanol-HCl induced ulcer) Fig. 2: Ulcer inhibition by the test drug at 400mg/kg (Ethanol- HCl induced ulcer) Fig. 3: Ulcer inhibition by the standard drug Omeprazole (Ethanol - HCl induced ulcer) Fig. 4: Ulcer inhibition by the test drug at 200mg/kg (Modified pyloric ligation model) Fig. 5: Ulcer inhibition by the test drug at 400mg/kg (Modified pyloric ligation model) Fig. 6: Ulcer inhibition by the standard drug Omeprazole (Modified pyloric ligation model) Pharmaceutical and Biological Evaluations 6

In the above Modified pyloric ligation model the initial damage by aspirin aggravates the further damage caused by pyloric ligation, which potentiates acid secretion and augmented mucus production in increased pepsin activity. The ligation of the pyloric end of the stomach causes accumulation of gastric acid in the stomach. This increase in the gastric acid secretion causes ulcers in the stomach. The lesions produced by this method are located in the lumen region of the stomach. The omeprazole and the extracts significantly decreased the total acidity and free acidity; this suggests that it is having an antisecretory effect. The above investigation revealed that the methanol leaf extracts of A. officinalis showed significant antiulcer activity in both the models, when compared with Omeprazole. The percentage of ulcer inhibition and the ulcer index measured shows strong evidence for the antiulcer activity of the leaf extract. Acknowledgement Authors are thankful to the H.O.D., U.D.P.S., for providing the necessary facilities to carry out the research work. References 1. Tandon BN. Digestive disease in India. Available from: http://www.isg.org/archive2.htm l. 2003. [accessed on 2008 Dec 16] 2. Parmar NS, Desai JK. A review of the current methodology for the evaluation of gastric and duodenal anti-ulcer agents. Indian J Pharmacol 1993; 25: 120 35. 3. Saxena HO, Brahmam M. The Flora of Orissa, Orissa Forest Development Corporation, Vol-III, Bhubaneswar. 1996; p. 1400. 4. Nadkarni KM. Indian Materia Medica, Popular prakashan Pvt. Ltd. 3 rd edition, Vol-3. 1982; p.466. 5. Kritikar KR, Basu BB. Indian Medicinal Plants. 2 nd ed., reprint. Jayyed Press. New Delhi. 1975; p. 6. 6. Ramanathan T, Thirunavukkarasu P, Ramkumar L, Shanmugapriya R. Antiulcer effect of Avicennia officinalis leaves in albino rats. World Applies Sciences Journal. 2009; 9(1): 55-8. 7. Mukherjee PK. Quality control of herbal drugs. Business Horizons. New Delhi, India. 2002; p.398. 8. Pani SR, Panda PK, Mishra S, Sahoo S, Nephroprotective effect of Bauhinia variegata (Linn.) whole stem extract, against Cisplatin induced Nephropathy in rats. Indian J Pharmacol. 2011; 43: 200-2. 9. Mizui T, Douteuchi M. Effect of polyamines on acidified ethanol induced gastric lesions in rats. Jap. J. Pharmacol. 1983; 33: 939-45. 10. Sanmugapriya E, Venkataraman S. Antiulcerogenic potential of Strychnos potatorum Linn. Seeds on Aspirin plus pyloric ligation-induced ulcers in experimental rats. Journal of Herbal Medicine. 2007; 4(5): 360-5. 11. Goel RK, Chakrabarti A, Sanyal AK. The effect of biological variables on the anti-ulcerogenic effect of vegetable plantain banana. Planta Med 1984; 51: 85 8. 12. Shay H, Kuarov SA, Fele SS, Meranze D, Gruenstein H, Siplet H. A simple method for uniform production of gastric ulceration in rat. Gastroenterol 1945; 5: 43-61. 13. Parmar NS, Desai JK. A review of the current methodology for the evaluation of gastric and duodenal anti-ulcer agents. Indian J Pharmacol 1993; 25: 120 35. Pharmaceutical and Biological Evaluations 7

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