EVALUATION OF ANTI ULCER ACTIVITY OF FICUS PUMILA L. LEAF EXTRACT IN ALBINO RATS

Abstract

The objective of this study was to investigate the gastro protective activity of ethanolic extract of leaves of Ficus pumila L. (Moraceae) in different experimental models of gastric ulcer in rats. Two doses (200 mg/kg and 400 mg/kg) of the extracts were used for the study. The experimental ulcers were induced by different models such as pyloric ligation, ethanol and cold restrained ulcer models. Omeprazole (10 mg/kg) and sucralfate 100 mg/kg) were used as the standard drugs. All drugs were administered by the oral route. The ulcer index and percentage of ulcer protection were measured. Phytochemical tests and various parameters such as free acidity, total acidity, total hexose, Hexosamine, Fucose and total protein were measured. The result showed that administration of the extract of Ficus pumila L. to the Albino rats significantly decreases the ulcer index with increase in the percentage of ulcer protection in a dose dependent manner when compared with control groups. Phyto-chemical analysis revealed the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes. It could be concluded that the ethanolic extract of leaves of Ficus pumila possess significant, dose dependent anti ulcer activity. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

The objective of this study was to investigate the gastro protective activity of ethanolic extract of leaves of Ficus pumila L. (Moraceae) in different experimental models of gastric ulcer in rats. Two doses (200 mg/kg and 400 mg/kg) of the extracts were used for the study. The experimental ulcers were induced by different models such as pyloric ligation, ethanol and cold restrained ulcer models. Omeprazole (10 mg/kg) and sucralfate 100 mg/kg) were used as the standard drugs. All drugs were administered by the oral route. The ulcer index and percentage of ulcer protection were measured. Phytochemical tests and various parameters such as free acidity, total acidity, total hexose, Hexosamine, Fucose and total protein were measured. The result showed that administration of the extract of Ficus pumila L. to the Albino rats significantly decreases the ulcer index with increase in the percentage of ulcer protection in a dose dependent manner when compared with control groups. Phyto-chemical analysis revealed the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes. It could be concluded that the ethanolic extract of leaves of Ficus pumila possess significant, dose dependent anti ulcer activity.

KEYWORDS: Anti-ulcer, Ficus pumila L., pyloric ligation, ethanolic extract, ethanol, cold restraint Ulcer.

(ProQuest: ... denotes formulae omitted.)

INTRODUCTION

The ulcer is a lesion of the gastric mucous membrane and it occurs probably due to an imbalance between the aggressive (acid, pepsin, bile and H. pylori) and the defensive (gastric mucus and bicarbonate secretion, prostaglandins, nitric oxide, innate resistance of the mucosal cells) factors (Rao et al., 2004). Gastric and duodenal ulcers are the two types of ulcer distinguished, which affects a considerable number of people in the world (Ineu et al., 2008). The infection with Helicobacter pylori and the use of non-steroidal anti-inflammatory drugs (NSAIDs) are the two essential causes for the development of gastric ulcer (O'Malley 2003). Outwards of these well identified causes certain other factors can increase the ulcer development risk such as alcohol consumption, cigarette smoking, psychological stress and dietary factors. It has been suggested that reactive oxygen species, primarily super-oxide anions, hydroxyl radicals, and lipid peroxides are the harmful species known to cause gastric ulcer development (Smith et al., 1996). Plant extracts, containing a wide variety of antioxidants such as phenolic and flavonoid compounds are some of the most attractive sources of new drugs and have been shown to produce promising results in the treatment of gastric ulcers (Hiruma-Lima et al., 2001).

Herbal medicine is fast emerging as an alternative treatment to synthetic drugs for treatment of ulcer possibly due to lower costs, availability, fewer adverse effects and perceived effectiveness (Thirunavukkarasu et al., 2009) & plants are more potent healers because they promote the repair mechanisms in the natural way (Veda Vidya T et al., 2012). In the regions of Tamil Nadu, many Ficus species like F. religiosa & F. virens are considered as the abode of spirits and worshiped in many forms accrediting them as the sthalavrikshas. (Umavathi R et al., 2012). In the present study one another plant from the Genus Ficus, named Ficus pumila L. of the Moraceae family was selected, which is a scandent shrub with evergreen coriaceous leaves that is normally grown between the trees as well as on fragmented surface. The leaves of the plant has been traditionally consumed by some Okinawan elders either as a beverage or used as an invaluable medicinal herb by the folks to treat diabetes, dizziness, high blood pressure, and neuralgia (Mitsuhashi, 1988; Tobinaga, 1989). Several studies have been performed on the composition of Ficus pumila L.; phytochemical analysis was performed and confirmed the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes. The important constituents isolated in the previous study were apigenin, luteolin, rutin, genistein, hesperidin, astragalin, isoquercitrin, and chrysin (Abraham et al., 2008). Although many other species of this genus such as Ficus deltoidea (Fathim Zahra et al., 2009), Ficus bengalenesis (Mayank Krishna et al., 2011), Ficus nervosa ( Rama Devi et al., 2012), Ficus religiosa (Sarmistha Saha et al., 2010), Ficus arnottiana (Gregory Marslin et al.,2009), Ficus indica (L.)Mill (Galati et al., 2002), Ficus hispida (Sivaraman et al., 2010), Ficus trichopoda (Balogun et al.,2011), Ficus glomerata (Rao et al., 2008), Ficus asperifolia (Raji Y et al., 2011) has been reported for anti-ulcer activity. However, the anti ulcer activity of Ficus pumila L. had never been investigated, thus the present study was initiated to evaluate the anti ulcer activity of ethanolic extract of leaves of Ficus pumila L. in Albino rats.

MATERIALS AND METHODS

Collection of Plant material and preparation of extract

The leaves of Ficus pumila L. were collected from the campus of Nandha college institution- Erode (Tamilnadu). The plant was identified and authenticated by the taxonomist Dr. G.V.S. Murthy, Botanical Survey of India, Tamilnadu Agricultural University Campus (TNAU), Coimbatore. The voucher specimen (BSI/SRC/5/23/2012-13/Tech-448) has been deposited in the herbarium of TNAU for future reference. The leaves were shade dried, powdered and were extracted using 70% ethanol as the solvent in a soxhlet apparatus until complete extraction. Solvent evaporation under reduced pressure was carried out to get semisolid extract which was used for the studies.

Experimental animal

The study was conducted on Wistar Albino rats of 150-200 g and was maintained under standard conditions (room temperature 240C- 270C and humidity 60-65%). Animals were provided with standard rodent pellet (M/s. Hindustan Lever Ltd, Mumbai) and had free access to water. Rats of either sex were selected and grouped in to four of six animals each. All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics committee (688/2/C-CPCSEA) of Nandha College of Pharmacy, (Proposal No- NCP / IAEC / No: 9/2012-13) and were in accordance with the guidelines of the IAEC.

Phytochemical screening:

The freshly prepared crude ethanolic extract of Ficus pumila L. was qualitatively tested for the presence of major phytochemical constituents according to standard methods (Trease, G.E., Evans, W.C. -1996)

Anti ulcer studies

a) Pyloric ligation induced ulcers:

Four groups of albino Wister male rats (n = 6) were selected. In this model, Group 1 served as normal control (vehicle) received 0.5% Carboxy methyl cellulose (CMC), p.o, and group 2 Omeprazole (10 mg/kg, p.o), whereas groups 3 and 4 animals received ethanolic extract of leaves of F. pumila L. (200 and 400 mg/kg, p.o. respectively) daily for 3 days. Animals were fasted overnight prior to starting the experiment and water ad libitum. Pyloric ligation was performed by ligating the pyloric end of the stomach of rats on 3rd day under Phenobarbital anaesthesia (35 mg/kg i.p), after 30 min of administration of ethanolic extract of F. pumila or Omeprazole. Animals were allowed to recover and stabilize in individual cages and were deprived of water during postoperative period (Ramaswami et al., 2010).

After 4 h of pyloric ligation, rats were sacrificed, stomach was removed and gastric juice was collected for performing gastric secretion study and for ulcer scoring. The gastric juice was collected and centrifuged. The volume and pH was recorded and subjected to bio-chemical estimations like free acidity and total acidity (Hawk et al.1947), total proteins (Lowry et al., 1951), total hexoses (Winzler et al., 1958), hexosamine (Dische et al .,1950) and fucose (Dische et al., 1948), ulcer was scored as follows.

Mean ulcer score for each animal will be expressed as ulcer index. The percentage of ulcer protection was determined as follows: Ulcer index (UI) was measured by using following formula

...

Where, UI = Ulcer Index; UN = Average number of ulcers per animal; US = Average number of severity score; UP = Percentage of animals with ulcers.

The following Table indicating Ulcer Score and Descriptive Observation.

Percentage inhibition of ulceration was calculated as below:

...

Gastric Secretion Study

The gastric juice was collected 4 h after pyloric ligation and the gastric volume and pH were measured. The collected gastric juice was centrifuged for 5 min at 2000 rpm. The volume of the supernatant was expressed as ml/100 g body weight. Free acidity and total acidity (Hawk et al., 1947) were determined by titrating with 0.01 M NaOH using Toepfer's reagent and phenolphthalein as indicator. Dissolved mucosubstances like total hexoses (Winzler et al., 1958), hexosamine (Dische et al., 1950), fucose (Dische et al.,1948) and total protein (Lowry et al., 1951) also were estimated.

b) Ethanol-induced ulcer

The overnight fasted rats were divided into four groups of six each. All the groups of rats were given treatments as follows by oral route: group 1 received 1 ml/kg of 5% CMC (control group), group 2 received 100 mg/kg sucralfate, group 3 received 200 mg/kg, and group 4 received 400 mg/kg of ethanolic extract of F. pumila L. Thirty minutes later, ulcers were induced by administering 1 ml of absolute ethanol (99%) to each rat. One hour later all the rats were sacrificed and the stomach were excised, cut along the greater curvature and gently rinsed under tap water. The stomachs were stretched on a corkboard and a magnifying glass (10X magnification) was used to spot and count the craters (Ukwe et al., 2010). The ulcer index was obtained by the sum of a group's crater score and divided by magnification. Ulcer inhibition (UI) was calculated as above.

c) Cold restraint induced ulcers

Four groups of Wistar Albino rats (n = 6) were selected. In this model, Group 1 served as Normal control (vehicle) received 0.5% CMC, p. o., and Group 2 Omeprazole (10 mg/kg, p.o), whereas Groups 3 and 4 animals received ethanolic extract of F. pumila (200 and 400 mg/kg, p.o. respectively) daily for 3 days. Animals were fasted overnight prior to start of the experiment, and water ad libitum. On day 3, after 30 min of administration of ethanolic extract of F. pumila or Omeprazole, rats were immobilized in a stress cage and were placed at 4-60C in an environmental cage (Ramaswami et al., 2010) The animals were sacrificed 2 h later and ulcer index was calculated following the method as described earlier.

Statistical analysis

The values were expressed as mean ± SEM. The statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Dunnet's't' - test. P values < 0.05 were considered significant.

RESULTS

a) Phytochemical analysis

The phytohemical analysis of the ethanolic extract of F. pumila L. revealed the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes.

b) Pylorus ligation induced model

The antiulcer activity of ethanolic extract of F. pumila L. was carried out by pylorus ligation induced ulcer model (Table 1). F. pumila L. showed significant dose dependent antiulcer effect at the dose of 200 mg/kg and 400mg/kg (p.o) with an ulcer index of (1.68 ± 0.14), (3.43 ± 0.09) and % ulcer protection (72.91), (79.56) respectively. This shows the decrease in ulcer index and increase in % of ulcer protection. It was compared with the standard drug Omeprazole. The bio-chemical parameter showed that there was a decrease in gastric volume, free acidity, total acidity and total protein while an increase in total hexose, hexosamine and fucose (Table 2 and 3).

c) Ethanol induced model

The antiulcer activity of ethanolic extract of F. pumila L. was carried out by ethanol induced ulcer model (Table 4). F. pumila L. treated groups were compared with sucralfate (P < 0.01). Pre-treatment of rats with F. pumila L. extracts produced a dose dependent protection in the ethanol induced ulceration model as compared to control group. F. pumila L. showed significant antiulcer effect at the dose of 200 mg/kg and 400 mg/kg (p.o) dose. 200 mg/kg and 400 mg/kg dose produced an ulcer index of (11.12 ± 0.32), (6.69 ± 0.41) and % protection (50.22), (70.85) respectively. This shows the decrease in ulcer index and increase in % of ulcer protection. It was compared with the standard drug sucralfate (81.15).

d) Cold restraint induced model

The effect of ethanolic extract of F. pumila L. in cold restraint stress ulcer model was shown in Table 5. Hypothermic and Immobilization stress produced considerable ulcero-genicity in rats. The ulcers were in the form of hemorrhagic mucosal lesions in the stomach, which were confined to the rugae of glandular segment. The parameters studied included ulcer index and % of ulcer protection. F. pumila L. treated groups were compared with Omeprazole (P < 0.01). F. pumila L. showed a significant dose dependent antiulcer effect at the dose of 200 mg/kg and 400 mg/kg (p.o).200 mg/kg and 400 mg/kg were produced an ulcer index of (6.46 ± 0.25) and (3.80 ± 0.29) and % ulcer protection of (68.30) and (81.35) respectively. This shows significant decrease in ulcer index and increase in % ulcer protection. It was compared with the standard drug Omeprazole which showed significant % of ulcer protection (86.90).

DISCUSSION

The etiology of peptic ulcer is unknown in most of the cases, yet it is generally accepted that it results from an imbalance between aggressive factors and defensive factors (Akah et al., 1998). To regain the balance, different therapeutic agents including plant extracts may be used (Gurbaz et al.,2003, Sairam . et al., 2003). The causes of gastric ulcerulceration by pyloric ligation are believed to be due to stress induced increase in gastric hydrochloric acid secretion and/or stasis of acid, and the volume of secretion is also an important factor in the formation of ulcer due to exposure of the unprotected lumen of the stomach to the accumulating acid (Raju et al .,2009). Pyloric ligation induces ulcers by auto digestion of the gastric mucosa and breakdown of the gastric mucosal barrier. These factors are associated with the development of upper gastrointestinal damage including lesions, ulcers and life threatening perforation and haemorrhage. Gastric wall mucus, an obligatory component of which is hexosamines, is thought to play an important role as a defensive factor against gastrointestinal damage (Davenport 1968). The determined gastric wall mucus was used as an

indicator for gastric mucus secretion, while mucosal hexosamine content was used as an indicator for gastric wall mucus synthesis (Lukie et al.,1972). In the present study, gastric wall mucus and hexosamine contents in ethanol induced ulcerated rats were markedly lowered when compared to those of the non-ulcerated group. It was found that pre-treatment with extract of F. pumila L. increased both gastric mucus and Hexosamine contents significantly. This finding indicates that the extract of F pumila L. can preserve both gastric mucus synthesis and secretion in the experimental rats.

Ethanol has also been reported to cause disturbances in gastric secretion, damage to the mucosa, alterations in the permeability, gastric mucus depletion and free radical production. This is attributed to the release of superoxide anion and hydroperoxy free radicals, during metabolism of ethanol as oxygen derived free radicals has been found to be involved in the mechanism of acute and chronic ulceration in the gastric mucosa (Jude et al., 2009) induced gastric lesion formation may be due to stasis in gastric blood flow which contributes to the development of the haemorrhage and necrotic aspects of tissue injury. Alcohol rapidly penetrates the gastric mucosa apparently causing cell and plasma membrane damage leading to increased intra cellular membrane permeability to sodium and water.

The massive intracellular accumulation of calcium represents a major step in the pathogenesis of gastric mucosal injury. This leads to cell death and exfoliation in the surface epithelium (Raju et al., 2009).

It was observed in this study that the extract significantly reduced ethanol induced ulcer. This may be due to cyto-protective effect of the extract via antioxidant effects. The extract shows protection against characteristic lesions produced by ethanol administration, this antiulcer effect of F. pumila L. may be due to both reductions in gastric acid secretion and gastric cyto-protection. The antiulcer property of F. pumila L. in pyloric ligation model is evident from its significant reduction in free acidity, total acidity, number of ulcers and ulcer index. F. pumila L. treated animals significantly inhibited the formation of ulcers in the pylorus ligated rats and also decreased both the concentration and increased the pH, which suggests that F. pumila L. can suppress gastric damage induced by aggressive factors.. The significant increase in the antiulcer activity of F. pumila L. could be attributed to the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes. Flavonoids are among the cytoprotective materials for which anti-ulcerogenic efficacy has been extensively confirmed. It suggests that, these active compounds would be able to stimulate mucus, bicarbonate and the prostaglandin secretion and counteract with the deteriorating effects of reactive oxidants in gastrointestinal lumen (Sakat et al .,2009). So the antiulcer activity of F. pumila L. may be attributed to its flavonoid content. The results of the present study suggest that the ethanolic extract of F. pumila L. leaves may be beneficial in the treatment of gastric lesions. Further studies are required to identify the active constituents and elucidation of the mechanism of action.

Cold restrained stress provides both emotional stress as well as physiological stress to the animal, which induces ulcer due to the auto digestion of gastric mucosal barrier, accumulation of HCl and generation of free radicals . The role of the free radicals in gastric ulcerations is well-documented (Cochran et al., 1983)

CONCLUSION

From this study, we can conclude that the ethanolic extract of F. pumila L. leaf extract have significant anti-ulcer activity in animal models. It has muco-protective activity and gastric anti-secretary when compared with that of the reference drug Omeprazole and Sucralfate. The anti-ulcer activity is probably due to the presence of flavonoids. Further studies are being carried out to characterize and explore the biological activity of the compounds present in the extract.

ACKNOWLEDGEMENT

We are deeply thankful to Mr. Nandha Kumar Pradeep, Secretary, Nandha College of Pharmacy and Research Institute for support and institutional facilities.

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Conflict of Interest: None Declared