J Nat Med DOI 10.

1007/s11418-011-0522-1

NOTE

Anti-inammatory and gastromucosal protective effects of Calotropis procera (Asclepiadaceae) stem bark
Nagesh Tour Gokul Talele

Received: 5 August 2010 / Accepted: 27 January 2011 The Japanese Society of Pharmacognosy and Springer 2011

Abstract This study was aimed at evaluating the antiinammatory and gastromucosal protective effect of chloroform extract (CH) and hydroalcoholic extract (HE) of the stem bark of Calotropis procera obtained successively by cold maceration. The anti-inammatory effect of the CH and HE extracts of the stem bark of Calotropis procera against carrageenan-induced paw oedema and also its gastromucosal protective effect has been studied by using two acute models: aspirin (100 mg/kg, p.o.) and ethanol (96%) in albino rats. CH and HE extracts showed signicant anti-inammatory activity at 200 and 400 mg/kg, while CH extract at 400 mg/kg was also found to have a signicant gastromucosal protective effect. As part of investigations to obtain compounds with anti-inammatory and gastromucosal protective effects in this work, a bioassay was carried out with fractions obtained from the CH extract with n-hexane (NF1), 1-butanol (BF1), ethyl acetate (EF1) and chloroform (CF1). The HE extract of the stem bark was fractionated with n-hexane (NF2), 1-butanol (BF2), ethyl acetate (EF2), chloroform (CF2) and water (WF2). The fractions were evaluated for their anti-inammatory and gastromucosal protective effects. Fractions NF1, CF1, BF2 and EF2 (20 mg/kg) showed signicant antiinammatory activity, while NF1 and BF2 (20 mg/kg) also showed gastromucosal protective effects. The results obtained for gastromucosal protective effects were also well supported by histopathological examination of the open excised rat stomach.
N. Tour (&) R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dhule 425405, Maharashtra, India e-mail: tournagesh@yahoo.co.in G. Talele Nashik Gramin Shikshan Prasarak Mandals College of Pharmacy, Anjaneri, Nashik 422005, Maharashtra, India

Keywords Calotropis procera Stem bark Asclepiadaceae Anti-inammatory Gastromucosal protection Healing effect

Introduction Currently, varieties of steroidal and nonsteroidal antiinammatory drugs (NSAIDs) are used for treating inammatory diseases. In general, NSAIDs act by inhibiting the metabolism of arachidonic acid by both the cyclooxygenase and lipoxygenase enzyme pathways [1]. Gastrointestinal bleeding and ulceration are the most common and severe adverse effects associated with NSAIDs [2], and thus safer compounds are needed. Although there are many products used for the treatment of gastric ulcers, most of these drugs produce several adverse reactions [3]. To investigate the effects of drugs on the acute phase of inammation, models using pro-inammatory agents such as carrageenan, dextran, formaldehyde, serotonin, histamine and bradykinin in rat paws are employed [4]. Carrageenan, a mucopolysaccharide, is perhaps the most commonly used and well-studied of these phlogistics [5], producing a maximal edema in 3 h. While the carrageenan model is typically associated with activation of the cyclooxygenase pathway and is sensitive to glucocorticoids and prostaglandin synthesis antagonists, the early phase of the carrageenan response is due to the release of serotonin and histamine [6]. Due to the growing interest in alternative therapies in recent years, the use of natural products, especially those derived from plants, is in demand [7, 8]. Calotropis procera (Ait.) R.Br. (Asclepiadaceae) stem bark extract is one such herbal drug, currently investigated in the present study primarily to evaluate its anti-inammatory and gastromucosal protective potential in rats. Calotropis procera is a

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medicinal plant distributed in northern, western and central India, and commonly known as Madar [9]. The latex of the plant is used for treating epilepsy, inammation, painful joints and swellings; the leaves are used as an antibacterial and antifungal and to alleviate ear pain; the root bark is used in skin diseases and as an anthelmintic; and the owers are used in loss of appetite. The plant contains the cardenolide, proceragenin [10], and is used in treating eye troubles [11]. Because of the potential medicinal value of Calotropis procera, interest in this plant is justiable for anti-inammatory and gastromucosal protective effects.

TLC, HPTLC and HPLC Qualitative evaluation of the extracts and their fractions were performed by thin-layer chromatography (TLC) on silica gel G plates. The solvent system for CH was benzene:ethylacetate (9:1) with Rf values 0.69, 0.53 and 0.21 for lupeol, b-sitosterol and oleanolic acid, respectively, and for HE, tolune:acetone:formic acid (6:6:1) with Rf values 0.59 and 0.46 for gallic acid and epicatechin, respectively. HPTLC was carried out with TLC silica gel 60 F254 aluminium precoated plates (Merck) using the same solvent systems as in the TLC. HPTLC (CAMAG) ngerprinting of the CH, NF1, BF1, EF1 and CF1 fractions was carried out using lupeol, b-sitosterol, oleanolic acid and betulinic acid as markers. Gallic acid and epicatechin were used as markers in an attempt to characterize the constituents in HE, NF2, BF2, EF2, CF2 and WF2. High-performance liquid chromatography (HPLC) was carried out by using an Exsil ODS column (250 9 4.6 mm, 5 lm particle size) revealing the presence of lupeol, b-sitosterol and oleanolic acid (2.0, 0.14 and 1.5% w/w, respectively) in the CH extract. Gallic acid and epicatechin were detected in the HE extract (1.6 and 0.19% w/w, respectively). The NF1 fraction showed the presence of lupeol and oleanolic acid whereas the CF1 fraction showed the presence of b-sitosterol. Epicatechin and gallic acid was detected in the BF2 and EF2 fractions, respectively (Figs. 1, 2). Animals Albino rats of Wistar strain of either sex weighing between 150 and 200 g were used. They were housed in standard cages at room temperature (25 2C) and provided with food and water ad libitum. The study was conducted after obtaining Institutional Ethical Committee clearance (RCPIPER/IAEC/ 2009-10/04). Selection of dose of the extract The LD50 was determined according to OECD guidelines for xing the dose for biological evaluation. The LD50 of the extract according to OECD guidelines falls under class four values with no signs of acute toxicity at 2000 mg/kg. The biological evaluation was carried out at doses of 100, 200 and 400 mg/kg body weight. Anti-inammatory activity of the extracts of Calotropis procera Carrageenan-induced rat paw edema Eight groups of six animals per group were used. The plant extract was administered orally at doses of 100, 200 and

Materials and methods Plant material Calotropis procera stem bark was collected from Dhule district, Maharashtra, India, during the month of May 2009. It was identied and authenticated by T. Chakraborty, Scientist-D, Joint Director, Botanical Survey of India, Pune, India. A voucher specimen has been deposited at the Herbarium of the Centre (V. No: CAPNAS1). Preparation of the extracts Stem bark was collected, shade-dried and powdered mechanically. About 1000 g of the stem bark powder was subjected to successive extraction with 2000 ml of chloroform and 2000 ml of hydroalcohol (ethanol:water 60:40) by maceration at room temperature for 48 h using a mechanical shaker. The extracts were dried at 40C under vacuum by using a Rota Evaporator (BUCHI Rotavapor R-215) and the yields were 5 and 6.6%, respectively. The chloroform extract was fractionated with n-hexane (NF1), 1-butanol (BF1), ethyl acetate (EF1) and chloroform (CF1) by using the solidliquid partition technique. The fractions were freeze-dried and were equivalentto 35.06, 24.45, 8.34 and 7.95%, respectively, of the dry CH. The hydroalcoholic extract (HE) of the stem bark was fractionated by using the liquidliquid partition technique with n-hexane (NF2), 1-butanol (BF2), ethyl acetate (EF2), chloroform (CF2) and water (WF2). The fractions were freeze-dried and were equivalentto 4.75, 10.25, 5.6, 15.9 and 46%, respectively, of the dry HE. Phytochemical screening In a preliminary phytochemical study, the chloroform extract showed the presence of triterpenoids and steroids, while successive hydroalcoholic extracts showed the presence of polyphenols and tannins.

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400 mg/kg, as was indomethacin at 10 mg/kg. Control rats received distilled water. The administration of extract and drug was carried out 30 min prior to injection of 0.05 ml of 1% carrageenan in the right hindpaw subplantar of each rat [12]. Remaining groups were treated with the CH and HE extracts at the oral dose of 100, 200 and 400 mg/kg, 1 h before carrageenan injection. The paw volume was measured using a plethysmometer, before injection and 6 times at 1-h intervals [13]. The antiinammatory activities in animals receiving CH and HE extracts were compared with those in the indomethacin and control groups. Gastromucosal protective effects of the extracts of Calotropis procera Aspirin-induced gastric mucosal lesions in albino rats Albino rats were divided into eight groups of six rats each. All the rats were starved for 24 h. After the fasting period, aspirin (100 mg/kg, p.o.) was given. All samples of the plant extracts were given 30 min prior to aspirin at three different doses, i.e. 100, 200 and 400 mg/kg. The animals were killed 5 h after the treatment. The stomach was cut open in the greater curvature and lesion scoring was done using a magnifying lens. The lesion was scored according to its severity in comparison with that of the standard as described in Lesion score, below. Histopathological studies were performed to conrm the lesion score [14]. The gastromucosal protective effects of the extracts were compared to those of ranitidine (100 mg/kg). Ethanol-induced gastric mucosal lesions in albino rats Rats fasted for 24 h were used. All samples of the plant extracts were given 30 min prior to ethanol treatment at three different doses, i.e. 100, 200 and 400 mg/kg. The animals were killed under anaesthesia using ether, 1 h after ethanol treatment. The stomach of each animal was excised and opened along the greater curvature. Lesion score was recorded as described below [15]. The gastromucosal protective effects of the extracts were compared to those of ranitidine (100 mg/kg). Lesion score The gastric mucosa was examined for lesions using a magnifying lens and the lesion scored according to its severity in comparison with that of the standard. Lesion score was recorded as follows: 0, normal, no lesion; 1, isolated haemorrhagic spot; 2, dense haemorrhagic spot; 3, small lesion; 4, large lesion; 5, perforation [16].

Fig. 1 HPLC prole (Exsil ODS column, 250 9 4.6 mm, 5 lm particle size) of chloroform extract showing presence of (a) lupeol (ow rate 0.7 ml/min), (b) b-sitosterol (ow rate 0.3 ml/min), (c) oleanolic acid (ow rate 0.2 ml/min). Mobile phase: CH3CN:H2O (90:10), detection: 210230 nm

Fig. 2 HPLC prole of hydroalcohol extract showing presence of epicatechin and gallic acid. Mobile phase: 0.1% H3PO4:CH3CN (85:15), detection: 280 nm, ow rate: 0.5 ml/min

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Histopathological evaluation of aspirin-induced and ethanol-induced gastric mucosal lesions Stomachs were immersed in a 10% formalin solution for histopathological examination following the assessment of lesion score. The central part of the damaged tissue (if present) was cut in half along the long diameter. If the stomach was protected from the damage then the section was taken from the basal part. After the standard processing, the wet tissue was embedded in parafn and cut into 5-lm thick sections in a rotary micrometer. The sections were stained with haematoxylineosin, mounted with Canada balsam, and examined under the microscope for histopathological changes such as oedema, inammation, inltration and erosion. Anti-inammatory activity of the fractions obtained from extracts of Calotropis procera Albino rats were divided into eleven groups of six rats each. Anti-inammatory activity was evaluated as for the plant extracts. All samples of the fractions were given orally 1 h before carrageenan injection at a dose of 20 mg/kg. Gastromucosal protective effects of the fractions obtained from extracts of Calotropis procera Aspirin-induced gastric mucosal lesions in albino rats All rats were starved for 24 h. After the fasting period, aspirin (100 mg/kg, p.o.) was given. All samples of the fractions were given 30 min prior to aspirin at a dose of 20 mg/kg. Lesion score was recorded as described above [14]. Ethanol-induced gastric mucosal lesions in albino rats Rats fasted for 24 h were used. All samples of the fractions were given 30 min prior to ethanol at a dose of 20 mg/kg. Lesion score was recorded as described above [15]. Statistical analysis The data on anti-inammatory activity are reported as mean standard error of the mean (SEM), and were compared using one-way analysis of variance (ANOVA), followed by Dunnetts test. The data on gastromucosal protective effect are reported as median (minimum value, maximum value), and were compared using the Kruskal Wallis test, followed by Dunns test. The statistical analysis was carried out by Graphpad Prism 5 software and p values \0.05 were considered signicant.

Results Anti-inammatory effects of extracts on carageenan-induced inammation Despite the fact that they have weaker anti-inammatory effects than indomethacin, both CH and HE of C. procera were found to have anti-inammatory effects at the doses of 200 and 400 mg/kg. The most potent drug was found to be indomethacin (55.6%), while the most potent extract was found to be CH of C. procera at doses of 200 and 400 mg/kg body weight (bw), with 52.0 and 66.0% acute anti-inammatory effects, respectively, obtained 2 h following carrageenan injection. The same extract in a dose of 100 mg/kg bw exerted only 23.0% anti-inammatory effect. For the HA of C. procera, acute anti-inammatory effects of 21.4% for 100, 43.6% for 200 and 52.0% for 400 mg/kg doses were found 2 h following carrageenan injection (Table 1). Anti-inammatory effects of fractions on carageenan-induced inammation In the acute phase of inammation, carrageenan-induced paw-volume increased, and the effects of the indomethacin and C. procera fractions were evaluated. Indomethacin showed 49.1% anti-inammatory activity 2 h after carrageenan injection. The fractions NF1, CF1, BF2, and EF2 showed inhibitions of 33.9, 30.5, 42.2, and 37.0%, respectively. 2 h following carrageenan injection (Table 2). Gastromucosal protective effect of extracts on aspirin-induced gastric lesions The doses of 200 and 400 mg/kg of the CH and HE showed negligible lesion score. Inhibition of 59.0 and 82.3% was observed with 200 and 400 mg/kg of the CH. Inhibition of 53.0 and 70.7% was observed with 200 and 400 mg/kg of the HE. Lesion scores were conrmed by histopathological studies. There was no ulceration and cell necrosis. The gastric mucosa was normal, which was comparable to the effect of ranitidine (Table 3). Gastromucosal protective effects of extracts on ethanol-induced gastric mucosal lesions Pretreatment with CH and HE offered protection against gastric mucosal lesions caused by ethanol (96%, 1 ml per 200 g bw) when compared to ranitidine (Table 3). CH extract showed 47.6 and 78.9% inhibition at 200 and 400 mg/kg; HE extract showed 42.3 and 63.1% inhibition at 200 and 400 mg/kg.

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Table 1 The effects of Calotropis extracts and indomethacin (Indom.) in carrageenan-induced acute paw volume (ml) 1h 2h 3h 4h 5h 6h

J Nat Med

Dose (mg/kg)

0h

Control 1.50 0.03 1.53 0.003 (48.7) 1.38 0.18* (27.3) 1.39 0.03* (36.4) 1.45 0.03 (43.9) 1.51 0.008 (21.3) 1.37 0.04* (33.4) 1.46 0.02 (37.3) 1.54 0.04* (43.6) 1.57 0.01* (52.0) 1.75 0.08* (33.6) 1.80 0.01* (38.7) 1.84 0.04* (20.2) 1.85 0.003* (30.8) 1.79 0.02 (21.4) 2.02 0.04 (14.1) 2.06 0.05 (5.0) 1.48 0.008* (66.0) 1.77 0.01* (43.4) 1.79 0.004* (37.6) 1.50 0.06* (52.0) 1.74 0.09* (37.3) 1.82 0.02* (25.1) 1.80 0.02* (26.7) 1.71 0.003* (45.5) 2.17 0.02* (-8.3) 1.90 0.01* (12.4) 1.80 0.07* (34.1) 1.67 0.21* (23.0) 1.90 0.08* (15.9) 1.92 0.08* (9.9) 2.02 0.13 (-3.7) 1.66 0.02* (55.6) 1.77 0.06* (53.4) 1.70 0.003* (58.2) 1.67 0.01* (61.0) 1.86 0.08 2.06 0.05 2.02 0.05 2.00 0.05 2.01 0.10 1.57 0.04* (72.3) 1.96 0.02 (4.3) 1.75 0.01* (32.5) 1.62 0.10* (56.4) 2.13 0.02* (-2.5) 1.86 0.01* (17.7) 1.74 0.06* (41.5)

1% (0.05 ml)

1.10 0.05

Indom.

10

1.32 0.08

CH

100

1.09 0.03

200

1.14 0.01

400

1.22 0.01

HA

100

1.20 0.01

200 400

1.11 0.01 1.21 0.01

Values are expressed as mean SEM for six rats. Percentage inhibitions are in brackets

* p \ 0.05 compared with the values before injection for each group

Table 2 The effects of Calotropis fractions and indomethacin (Indom.) in carrageenan-induced acute paw edema 1h 1.43 0.09 1.53 0.03* (41.2) 1.59 0.13* (14.3) 1.45 0.02 (-12.7) 1.32 0.01* (-17.5) 1.49 0.02 (11.1) 1.43 0.01 (-19.6) 1.56 0.03* (20.7) 1.36 0.02 (19.0) 1.34 0.02 (-13.3) 1.53 0.04* (15.2) 1.73 0.02 (30.5) 1.71 0.004* (11.2) 1.74 0.12 (42.2) 1.57 0.02* (37.0) 1.76 0.10 (-3.7) 1.70 0.03* (42.2) 1.62 0.01* (10.43) 1.80 0.05 (5.4) 1.81 0.18 (33.9) 1.73 0.02* (49.1) 1.86 0.09 2.06 0.05 1.77 0.02* (55.6) 1.92 0.02* (36.6) 1.93 0.02* (12.0) 1.76 0.16* (14.8) 1.91 0.02* (27.0) 1.80 0.03* (21.1) 1.89 0.10* (39.7) 1.76 0.03* (31.0) 1.77 0.09* (17.9) 1.89 0.05* (34.9) 2h 3h 4h 1.94 0.02 1.70 0.02* (57.5) 1.82 0.07* (39.3) 1.96 0.02 (-3.8) 1.73 0.17* (6.4) 1.80 0.01* (28.7) 1.85 0.07 (3.8) 1.81 0.04* (39.8) 1.79 0.01* (17.0) 1.72 0.11* (11.3) 1.93 0.02 (20.7) 5h 1.87 0.03 1.67 0.01* (57.9) 1.74 0.13* (44.1) 1.92 0.05 (-8.1) 1.76 0.06* (-5.8) 1.77 0.06* (27.3) 1.67 0.02* (18.1) 1.75 0.11* (42.2) 1.67 0.02* (25.7) 1.82 0.08 (-9.3) 1.88 0.02 (19.6) 6h 1.86 0.03 1.58 0.06* (68.5) 1.70 0.16* (48.3) 1.91 0.03 (-9.1) 1.68 0.13* (3.2) 1.66 0.03* (40.0) 1.76 0.01 (5.5) 1.77 0.10 (39.2) 1.59 0.01* (35.0) 1.76 0.07 (-4.2) 1.94 0.03 (9.9)

Dose

0h

Control

1% (0.05 ml)

1.10 0.05

Indom.

10

1.34 0.07

NF1

20

1.31 0.05

BF1

20

1.09 0.04

EF1

20

0.94 0.05

CF1

20

1.21 0.05

NF2

20

1.04 0.02

BF2

20

1.31 0.05

EF2

20

1.09 0.03

CF2

20

0.98 0.02

WF2

20

1.26 0.27

Values are expressed as mean SEM for six rats. Percentage inhibitions are in brackets

* p \ 0.05 compared with the values before injection for each group

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J Nat Med Table 3 Effects of Calotropis extracts on % inhibition of gastric mucosal lesions induced by aspirin and ethanol Treatment Dose (mg/kg) Aspirin-induced gastric lesion 100 mg/kg (aspirin) Lesion index Vehicle Ranitidine CH 100 100 200 400 HE 100 200 400 3.00 (2.00, 3.00) 0* 2.00 (1.00, 3.00) 1.00 (0, 2.00) 0.50 (0, 1.00)* 2.00 (1.00, 3.00) 1.00 (0, 3.00) 1.00 (0, 2.00) Inhibition (%) 100 29.3 59.0 82.3 23.3 53.0 70.7 Ethanol-induced gastric lesion 1 mL/200 g body weight (96% ethanol) Lesion index 3.50 (2.00, 4.00) 0 (0, 1.00)* 2.00 (2.00, 3.00) 2.00 (1.00, 2.00) 1.00 (0, 1.00)* 2.00 (1.00, 4.00) 2.00 (1.00, 2.00) 1.00 (0, 2.00) Inhibition (%) 89.6 26.5 47.6 78.9 26.5 42.3 63.1

Results are median (minimum value, maximum value) for six rats. Statistical comparison was performed using KruskalWallis test followed by Dunns test * Statistically signicant at p \ 0.05 (compared with control)

Table 4 Effects of Calotropis fractions on % inhibition of gastric mucosal lesions induced by aspirin and ethanol Treatment Dose (mg/kg) Aspirin-induced gastric lesion 100 mg/kg (aspirin) Lesion index Vehicle Ranitidine NF1 BF1 EF1 CF1 NF2 BF2 EF2 CF2 WF2 100 20 20 20 20 20 20 20 20 20 3.00 (2.00, 3.00) 0* 0 (0, 1.00)* 2.00 (1.00, 3.00) 1.00 (1.00, 2.00) 0.50 (0, 2.00) 1.50 (1.00, 3.00) 0 (0, 1.00)* 0 (0, 2.00)* 2.00 (2.00, 3.00) 2.00 (1.00, 3.00) Inhibition (%) 100 87.6 25.1 51.3 74.9 37.5 93.7 81.3 12.7 25.1 Ethanol-induced gastric lesion 1 mL/200 g body weight (96% ethanol) Lesion index 3.00 (2.00, 4.00) 0 (0, 1.00)* 0.50 (0, 1.00)* 2.00 (1.00, 3.00) 2.00 (1.00, 2.00) 1.00 (0, 2.00) 2.00 (2.00, 3.00) 0.50 (0, 1.00)* 0.50 (0, 2.00) 2.00 (1.00, 2.00) 1.00 (0, 2.00) Inhibition (%) 89.0 83.3 27.7 44.3 72.3 22.3 83.3 77.7 44.3 72.3

Results are median (minimum value, maximum value) for six rats. Statistical comparison was performed using KruskalWallis test followed by Dunns test * Statistically signicant at p \ 0.05 (compared with control)

Gastromucosal protective effects of fractions on aspirin-induced gastric mucosal lesions Fractions NF1, CF1, BF2 and EF2 showed gastromucosal protective effects at 20 mg/kg when compared to the control, and results were comparable to that of ranitidine. Inhibitions of 87.6, 74.9, 93.7 and 81.3% were observed with NF1, CF1, BF2 and EF2, respectively, as given in Table 4. Gastromucosal protective effects of fractions on ethanol-induced gastric mucosal lesions The fractions NF1, CF1, BF2 and EF2 showed higher levels of gastromucosal protection at the dose of 20 mg/kg, similar to those in the aspirin-induced model. In addition,

WF2 also showed effective gastromucosal protection in ethanol-induced lesions. Inhibitions of 83.3, 72.3, 83.3, 77.7 and 72.3% were observed with NF1, CF1, BF2, EF2 and WF2, respectively (Table 4). Histological evaluation In the histopathological study of the rats with ethanolinduced and aspirin-induced lesions, congestion, oedema, cellular debris and damaged mucosal epithelium were found in the stomach membrane. Apparent epithelization was observed as protection against these histopathological changes in rats pretreated with CH, HE and NF1, CF1, BF2 and EF2, which was similar to the result of ranitidine pretreatment.

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Discussion Anti-inammatory activity of extracts in carrageenan-induced inammation The oedema induced by carrageenan is manifested in two phases [17]. In the rst phase, a rapid rise in oedema occurs immediately after subplantar injection of carrageenan. In the second phase at around 6090 min, a strong increase in oedema occurs. The release of prostaglandins has been determined to be the main reason for second phase oedema [18], and some other mediators may be responsible for the rst phase oedema, probably platelet activation factor (PAF). A later experiment has shown that two or more mediators are released during carrageenan-induced oedema in two phases [19]. The CH and HE extracts showed signicant reduction in the inammation at 200 and 400 mg/kg in the rst phase (Table 1). This suggests that the possible mechanism of the anti-inammatory action of the extracts is on the rst phase, perhaps by inhibiting the mediator of inammation, probably by inhibiting the PAF receptors present in proinammatory cells such as mast cells and neutrophils. Gastromucosal protective effects of extracts in aspirin-induced gastric mucosal lesions It is well known that NSAIDs available in the market can induce gastric ulcers, and so these drugs cannot be used for long-term treatment of inammation. From Table 3, it is clear that the CH extract showed a signicant gastromucosal protective effect at 400 mg/kg in aspirin-induced gastric mucosal lesions. Gastromucosal protective effects of extracts in ethanol-induced gastric mucosal lesions Ethanol produced characteristic gastric mucosal lesions in control group, with the appearance of ulcers and petechial lesions. The gastric mucosal lesions formed by ethanolare caused by interference with the gastric defensive mechanisms [20]. The formation of gastric mucosal lesions by ethanol involves several mechanisms, which reduce gastric blood ow, thereby contributing to the development of necrosis and hemorrhage, and to the solubilization of mucus constituents in stomach. These actions result in an increased pepsin secretion and ux of Na? and K?, but a decrease in histamine and H? ions into the lumen [21]. From Table 3, it is clear that the CH and HE extracts of C. procera exhibited signicant gastromucosal protective effects, which may be related to (1) activation of alcohol dehydrogenase contained in gastric mucosa decreasing the amount of ethanol in the stomach [22]; (2) the gastric

mucosa damaged after ethanol administration releasing free radicals that can be neutralised [23]; and (3) an increase in the nonprotein sulfhydryl groups and mucus in the stomach, which decreased after ethanol administration [24]. Results in Table 3 indicate that extracts from C. procera display a gastromucosal protective effect, which is related to cytoprotective activity since it signicantly reduced ethanol-induced gastric mucosal lesions. To obtain compounds with anti-inammatory and gastromucosal protective effects, a bioassay was carried out with fractions of C. procera. The study was initiated by evaluating protective activities of the n-hexane, 1-butanol, chloroform, ethyl acetate and aqueous-soluble fractions against carrageenan-induced inammation (Table 2) and aspirin and ethanol-induced gastric mucosal lesions in rats (Table 4). NF1, CF1, BF2, EF2 fractions at 20 mg/kg showed signicant anti-inammatory activity. Among these fractions, the NF1 and BF2 fractions also showed signicant gastromucosal protective effects at the very low dose of 20 mg/kg in aspirin-induced as well as ethanol-induced gastric mucosal lesions. In addition, the EF2 fraction showed a signicant gastromucosal protective effect at 20 mg/kg in aspirin-induced gastric mucosal lesions. In conclusion, both CH and HE at doses of 200 and 400 mg/kg exhibited anti-inammatory activity. In addition CH at 400 mg/kg exhibited a signicant gastromucosal protective effect. Fractionation of these extracts reduced the complexity of the extract in order to have a better idea of the type of phytoconstituents responsible for the anti-inammatory and gastromucosal protective effects. Fractions NF1, CF1, BF2 and EF2 showed higher levels of cytoprotection and reduction in inammation at the dose of 20 mg/kg, which may be due to high triterpenoids, steroids, saponins and tannin content. However, the mechanisms behind these activities are still unclear. Therefore, further experiments are underway to determine which phytoconstituents and mechanisms are involved in the activities.
Acknowledgments The authors are thankful to the Principal, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Maharashtra, India for providing experimental facilities to carry out the work.

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