In vivo antiulcer activity of the aqueous extract of Bauhinia purpurea leaf

Journal of Ethnopharmacology 137 (2011) 1047–1054

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In vivo antiulcer activity of the aqueous extract of Bauhinia purpurea leaf Z.A. Zakaria a,∗ , E.E. Abdul Hisam b , M.S. Rofiee b , M. Norhafizah c , M.N. Somchit a , L.K. Teh d , M.Z. Salleh d a

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Pharamceutical Science, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia c Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia d Pharmacogenomics Center, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia b

a r t i c l e

i n f o

Article history: Received 10 March 2011 Received in revised form 12 July 2011 Accepted 12 July 2011 Available online 23 July 2011 Keywords: Bauhinia purpurea Fabaceae Aqueous extract Antiulcer Saponins Sugar-free polyphenols

a b s t r a c t Ethnopharmacological relevance: Bauhinia purpurea (Fabaceae) is a medicinal plant traditionally used to treat various ailments, including ulcers. In order to establish pharmacological properties of the leaf of Bauhinia purpurea, studies were performed on antiulcer activity of the plant’s aqueous extract. Materials and methods: The Bauhinia purpurea aqueous extract (BPAE) was prepared in the doses of 100, 500 and 1000 mg/kg. Antiulcer activity of BPAE was evaluated by absolute ethanol- and indomethacininduced gastric ulcer, and pyloric ligation models. Acute toxicity was also carried out. Results: BPAE, at the dose of 5000 mg/kg, did not cause any signs of toxicity to rats when given orally. Oral administration of BPAE exhibited antiulcer activity (p < 0.05) in all models used. However, the dosedependent activity was observed only in the absolute ethanol-induced gastric ulcer model. Histological studies supported the observed antiulcer activity of BPAE. In pyloric ligation assay, BPAE increased the gastric wall mucus secretion. Conclusions: The BPAE exhibits antiulcer activity, which could be due to the presence of saponins or sugar-free polyphenols, and, thus, confirmed the traditional uses of Bauhinia purpurea in the treatment of ulcers. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Plants have been a valuable source of new molecules and considered as an alternative strategy in search for new drugs. There is a rich abundance of plants used in traditional medicine known to possess antiulcer properties that may, after possible chemical manipulation, provide new and improved antiulcer drugs (Shah et al., 2006; Shokunbi and Odetola, 2008). One of the plants that have been studied in our laboratory for its antiulcer activity is Bauhinia purpurea L. Bauhinia purpurea belongs to the family of Fabaceae and commonly known as camel’s foot tree or purple orchid tree. In Malaysia, the tree is known as ‘tapak kerbau’ or ‘tapak kuda’ while in India, where the plant is widely used, Bauhinia purpurea is known as ‘kachnar’ or ‘khairwal’ (Zakaria, 2007; Zakaria et al., 2007a,b). The plant is native in Southern Asia, Southeast Asia, Taiwan and Province of China but can also be found in Australia, United States of America and Puerto Rico. Generally, the plant is used in traditional medicine for the treatment of dropsy, pain, rheumatism, convulsions, delirium, and septicaemia (Asolker et al., 2000) and, for curing body pain, fever,

∗ Corresponding author. Tel.: +60 3 89472654. E-mail addresses: [email protected], dr [email protected] (Z.A. Zakaria). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.07.038

ulcers, cancerous growth in the stomach and indigestion (Kirthikar and Basu, 2001; Janardhanan et al., 2003). In addition, the root, stem, bark and leaves are used against diseases such as jaundice, leprosy, and cough and also in several Ayurvedic medicine formulation (Parrota, 2001). Other than that, in India, the bark of Bauhinia purpurea is used traditionally as an astringent in diarrhoea treatment while its root is also used for treating carminative, diarrhoea, ulcer, boils and abscesses (Chopra et al., 1956; Kirtikar and Basu, 1984). In Pakistan, the fresh and dried flower buds of Bauhinia purpurea are used as a food material while the leaves, stems and roots are widely used to treat infections, pain, diabetes, jaundice, leprosy and cough and also in several medicinal formulations (Morais et al., 2005). Despite its claimed medicinal uses in Southern Asia, there is no documentation found for its uses as medicine in other continents. Several pharmacological studies have been carried out on different parts of Bauhinia purpurea. Generally, the plant exhibited antidiarrheal (Mukherjee et al., 1998), antispasmodic and antimicrobial (Silva et al., 2000), antinociceptive, anti-inflammatory and antipyretic (Silva et al., 2000; Zakaria et al., 2007a, 2009), in vitro antiproliferative and antioxidant (Zakaria, 2007; Zakaria et al., 2011), antimycobacterial, antimalarial, antifungal, cytotoxic, and anti-inflammatory (Boonphong et al., 2007), thyroid stimulating and anti-hypothyroidism (Panda and Kar, 1999; Jatwa

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and Kar, 2009), wound healing (Ananth et al., 2010), antinephrotoxicity (Lakshmi et al., 2009), and larvicidal (Rao Gururaj and Balasubramaniam, 1999) activities. Efforts to identify the bioactive compounds presence in various parts of Bauhinia purpurea lead to isolation of kaempferol, quercetin and isorhamnetin (Salatino et al., 1999) have pacharin and bauhiniastatins 1–4 (Yadava and Tripathi, 2000) and, dihydrodibenzoxepins, a dihydrobenzofuran, a novel spirochromane2,1 -hexenedione, and a new bibenzyl (Boonphong et al., 2007). In addition (Zakaria et al., 2007a), reported the presence of flavonoids, triterpenes, tannins and steroids. Despite various claims on Bauhinia purpurea medicinal uses, particularly its potential to heal ulcer, no attempt has been made, to our best knowledge, to scientifically confirm on this matter. Thus, the aimed of the present study was to evaluate the gastroprotective effect of Bauhinia purpurea leaf aqueous extract using various types of ulcerogenic models. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (180–220 g) were used. The animals were fed with standard pellet with free access to tap water and kept in standard conditions of 12 h dark/12 h light. Fasting was applied prior to all assays as standard drugs and extract were administered orally (by gavage) with distilled water (10 ml/kg) as the vehicle. 2.2. Plant material and extract preparation Leaves of Bauhinia purpurea were collected in Shah Alam, Selangor, Malaysia between February and July 2009. The dried leaves were grinded into small particles and soaked in distilled water (dH2 O) in the ratio of 1:20 (w/v) and left for 24 h. Then, the mixtures were filtered using cloth filter, cotton wool and Whatman No. 1 filter paper to obtain the supernatant, which was subjected to freeze drying process. The extraction processes was repeated three times up to 72 h using the same residue. This method was carried out according to Zakaria et al. (2007b) with slight modifications. 2.3. Acute toxicity studies The acute toxicity study of Bauhinia purpurea aqueous extract (BPAE) was performed in a single dose administration of 5000 mg/kg (p.o.) (Mohamed et al., 2011). Rats were fasted for 24 h before the administration of BPAE. A group that received dH2 O represented the control group. The toxicity signs and symptoms or any abnormalities were observed at 0, 30, 60, 120, 180 and 240 min after BPAE administration. The observation was continued once a day for the next 14 days. The number of rats that survived was recorded at the end of the study period. 2.4. Antiulcerogenic activity 2.4.1. ETH-induced ulcer The experiment was carried out according to Noor et al. (2006) with slight modifications. BPAE (100 mg/kg, 500 mg/kg and 1000 mg/kg), dH2 O (10 ml/kg) or omeprazole (30 mg/kg) was administered orally to 48 h fasted rats. After 30 min, ulcer was induced using 1 ml/200 g body weight absolute ethanol (ETH). Fifteen minutes later, the rats were anesthetized using diethyl ether and then euthanized by cervical dislocation. The stomachs were removed and opened along the greater curvature. All the stomachs were gently rinsed with water to remove the gastric contents and blood clots. After the identification of ulcer areas, the length

of ulcer was measured and the number of ulcer spots were considered equivalent to 1 mm2 of ulcer (Cho and Ogle, 1979). The ulcer area (UA) in mm2 was determined as the total sum of gastric lesions for each stomach in the group. The protection percentage was calculated using the following formula: Protection (%) =

UA control − UA pre-treated group × 100 UA control

2.4.2. IND-induced ulcer The experiment was carried out according to Nwafor et al. (2000) with slight modifications. BPAE (100 mg/kg, 500 mg/kg and 1000 mg/kg), dH2 O (10 ml/kg) or omeprazole (30 mg/kg) were administered orally to 48 h fasted rats. 30 min after treatment, all the rats received indomethacin (IND) (100 mg/kg) to induce gastric ulcer. After 4 h, the rats were anesthetized using diethyl ether and then euthanized by cervical dislocation. The stomachs were removed and opened along the greater curvature. All the stomachs were gently rinsed with water to remove the gastric contents and blood clots. The macroscopic analysis was done as described earlier. 2.4.3. Histopathological analysis Gastric tissue samples from each group were fixed in 10% formalin. Then, the formalin fixed specimens were embedded in paraffin and sectioned (3–5 ␮m) and further stained with haematoxylin and eosin dye. The sections were evaluated by light microscopy and photographed. This method was applied for both inducers. 2.4.4. Pylorus-ligation Pylorus ligation was carried out according to the method by Shay et al. (1945) with slight modifications. Thirty minutes after the oral administration of BPAE (100, 500 and 1000 mg/kg), dH2 O (10 mL/kg) or omeprazole (30 mg/kg) as positive control, pylorus ligation was performed. The rats were lightly anesthetized by diethyl ether and the abdomen was opened without damaging any blood supply. Then its pylorus was ligated. The abdomen was closed by suturing and the rats were allowed to recover for 4 h. After 4 h, the animals were anesthetized using diethyl ether and then euthanized by cervical dislocation. The abdomen was opened and a ligature was placed around the oesophagus junction. The stomachs were removed and the content was measured before drained into a centrifuge tube and subjected to centrifugation at 3000 rpm for 10 min. pH of the gastric secretion was recorded with a pH meter. The total acidity of the gastric secretion was determined by titration with 0.01 N NaOH and phenolphthalein as indicator. The total acidity is expressed as mequiv./l using the following formula: n × 0.01 × 40 × 1000 where n is volume of NaOH quantified, 40 is the molecular weight of NaOH, 0.01 is normality of NaOH and 1000 is the factor represented in litre. The gastric lesion was also examined as described earlier. 2.4.5. Determination of gastric wall mucus content Gastric wall mucus content was determined by the method described by Corne et al. (1974) with slight modifications. The stomach was opened along the greater curvature, weighed and immersed in 10 ml of 0.1% Alcian blue in 0.16 M sucrose/0.05 M sodium acetate, pH 5.8 for 2 h. Then, the excessive dye was removed by two successive rinsed in 0.25 M sucrose solution (15 min; 45 min). The remaining dye complexed with the gastric mucus were then extracted with 0.5 M MgCl2 for 2 h and shaken intermittently in every 30 min. The blue extract was then shaken vigorously and the optical density was measured using a spectrophotometer at 580 nm. The quantity of Alcian blue extract per gram of wet stomach was calculated from the standard curve.

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2.5. Phytochemical screening and HPLC profiling of the BPAE 2.5.1. Phytochemical screening of the BPAE The phytochemical screening of BPAE was carried out according to the methods described by Ikhiri et al. (1992). 2.5.2. Preparation of sample for HPLC analysis Ten mg of crude dried BPAE was dissolved in 1 ml methanol and filtered through a membrane filter with pore size of 0.45 ␮m prior to analysis. 2.5.3. HPLC analysis The HPLC profile of BPAE was analysed by means of a HPLC system (Waters Delta 600 with 600 Controller) with photodiode array detector (waters 996) (Milford, MA, USA). A Phenomenex Luna (5 ␮m) (Torrance, CA, USA) column was used (4.6 mm i.d. × 250 mm) and for elution of the constituents, two solvents denoted as A and B were employed. A was 0.1% aqueous formic acid and B was 0.1% formic acid in acetonitrile. Initial conditions were 85% A and 15% B with a linear gradient reaching 25% B at t = 12 min. This was maintained for 10 min after which the programme return to the initial solvent composition at t = 25 min and continued for 10 min. The flow rate used was 0.1 ml/min and the injection volume was 10 ␮l. The column oven was set at 27 ◦ C and the eluant was monitored at 254, 300 and 366 nm. The retention times and UV spectra of major peaks were analysed. The HPLC analysis was carried out in the Laboratory of Phytomedicine, Medicinal Plants Division, Forest Research Institute of Malaysia (FRIM), Kepong, Malaysia. 2.6. Statistical analysis The results were express as mean ± S.E.M. and analysed using One-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison tests. Results were considered significant when p ≤ 0.05. 3. Result 3.1. Percentage yield of BPAE and acute toxicity study Dried leaves of Bauhinia purpurea (800 g) was soaked in dH2 O to yield approximately 21.3 g (≈2.7%) BPAE. A single oral administration of BPAE at 5000 mg/kg did not produce any symptom or sign of toxicity in the treated animals after observation for 14 days. 3.2. ETH-induced ulcer

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Table 1 Effect of BPAE on ETH-induced gastric lesions in rats. Ulcer model

Ethanolinduced

Pre-treatment

Dose (mg/kg)

Ulcer area (mm2 )

Control Omeprazole

– 30 100 500 1000

99.0 41.3 86.8 25.5 24.5

BPAE

± ± ± ± ±

14.5a 10.3b 13.5a 10.6b 5.6b

Protection (%) – 58.3 12.3 74.2 75.3

Values are presented as mean ± S.E.M. (n = 6). Means with different letter differed significantly (p < 0.05) when compared to control group.

500 and 1000 mg/kg BPAE demonstrated significant reduction in the total ulcer area, which are recorded as 4.16, 5.16 and 7.00 mm2 , respectively when compared to the control group. The ED50 value obtained for this assay was approximately 74.3 mg/kg. 3.4. Histopathological studies of ulcer-induced stomach 3.4.1. BPAE effect on the ethanol-induced gastric ulcer in rats Histological observation of ethanol-induced stomach tissue pretreated only with dH2 O (control group) showed marked lesions and severe damage to the gastric mucosa, hemorrhagic erosion, oedema and leucocytes infiltration of the submucosal layer (Fig. 1). On the other hand, stomach tissues of ethanol-induced rats pretreated with 100 and 500 mg/kg BPAE demonstrated mild lesions to almost normal architecture of the mucosa with moderate to mild effects of hemorrhage and oedema, respectively (Table 2). Interestingly, the tissue after pretreatment with 1000 mg/kg BPAE exhibited almost normal mucosal architecture with mild effects of hemorrhage and oedema. Overall, tissues that received pretreatment with BPAE had comparatively better protection of the gastric mucosa in a dosedependent manner as indicated by reduction in ulcer area, reduced or absence of submucosal oedema and infiltration of leucocytes. For comparison purposes, stomach of omeprazole pretreated rats showed moderate protection of the mucosa with moderate hemorrhage and oedema developed. 3.4.2. BPAE effect on the IND-induced gastric ulcer in rats Histological examination of IND-induced stomach tissue pretreated only with dH2 O (control group) showed normal mucosa architecture but accompanied by mild hemorrhagic and moderate oedema (Fig. 2). On the other hand, stomach tissues of rats pretreated with 500 and 1000 mg/kg BPAE demonstrated mild oedema while groups pretreated with 100 mg/kg BPAE or omeprazole exerted normal mucosal architecture. Overall, pre-treatment with BPAE demonstrated significant antiulcer activity in a doseindependent manner.

Gross pathological studies of the stomachs removed from ETHinduced animals that were not pre-treated with any test solutions (control group) showed complete ulceration. On the other hand, pretreatment with BPAE, at the doses of 500 and 1000 mg/kg, exhibited significant reduction of gastric lesion with percentage of protection of 74.2% and 75.3%, respectively when compared against the control group. In term of ulcer area developed, both doses of BPAE produced low ulcer area that are 25.5 and 24.5 mm2 (Table 1), respectively and gives better protection when compared to the group of rats that was treated with a standard drug, omeprazole. The ED50 value obtained for this assay was approximately 363.6 mg/kg. The histopathological evaluation is summarized in Table 2.

3.5. Pylorus-ligation

3.3. IND-induced ulcer

The phytochemical screening of BPAE demonstrated the present of only saponins, but no flavonoids, triterpenes, alkaloids, tannins and steroids. The HPLC profile of BPAE analysed at three different wavelengths, namely 254, 300 and 366 nm was shown in

IND, at the dose of 100 mg/kg, showed severe gastric lesion as seen in the control group (Table 3). Pretreatment with 100 (Fig. 2b),

In the pylorus ligation model, only 500 mg/kg BPAE exerted a significant reduction in the total ulcer area when compared with the control group with only approximately 0.5 mm2 ulcer area recorded (Table 4). Omeprazole was found to produce approximately 2.8 mm2 ulcer area. The dose-independent activity of BPAE was suggested as the highest dose (1000 mg/kg) of BPAE failed to reduce the ulcer area. Pretreatment with 500 mg/kg BPAE also produced the highest gastric wall mucus content (Table 5). However, it did not elevate the pH nor decrease the total acidity. 3.6. Phytochemical constituents and HPLC Profile of the BPAE

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Table 2 Histopathological evaluation of BPAE on ETH-induced gastric lesion in rats. Pre-treatment group

Mucosal epithelium

Glands

Hemorrhage

Oedema

Inflammatory exudates

Leucocytes infiltrate

Cellular debris

Distilled water 30 mg/kg omeprazole 100 mg/kg BPAE 500 mg/kg BPAE 1000 mg/kg BPAE

+++ ++ + + +

− − − − −

+++ ++ ++ + +

++ ++ + + +

− − − − −

++ − − − −

+ − − − −

(−) normal; (+) mild effect; (++) moderate effect; (+++) severe effect.

Table 3 Effect of BPAE on IND-induced gastric lesions in rats. Ulcer model

Pre-treatment

Dose (mg/kg)

Ulcer area (mm2 )

Control Omeprazole

– 30 100 500 1000

11.5 0.0 4.2 5.2 7.0

Indomethacin-induced BPAE

± ± ± ± ±

0.9a 0.0c 1.3b 1.6b 1.4b

Protection (%) – 100.0 63.8 55.1 39.1

Values are mean ± S.E.M. (n = 6/group). Means with different letter differed significantly (p < 0.05) when compared to control group.

Fig. 1. Histological evaluation of antiulcer activity of BPAE against ETH-induced gastric lesions in rats. (a) Stomach of an ulcer control rat; (b) stomach of a rat pre-treated with 1000 mg/kg BPAE; (c) stomach of a rat treated with 500 mg/kg BPAE; (d) stomach of a rat treated with 100 mg/kg BPAE; (e) stomach of a rat pre-treated with 30 mg/kg omeprazole. Respective histopathological sections are shown down; (f) stomach of the control animal showing severe effect on mucosa with hemorrhagic erosion, oedema, moderate infiltrate leucocytes and cellular debris; (g) stomach of 1000 mg/kg-BPAE-treated animals showing almost normal mucosa with mild effect of hemorrhage and oedema; (h) stomach of 500 mg/kg BPAE-treated animals showing almost normal mucosa with mild effect of hemorrhage and oedema; (i) stomach of 100 mg/kg BPAE-treated animals showing mild effect on mucosa with moderate effect of hemorrhage and mild effect on oedema; (j) stomach of 30 mg/kg omeprazole-treated animals show moderate effect on mucosa with moderate hemorrhage and oedema.

Fig. 3a. Two peaks with the retention time (RT ) of 9.050 and 11.752 appeared in the chromatogram analysed at all wavelength demonstrated two max in the region of 240–280 nm and 300–380 nm (Fig. 3b), thus, suggesting the presence of flavonoids, most probably glycosides of a flavonol. 4. Discussion Peptic ulceration, considered to be one of the modern age epidemics, has been affecting approximately 10% of world pop-

ulation (Shah et al., 2006). Earlier study suggested that peptic ulcer is due to an imbalance between acid and pepsin along with the weakness of the mucosal barrier (Aebi, 1984). Due to these, it is commonly associated with damage of the stomach’s mucosal layer, which is simply generated via excess generation of exogenous and endogenous active oxygen and free radicals. Some of the main causes of gastric ulcers include chronic use of alcoholic beverages and anti-inflammatory drugs, as well as stress and Helicobacter pylori infection (Barocelli et al., 1997).

Table 4 Effect of BPAE on gastric juice parameters in pylorus ligation model in rats. Pre-treatment Control Omeprazole

BPAE

Dose (mg/kg)

Ulcer area (mm2)

Protection (%)

Volume (ml)

pH (unit)

Total acidity (mequiv./l)

– 30

7.5 ± 0.8b 2.8 ± 0.5a

– 62.7

7.7 ± 0.3b 3.7 ± 0.3a

1.3 ± 0.02a 3.3 ± 0.07b

3948.8 ± 239.1a 2430.0 ± 153.7b

100 500 1000

6.6 ± 0.8b 0.5 ± 0.2a 5.5 ± 0.7b

12.0 93.3 26.7

7.0 ± 0.9b 8.7 ± 0.5b 8.2 ± 0.8b

1.2 ± 0.01c 1.2 ± 0.03c 1.2 ± 0.03c

4920.8 ± 156.1c 4860.0 ± 140.4c 5042.2 ± 275.0c

Values are mean ± S.E.M. (n = 6/group). Means with different letter differed significantly (p < 0.05) when compared to control group.

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Fig. 2. Histological evaluation of antiulcer activity of BPAE against IND-induced gastric lesions in rats. (a) Stomach of an ulcer control rat; (b) stomach of a rat pre-treated with 1000 mg/kg BPAE; (c) stomach of a rat treated with 500 mg/kg BPAE; (d) stomach of a rat treated with 100 mg/kg BPAE; (e) stomach of a rat pre-treated with 30 mg/kg omeprazole. Respective histopathological sections are shown down; (f) stomach of the control animal showed severe mucosal damage with mild hemorrhagic and moderate oedema; (g) stomach of 1000 mg/kg BPAE-treated animals showing slight damage to the mucosa layer with the presence of moderate hemorrhage and oedema; (h) stomach of 500 mg/kg BPAE-treated animal showing mild hemorrhage and oedema; (i) stomach of 100 mg/kg BPAE-treated animals showing slight mucosal damage with mild hemorrhagic and moderate oedema; (j) stomach of 30 mg/kg omeprazole-treated animals also showed normal architecture.

In the acute toxicity study, the BPAE, at the dose of 5000 mg/kg, exhibited no signs of toxicity. According to toxicologists throughout the world, any test substance that is not lethal on acute administration at a concentration of 5000 mg/kg body weight is essentially non toxic (Gregory et al., 2009). Based on the toxicity study, the current doses regime (100, 500 and 1000 mg/kg) was chosen for the antiulcer study. The extract, at the doses range of 100–1000 mg/kg, exhibited a non-dose-dependent gastroprotective activity in all models of gastric ulcer used. ETH is one of the factors that may increase the risk of gastric ulcer formation (Ray et al., 1990) and, thus, is widely used to induce experimental gastric ulcer in animals (Sheeba and Asha, 2006). According to Bagchi et al. (1998) ETH increases super oxide anion and hydroxyl radical production and lipid peroxidation in the gastric mucosa, and together with other reactive metabolites react with most of the cell components. These lead to changes in the cells’ structures and functions, or contributing to other mechanisms that ultimately help enhanced oxidative damage. Earlier studies revealed that ETH induces gastric mucosal injury by causing extensive damage to mucosal capillaries resulting in increased vascular permeability, oedema formation and epithelial lifting (Szabo et al., 1985; Kato et al., 1990; Nordmann, 1994). Furthermore, patients with gastric ulcer are at high risk for gastric cancer development (Hansson et al., 1996). The ETH-induced ulcer model was used to screen drugs for cytoprotection activity (Bighetti et al., 2005) and the ability of BPAE to reduce ETH-induced gastric ulcer partly suggested the involvement of local and nonspecific mech-

Table 5 Effect of BPAE on gastric wall mucus secretion. Pre-treatment Distilled water Omeprazole

BPAE

Dose (mg/kg)

Gastric wall mucus (Alcian blue ␮g/g wet tissue)

– 30

58.3 ± 3.4 36.1 ± 0.5a

100 500 1000

50.8 ± 2.3b 104.9 ± 3.1c 79.2 ± 5.3b

Values are mean ± S.E.M. (n = 6/group). Means with different letter differed significantly (p < 0.05) when compared to control group.

anism called adaptive cytoprotection. Cytoprotection may occur due to the capacity of some compounds to induce prostaglandin production, which in turn stimulates mucus and bicarbonate synthesis (Robert et al., 1983). According to Rachchh and Jain (2008), other than free radicals, ulcers induced by chemicals like ETH are due to several contributing factors including effects on mucosal blood flow, platelet thrombi, damage to capillary endothelium, and release of arachidonate metabolites, leukotriene C4/D4 (LTC4/D4), and platelet activating factor (PAF). Thus, the protection afforded by BPAE in ETH model can be linked to decrease in vascular permeability and, in so doing, preventing damage to the capillary endothelium and release of arachidonate metabolites. The ability of BPAE to reduce ETH-induced gastric ulcer is further suggested to be attributed to its previously reported anti-inflammatory (Zakaria et al., 2007a) and antioxidant (Zakaria et al., 2011) effects. Tsukimi et al. (1996) reported that suppression of neutrophil infiltration during inflammation enhanced gastric ulcer healing. Administration of antioxidants, on the other hand, has been demonstrated to inhibit ETH-induced gastric injury in rat (Ligumsky et al., 1995; Abdulla et al., 2010). Thus, it is speculated that the BPAE’s antiulcer activity could be ascribed to its anti-inflammatory activity (Swarnakar et al., 2005). On the other hand, the IND-induced ulcer model was employed for screening antiulcerogenic activity because the model shows cytoprotection and gastric acid secretion activities (Bighetti et al., 2005). IND, a nonsteroidal anti-inflammatory drug (NSAID), induced gastroduodenal ulceration via its ability to suppress prostaglandin synthesis (Wallace, 2001). The suppression of prostaglandin, which plays vital role in stimulating the secretion of bicarbonate and mucus, maintaining mucosal blood flow and regulating mucosal cell turnover and repair, results in increase susceptibility to mucosal injury and gastroduodenal ulceration (Hayllar and Bjarnason, 1995). The ability of BPAE to reduce INDinduced gastric ulcer suggested that the extract might be capable, in part, of regulating the synthesis of prostaglandins or stimulating the secretion of bicarbonate and mucus. Since BPAE demonstrated significant reduction of the ulcer lesions under experimental models that simulate the chronic uses of alcoholic beverages and NSAIDs conditions, we decided to determine the pharmacological mechanism involved. For this purpose,

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Fig. 3. (a) The HPLC profile of BPAE at various wavelengths. (b) The UV spectra analysis of peak 12 (RT = 9.050 min) and peak 15 (RT = 11.752 min) of the BPAE at 366 nm exhibiting the two max at 240–280 nm and 300–380 nm, suggesting the presence of flavonoid glycosides.

the pyloric ligation model was chosen (Schubert, 1994). Pylorusligation is an important procedure in determining the possible changes in parameters relative to the gastric content (de Barros et al., 2008). Pylorus ligated-induced ulcer is thought to be due to increase presence of acid and pepsin in the stomach. The important criteria that determine the status of mucosal defense barrier against the unpleasant attack of acid-pepsin is the quality and quantity of gastric mucus secretion (Rachchh and Jain, 2008). Increased mucus secretion by the gastric mucosal cells can prevent gastric ulceration

via a number of mechanisms, e.g. lessening stomach wall friction during peristalsis and acting as an effective barrier to the back diffusion of hydrogen ions. In addition, the ligation of the pyloric end of the stomach causes accumulation of gastric acid in the stomach, thus, agents that decrease the gastric acid secretion and/or increase mucus secretion are effective in preventing the ulcers induced by this method (Khare et al., 2008). In this assay, several parameters such as gastric wall mucus content, and gastric content’s pH, volume and hydrogenionic concentration were evaluated in animals

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after pretreatment with BPAE at various doses. Despite exhibiting antiulcerogenic activity, the BPAE significantly reduced the pH and increased the total acidity without altering the gastric volume when compared to the control group. In contrast to the BPAE action, omeprazole reduced the gastric volume and total acidity while increasing the pH of the gastric content. In addition, the BPAE increased the gastric wall mucus production, which might explain the extract ability to exert antiulcer activity in the pyloric ligation test. The non-dose-dependent activity exerted by BPAE in the INDinduced gastric ulcer and pyloric ligation models was suggested to be due to the ‘therapeutic windows’ effect (Tripathi, 2001). According to Tripathi (2001) a decrease in a drug’s effectiveness can sometimes be attributable to the presence of high concentrations of its active principle. Thus, the concentrations/doses of a particular drug have to be within its therapeutic window in order to achieve its maximum curative effect. In term of its phytochemical constituents, generally Bauhinia purpurea contains major class of secondary metabolites such as glycosides, flavonoids, saponins, triterpenoids, phenolic compounds, oxepins, fatty acids and phytosterols (Kumar and Chandrashekar, 2011). In other studies, Bauhinia purpurea leaf was reported to contain – in the sequence of high to low – steroids, saponins, triterpenes and flavonoids (Zakaria et al., 2007a) while BPAE was found to contain only saponins, but was high in total phenolic content (Zakaria et al., 2011). The low quantity of flavonoids presence in the leaf of Bauhinia purpurea (Zakaria et al., 2007a) might justify our failure to detect flavonoids in BPAE despite the occurrence of high total phenolic content (Zakaria et al., 2011). Based on the UV spectra of HPLC trace, it clearly suggested the presence of flavonoids, most probably flavonoid glycosides. It is well known that flavonoid glycosides are not very soluble in tap water, but are soluble in methanol:water and acetone:water solvent systems (Vilegas et al., 1999; Yes¸ilada and Takaishi, 1999; Yadav and Bhadoria, 2005). Thus, it is suggested that the presence of flavonoids glycosides in BPAE was attributed to the tensioactive effect of saponins, which are present in relatively high quantities when compared to the flavonoids. In addition, we have earlier carried out the HPLC analysis of BPAE and the extract was found to contain at least 2 major peaks when detected at 200 nm with their respective retention time of 15.03 and 20.98 min (Zakaria et al., 2007a). When comparison was made with flavonoids, namely catechin, rutin and fisetin, those major peaks failed to show any match with any of the standard flavonoids, further suggesting the presence of flavonoids of glycosyl derivatives. The presence of flavonoid glycosides in the leaf of Bauhinia purpurea have been reported earlier (Yadav and Bhadoria, 2005). Thus, it is suggested that saponins and flavonoid glycosides were responsible for the BPAE antiulcer activity (Vilegas et al., 1999; Yes¸ilada and Takaishi, 1999). Other than that, since phenolic compound are the major contributors to the antioxidant activities of plants (Abdulla et al., 2010) and are presence in BPAE, it is plausible to suggest the antiulcer activity of BPAE could be attributed to the extract’s polyphenolic antioxidant properties. In conclusion, the aqueous extract of Bauhinia purpurea possesses antiulcer activity, which is attributed to its antioxidant potential and the presence of saponins, flavonoid glycosides and high polyphenolic compounds. Thus, this finding confirms the traditional use of Bauhinia purpurea in the treatment of gastric ulcer.

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