protective effects of punica granatum in experimentally-induced gastric...

Introduction

Anti-ulcer drugs have been used as part of the treatment for ulcers since ancient times. Ulcers are sore or raw areas that form in the lining of the stomach or the duodenum (the upper part of the intestine). Most ulcers are caused either by infection with a bacterium called Helicobacter Pylori or by long-term use of aspirin or other Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). In either case, damage occurs to the barrier of mucus that normally protects the stomach and duodenum from the powerful acids and enzymes that are produced during digestion. The acids and enzymes affect the exposed tissue and cause ulcers. Reduction of gastric acid production and re-inforcement of gastric mucosal protection have been the major approaches for the treatment of peptic ulcer disease (Hoogerwerf and Pasricha 2001).

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly approved medications for the inflammation of body tissues, such as in tendinitis and bursitis. NSAIDs are the most common drug of use throughout the world. The major side-effects of NSAIDs are related to the gastrointes-tinal system. Approximately 10–50% of patients are unable

to stand NSAID treatment and15% of patients on long-term NSAID treatment develop peptic ulcers.

Medicinal plants have been playing an important role in the health care of human beings since ancient times. Several drugs have revolutionized ulcer therapy, but efficacy of these drugs is still a matter of debate; however, results of clinical studies show that there are incidences of relapse and adverse effects during therapy. This has been the rationale for the development of new anti-ulcer drug and has been extended to herbal drugs in search of novel molecules that could show better protection and decrease the incidence of relapse (Dharmani et al. 2005).

Punica granatum (PG) has been frequently described in an Unani system of medicine for the treatment of gastrointestinal disorders. It has been used as a liver and heart tonic and has benefits in stomach ailments. PG has also been incorporated as a constituent of several Unani compound formulations (Anonymous 1987). Pomegranate has a history of herbal use dating back more than 3000 years (Bown 1995). All parts of the plant contain unusual alkaloids, known as ‘pelletierines’, which paralyse tapeworms so that they are easily expelled

(Received 04 March 2010; revised 21 June 2010; accepted 01 July 2010)

ISSN 1537-6516 print/ISSN 1537-6524 online © 2010 Informa Healthcare USA, Inc.DOI: 10.3109/15376516.2010.508079 http://www.informahealthcare.com/txm

R E S E A R C H A R T I C L E

Protective effects of Punica granatum in experimentally-induced gastric ulcers

Mohd Sarfaraz Alam1, Mohd Aftab Alam1, Sayeed Ahmad2, Abul Kalam Najmi3, Mohd Asif1, and Tamanna Jahangir2

1Department of Ilmul Advia, Faculty of Medicine, 2Department of Pharmacognosy and Phytochemistry, and 3Department of

Pharmacology, Faculty of Pharmacy, Jamia Hamdard, New Delhi – 110062, India

AbstractIn the present investigation standardized aqueous methanolic extract of Punica (AMP) was used for its pos-sible ulcer protective activity in wistar rats against different experimental models. Preliminary phytochemical screening of AMP reveals the presence of saponin, tannins, and flavonoids, which may be responsible for its activity. HPTLC finger prints of AMP showed the presence of 12 spots at different (retention factor) rf values. Oral administration of AMP (490 and 980 mg/kg bw) significantly reduced the ulcer lesion index produced by alcohol, indomethacin, and aspirin, at both doses in rats. Further, in pylorus-ligated rats AMP significantly reduces the ulcer lesions, gastric volume, and total acidity. It prevents the ulceration by increasing the pH and mucus secre-tion in pylorus ligated rats. The present study shows the anti-ulcer activity of AMP in experimentally-induced gastric ulcers.

Keywords: Gulnar farsi; Punica granatum; anti-ulcer; Unani

Toxicology Mechanisms and Methods, 2010; 20(9): 572–578

Address for Correspondence: Dr Sayeed Ahmad, Assistant Professor, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi – 110062, India. E-mail: [email protected]

Toxicology Mechanisms and Methods

2010

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21 June 2010

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© 2010 Informa Healthcare USA, Inc.

10.3109/15376516.2010.508079

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http://informahealthcare.com/doi/abs/10.3109/15376516.2010.508079

mailto:[email protected]

Protection by punica 573

from the body by using a laxative (Bown 1995). The plant is also rich in tannins, which makes it an effective astringent. It is used externally in the treatment of vaginal discharges, mouth sores and throat infections (Bown 1995). LD50 of the extract was found to be 1321 ± 15 mg/kg in mice (Qnais et al. 2007).

The objective of the present work was to study the possible anti-ulcer activity of PG against different models of gastric lesions, induced by alcohol, indomethacin, aspirin and pyloric ligation in rats.

Materials and methods

Plant materialThe dried flowers of PG were procured from the local drug market (Khari Bawli, Delhi) and authenticated at the National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. The voucher specimen was deposited at the University Herbarium.

Experimental animalsMale Wistar rats weighing 200–250 g were procured from Jamia Hamdard animal house facility. The animals were kept under standard laboratory conditions and fed a diet supplied by Amrut Lab Animal-Feed (Pehladpur, New Delhi). Water was allowed ad libitum. The protocol was previously approved by the animal ethics committee (Protocol no. 173/CPCSEA). The protocol was also approved by Jamia Hamdard ethical committee for animals.

ChemicalsAspirin (Reckitt & Colman, Mysore of India), Methanol (S.D. Fine Chemicals Ltd, Bombay), Petroleum ether (60–80°C) (Central Drug House, Bombay), Chloroform (Thomsan Baker Limited, Bombay), Indomethacin (Jagsonpal Pharmaceuticals, New Delhi), and Alcohol (96%, V/V). All other chemicals used in the study were obtained commer-cially and were of analytical reagent grade.

Dosage route of administrationAnimals were administered orally (10 ml/kg) Aqueous methanolic extracts (AM) of test drug. Dosage of AM extract was determined by the extractive values of crude drugs as mentioned in Unani literature (Ghani 1917; Baitar 1999) sus-pended in 2% v/v tween 80 in distilled water.

Physico-chemical standardizationDifferent physico-chemical values such as moisture content; pH values; extractive values such as cold, hot, and successive extractive values using petroleum ether, chloroform, alco-hol, and water; percentage of total ash, acid-insoluble ash, water-soluble ash, and water-insoluble ash of dried and pow-dered flowers of Punica were determined as per the method described in Indian Pharmacopoeia (Anonymous 1966).

Preparation of aqueous methanolic extractThe dried and powdered flowers of PG (200 g) were extracted with methanol (80%) in a Soxhlet apparatus for 12 h. AM

extract was evaporated to dryness under reduced pres-sure, which yields 81.61 g of extract (AM, 40.8%). This AM extract was used for phytochemical analysis and HPTLC finger printing for standardization of extract. The AM was suspended in 2% v/v tween 80 in distilled water and used for pharmacological study.

Preliminary phytochemical screening and HPTLC fingerprinting of AMPThe presence or absence of different phytoconstituents like saponin, alkaloids, sugar, tannins, glycosides, flavanoids, phe-nolics, and proteins, etc., were detected by usual prescribed methods (Peach and Tracy 1955).

A known quantity of the extract AM was dissolved in methanol (10 mg/ml) and applied (5 µl each) on a silica gel G F

254 TLC plate (10 cm × 10 cm with 0.2 mm thickness,

E. Merck, Germany) in duplicate with a bandwidth of 6 mm using a Camag Linomat V (Switzerland) applicating device. The chromatogram was developed in a Twin trough cham-ber using the solvent system Chloroform:Methanol (5:1) and scanned in a Scanner III at 254 nm wavelength using deuterium and tungsten lamp in absorbance mode.

Anti-ulcer activityAlcohol-induced gastric lesionsAnimals were randomly divided into five groups, of six animals each, which were fasted for 24 h with water ad libi-tium. Groups I and II received vehicle (2% v/v tween 80 in distilled water) 10 ml/kg p.o., group III received omeprazole 20 mg/kg (suspended in 2% v/v tween 80 in distilled water) as a standard drug (Lambers et al. 1984). Groups IV and V received test drug AM 490 and 980 mg/kg bw (suspended in 2% v/v tween 80 in distilled water), respectively, at zero hour. After 30 min of oral administration, ulceration was induced by absolute ethanol 1 ml orally for each animal in all groups except group I according to the method described by Robert (1979). The animals were sacrificed and stom-achs were removed along the greater curvature, and sum of length of lesions was evaluated for ulcer index. The erosion produced by alcohol was measured and given a score as: erosion of 1 mm or less given score 1, 1–2 mm given score 2, and if more than 2 mm score is 3. The overall score was divided by a factor of 10, which was designated as the ulcer index (Main and Whittle 1975).

Indomethacin-induced gastric ulcerThe study was carried out as per the method described by Djahanguiri (1969); animals were divided into five groups (six each), which were fasted for 24 h following water ad libitium. Groups I and II received vehicle 10 ml/kg, Group III received omeprazole 20 mg/kg, whereas Groups IV and V received test drug aqueous methanolic extract (AM) 490 and 980 mg/kgbwt, respectively, at zero hour. After 1 h of oral administra-tion, ulceration was induced by indomethacin 20 mg/kg sus-pended in vehicle (2% v/v tween 80 in distilled water) in all groups except Group I. After 5 h animals were sacrificed by an overdose of anesthesia ether vapors and the stomachs were

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574 M. S. Alam et al.

removed, opened, and sum of length of lesion was evaluated for ulcer index as described by Main and Whittle (1975).

Aspirin-induced gastric ulcerThe aspirin-induced gastric ulcer protocol was based on the method described by Parmer et al. (1984); animals were divided into five groups, of six animals each, which were fasted for 24 h following water ad libitium. Groups I and II received vehicle 10 ml/kg, Group III received omeprazole 20 mg/kg, whereas Groups IV and V received test drug aqueous methanolic extract (AM) 490 and 980 mg/kgbwt, respectively, at zero hour. After 45 min of oral administra-tion, ulceration was induced by aspirin 150 mg/kg sus-pended in vehicle (2% v/v tween 80 in distilled water) except Group I. The animals were sacrificed after 5 h and stomachs were removed, opened, and sum of length of lesions were evaluated for ulcer index as described by Main and Whittle (1975).

Effect of AM on volume of gastric juice, pH, total acidity, gastric ulcer, and gastric wall mucus in pylorus ligated ratsAnimals were randomly divided into four groups of eight rats each. Control group (Group I) received vehicle 10 ml/ kg orally. Group II serves as reference control, which were treated orally with omeprazole 10 mg/kg suspended in vehicle. Groups III and IV were administered orally with 490 and 980 mg/kgbwt of AM extract, respectively. After 30 min of their respective treatment, pylorus was ligated as described by Shay et al. (1945). The rats were anesthetized with ether and the abdomen was opened through a midline incision. The pylorus was secured and ligated with silk sutures, after which the abdominal wound was closed and the animals were allowed to recover from anesthesia. After ligation of pylorus, drinking water was withheld and gastric juices were collected for a period of 4 h. The rats were sacrificed and stomachs were removed after clamping the esophagus. The gastric contents were collected through the esophagus and volume of secretion and pH was measured.

Gastric content was centrifuged at 3000 rpm for 20 min and supernatant was subjected to analysis for titrable acidity. For the determination of total acidity of the samples known, volume of the gastric juice was titrated with 0.01 N sodium hydroxide to pH 8.5 using phenolphthalein as an indicator (Parmer et al. 1984). The values of total acidity were expressed as milli-equivalents per liter at 4 h (meq/l).

The stomachs were opened along the greater curvature, washed with distilled water with care, and examined for ulcer lesions.

The glandular segments from the stomach, which had been opened along their greater curvature, were removed, weighed, and gastric wall mucus was determined (Riggs and Stadic 1943; Corne et al. 1974). Each segment was transferred immediately to 10 ml of 0.1%, w/v alcian blue solution (in 0.16 M sucrose solution, buffered with 0.05 M sodium acetate adjusted to pH 5.8 with HCl). The dye uncomplexed with mucus was removed by two successive washes with 0.25 M sucrose at 15 and 45 min,

whereas the dye complexed with mucus was diluted by 0.5 M magnesium chloride by immersion for 2 h. The resulting blue solution obtained after 2 h immersion was shaken vigorously with an equal volume of diethyl ether and the emulsion thus obtained was centrifuged at 3000 rpm for 10 min. The absorb-ance of the aqueous layer against blank 0.5 M MgCl

2 solution

was recorded at 605 nm and amount of alcian blue recovered per gram of net glandular tissue was calculated using stand-ard calibration curve. Standard solutions of alcian blue were prepared (5–25μg/ml) and their Optical Densities (ODs) were recorded at 605 nm against 0.5 M MgCl

2 blank. The standard

calibration curve of alcian blue was prepared using OD against concentration (μg/ml).

The gastric wall mucus, total acidity, and volume of secre-tion of the samples of gastric juice having a pH more than 2.5 or contaminated with blood were not included in the study.

Results

Physico-chemical standardization, preliminary phytochemical screening, and HPTLC fingerprintThe physico-chemical values like moisture content, pH values, extractive values, and ash values of dried and pow-dered flowers of punica were determined and tabulated in Table 1. Phytochemical screening of AM showed positive results for saponin, tannin, flavonoids, proteins, phenolics, and carbohydrates.

HPTLC fingerprints of AM showed the presence of 12 spots with their retention factor value 0.01, 0.08, 0.22, 0.31, 0.40, 0.50, 0.52, 0.58, 0.63, 0.69, 0.89 and 0.97 in the solvent system Chloroform:methanol (5:1) at 254 nm wavelength.

Table 1. Physico-chemical constants of PG flowers.

Sl no. Parameters Meana ± SD

1. Moisture content (% w/w) 13.440 ± 0.259

2. pH of powdered drug

2.a. 10%-soln. 3.722 ± 0.007

2.b. 5% -soln. 3.548 ± 0.117

3. % Extractive value (Cold extraction)

3.a. Petroleum ether 0.323 ± 0.017

3.b. Chloroform 0.720 ± 0.014

3.c Alcohol 13.142 ± 0.283

3.d. Water 13.827 ± 0.070

4. % Extractive value (Hot extraction)


4.b. Chloroform 1.868 ± 0.076

4.c Alcohol 36.265 ± 0.263

4.d. Water 41.810 ± 0.132

5. % Extractive value (Successive extraction)


5.b. Chloroform 1.475 ± 0.034

5.c Alcohol 33.640 ± 0.222

5.d. Water 35.298 ± 0.081

6. Ash values

6.a. Total Ash 5.303 ± 0.087

6.b. Acid insoluble Ash 0.705 ± 0.018

6.c Water-soluble Ash 3.813 ± 0.033

6.d. Water insoluble Ash 1.510 ± 0.023

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Prevention of alcohol-induced gastric ulcer in rats by AMThe pre-treatment with AM P showed a dose-dependent reduction in the severity of ulcer lesions (Table 2). Test drug AM 980 mg/kg bw showed an ulcer index of 0.35 ± 0.08 (p < 0.01) with a significant inhibition of ulceration (87.5%) as compared to 2.80 ± 0.11 in toxic control, whereas AM 490 mg/kg bw and standard drug omeprazole (20 mg/kg) exhibited 65% and 49.64% inhibition in the ulcer index, respectively.

Prevention of indomethacin-induced gastric ulcer in rats by AMPre-treatment with AMP reversed ulcer lesions at both doses (Table 3). The test drug AM 980 mg/kg bw showed an ulcer index of 0.40 ± 0.06 (p < 0.01) with a significant inhibition of ulceration (80.32%) as compared to 2.80 ± 0.11 in toxic con-trol, whereas standard drug and AM 490 mg/kgbw illustrated 69.55% and 40.48% inhibition, respectively.

Prevention of aspirin-induced gastric ulcer in rats by AMPAMP treatment maintained anti-ulcer activity, when tested against aspirin-induced ulcers, and exhibited significant reduction in ulcer lesions (Table 4). AM 980 mg/kgbw dem-onstrated 83.93% inhibition of ulceration as compared to toxic control, whereas standard drug and AMP 490 mg/kgbw showed 78.20% and 73% inhibition in the ulcer index, respectively.

Effect of AMP on volume of gastric juice, pH, total acidity, gastric ulcer, and gastric wall mucus in pylorus ligated ratsAMP, when tested against a pylorus ligated model, shows significant, dose-dependent reduction in the severity of lesions (Table 5). AMP 980 mg/kgbw showed a 66.01% inhibition as compared to control, whereas standard drug (omeprazole 20 mg/kg) and AMP 490 mg/kgbw exhib-ited 49.24% and 41.12% inhibition, respectively, in ulcer lesions.

AMP-treated pylorus ligated rats demonstrated significant reduction in the volume of gastric acid secretion and total

acidity of gastric juice; it was also found to increase the pH of gastric juice to some extent (Table 6).

Administration of AMP 980 mg/kgbw was found to reduce the volume of gastric acid up to 40.04% as compared to control, whereas standard drug and AMP 490 mg/kgbw exhibited 30.03% and 24.40% reduction in gastric volume, respectively.

Pylorus ligated rats on treatment with AM 980 mg/kgbw showed a significant increase in pH (2.64 ± 0.52, p < 0.01) as compared to control (1.64 ± 0176), whereas AMP 490 mg/kgbw and standard drug (omeprazole 20 mg/kg) could not produce a significant increase in pH of gastric juice.

Prophylactic treatment of animals with AMP 980 mg/kgbw reduced the total titrable acidity up to 50.83% as compared to control, whereas Omeprazole 20 mg/kg and AMP 490 mg/

Table 2. Alcohol-induced gastric ulcer in rats and its prevention by AMP.

Group (treatment, dose, and route)Ulcer Index(mm),

M ± SE Inhibition(%)

I–Control (vehicle, 10 ml/kg, p.o.) 0

II–Toxic Control (alcohol, 1 ml each, p.o.)

2.80 ± 0.115

III–Omeprazole (alcohol, 1 ml each, p.o. + omeperazole 20 mg/kg, p.o.)

1.41 ± 0.083** 49.64

IV–AM1 (alcohol, 1 ml each, p.o. + AM 490 mg/kgbwt, p.o.)

0.98 ± 0.094** 65.00

V–AM2 (alcohol, 1 ml each, p.o. + AM 980 mg/kgbwt, p.o.)

0.35 ± 0.084** 87.50

Group II was compared with all the other groups by one way ANOVA fol-lowed by Dunnet’s post-hoc test. p < 0.05 was considered significant.** p < 0.01.

Table 3. Indomethacin-induced gastric ulcer in rats and prevention by AMP.



I–Control (Vehicle, 10 ml/kg, p.o.) 0

II–Toxic control (Indomethacin, 20 mg/kg, p.o.)

2.033 ± 1.47

III–Omeprazole (Indomethacin, 20 mg/kg, p.o. + omeperazole 20 mg/kg, p.o.)

0.616 ± 0.09** 69.55

IV–AM1 (Indomethacin, 20 mg/kg, p.o. + AM 490 mg/kgbwt, p.o.)

1.21 ± 0.087** 40.48

V–AM2 (Indomethacin, 20 mg/kg, p.o. + AM 980 mg/ kgbwt, p.o.)

0.40 ± 0.06** 80.32


Table 4. Aspirin-induced gastric ulcer in rats and prevention by AMP.



I–Control (Vehicle, 10 ml/kg, p.o.) 0

II–Toxic control (Aspirin, 150 mg/kg, p.o.)

1.45 ± 0.084

III–Omeprazole (Aspirin, 150 mg/kg, p.o. + Omeperazole 20 mg/kg, p.o.)

0.316 ± 0.054** 78.20

IV–AM1 (Aspirin, 150 mg/kg, p.o. + AM 490 mg/kg, bwt p.o.)

0.380 ± 0.047** 73.79

V–AM2 (Aspirin, 150 mg/kg, p.o. + AM 980 mg/kgbwt, p.o.)

0.233 ± 0.042** 83.93


Table 5. Effect of AMP on gastric ulcer in pyloric ligated rats.

Group (treatment, dose, and route)

Ulcer Index(mm), M ± SE Inhibition(%)

I–Control (Vehicle, 10 ml/kg, p.o.) 2.463 ± 0.146

II–Omeprazole (Omeperazole, 20 mg/kgbw, p.o.)

1.250 ± 0.121** 49.24

III–AM1 (AM, 490 mg/kgbw, p.o.) 1.45 ± 0.143** 41.12

IV–AM2 (AM, 980 mg/kgbw, p.o.) 0.837 ± 0.075** 66.01


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kgbw showed 43.74% and 34.37% lowering in total titrable acidity, respectively.

The gastric wall mucus was calculated as microgram of alcian blue per gram of glandular tissue from the standard calibration curve (r2 = 0.999, y = 0.1006x), and it was observed that there was no significant improvement in total gastric bar-rier after treatment with AMP as well as with standard drug, as indicated in Table 7.

Discussion

Peptic ulcer is one of the most common gastrointestinal diseases. It is more common among male adults and in low socio-economic groups of people. Peptic ulcers are the result of over-production of gastric acid and decrease in gastric mucosal protection mechanism. Hence, the potential anti-ulcerogenic and ulcer-healing drugs are known to possess the property of decreasing the offensive factors or of increasing the defensive factors. Gastrointestinal disorders have been attributed due to various causes like stress, hormones, synthetic drugs, alcohol, smoking, and ingestion of certain foods (Mc Guigan 1994). The involvement of the pathogenic organism was later confirmed with the discovery of H. pylori (Flenstom and Turnberg 1984) and has been implicated in the antral gastritis, peptic ulcer, gastric malignancy, and ulcer dyspepsia (Marshall 1995).

The imbalance between mucus bicarbonate secretion and prostaglandins was also reported to be involved in the ulcer pathogenesis (Hoogerwerf and Pasricha 2001). Different therapeutic agents including plant extracts are used to inhibit the gastric acid secretion or to boost the mucosal defense mechanism by increasing mucus production, stabilizing the

surface epithelial cells or interfering with the PGs synthesis

(Afifa et al. 1997).In the Unani System of Medicine several single and com-

pound formulations containing Gulnar Farsi (Punica grana-tum) as an important ingredient are frequently prescribed since long, for the treatment of gastrointestinal disorders and are used as Muqauvie meda (stomach), Mujaffif (Desiccant), Muhallil (Resolvent), Habis (Retentive), Hazim (Digestive), Manequi (Anti emetic), and Kasir-e-riyah (Carminative) (Anonymous 1987).

There was no scientific report available on the traditional claim (useful in peptic ulcer disorders) of the flowers of the plant. Therefore, we investigated the anti-ulcerogenic effects of aqueous methanolic extract on different models of gas-tric lesions induced by alcohol, aspirin, indomethacin, and pylorus ligation in rats to determine the anti-ulcer proper-ties of Punica. A literature survey revealed that the Punica granatum showed anti-fertility, antibacterial, anti-helmentic (Satyavati et al. 1987), antioxidant (Schubert et al. 1999), hyo-glycemic (Jafri et al. 2000), molluscidal (Tripathi and Singh 2000), anti-diarrhoeal (Das et al. 1999), and gastroprotective activity (Gharzouli et al. 1999).

Ethanol-induced ulcers are due to a direct necrotizing effect of ethanol on gastric mucosa (Miller and Henagan 1984). Ethanol causes necrosis of superficial epithelial cells on gastric mucosa and erosion (Oates and Kakkinen 1988). Hence, a cytoprotective agent, which increases mucus secretion, will be effective in this model. The products of the 5-lipo-oxygenase pathway may also play a key role in the development of ulcer, induced by irritant agents such as ethanol (Lange et al. 1985). In the present study, it was observed that AM extract significantly reduced the ulcer index highlighting its cytoprotective effect.

Aspirin model was used in this study, considering the different mechanism by which it induces gastric ulcera-tion. The reason being attributed principally to inhibition of biosynthesis of cytoprotective prostaglandin’s (PGE’s and PGI

2) by inhibition of cycloxygenase pathway of arachidonic

acid metabolism, results in over production of leukotrienes and other products of 5-lipooxygenase pathway (Rainford and Whitehouse 1980). We observed that AM 980 mg/kg, 490 mg/kg.bwt and omeprazole 20 mg/kg showed signifi-cant inhibition of ulceration by 83.93%, 73.79% and 78.20%, respectively, which further confirmed a cytoprotective effect of AM.

The ulcerogenic effect of NSAIDs seems to be related to the inhibition of endogenous PGs synthesis; although it has also been established that indomethacin modifies

Table 6. Effect of AMP on gastric volume, pH, and total acidity in pyloric ligated rats.

Group (treatment, dose, and route)

Volume of gastric juice (ml/100 g),

M ± SE pH, M ± SE

Titrable acidity (meq/l/4 h),

M ± SE

I–Control (Vehicle, 10 ml/kg, p.o.)

1.918 ± 0.08 1.64 ± 0.176 203.85 ± 10.14

II–Omeprazole (Omeperazole, 20 mg/kg, p.o.)

1.342 ± 0.05** 2.26 ± 0.232ns 114.69 ± 5.90**

III–AM1 (AM 490 mg/kgbw, p.o.)

1.45 ± 0.05** 1.95 ± 1.84ns 133.78 ± 6.87**

IV–AM2 (AM 980 mg/kgbw, p.o.)

1.15 ± 0.12** 2.64 ± 0.152** 100.24 ± 13.44**

Group II was compared with all the other groups by one way ANOVA fol-lowed by Dunnet’s post-hoc test. p < 0.05 was considered significant.** p < 0.01, ns = non-significant.

Table 7. Effect of AMP on gastric wall mucus in pyloric ligated rats.

Group (treatment, dose and route) Gastric wall mucus (μg of alcian blue/gm glandular tissue), M ± SE

I–Control (Vehicle, 10 ml/kg, p.o.) 52.95 ± 2.133

II–Omeprazole (Omeperazole, 20 mg/kg, p.o.) 56.03 ± 0.964ns

III–AM1 (AM, 490 mg/kgbw, p.o.) 53.76 ± 3.37ns

IV–AM2 (AM, 980 mg/kgbw, p.o.) 56.29 ± 2.03ns

Group II was compared with all the other groups by one way ANOVA followed by Dunnet’s post-hoc test. p < 0.05 was considered significant.p > 0.05, ns = non-significant.

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some protective mechanisms of the gastric mucosa, includ-ing gastric secretion and the permeability of the gastric mucosal barrier (Rainford 1978). In addition, indomethacin is also known to include a relative increase in leukotriene C

4 at the cost of reduced PGE

2 levels which may induce

mucosal vasoconstriction and enhance NSAID-induced injury (Hawkey 1989). In the present investigation, AM showed significant prevention of gastric lesions in the indomethacin-induced rats and it also reduced the gastric acid secretion.

It has been postulated that histamine may be involved in the formation of pylorus ligation ulcers which play a mediating role in the gastric secretion stimulated by gastrin, vagal excita-tion, and cholinergic agents (Glick et al. 1966). In this study AM significantly reduced the gastric ulceration in pylorus ligated rats, AM 980 mg/kg showed inhibition of ulceration up to 66.01%. The protective mechanism of AM on gastric mucosa involved inhibition of secretion and total acidity as well as an increase in pH of gastric juice, whereas it cannot produce a significant role on the production of mucous.

The anti-ulcerogenic activity of AM may be attributed due to the presence of saponins (Yesilada and Takaishi 1999; Yesilada et al. 2000), tannins (Ramirez and Roa 2003; Ariga 2004), and flavonoids (Havsteen 2002; Zahorodnyi 2003; Min et al. 2005; Sannomiya et al. 2005; Ustun et al. 2006). Phenolics are mainly responsible for the antioxidant property (Hamauzu et al. 2006) and possibly attributing its cytoprotective property. The flowers of Punica granatum have been reported to contain flavonoids like pelargonidine-3,5-diglucoside, delphenidine-diglycoside, malvidine-pen-tosglycoside, and isoquercetrine (Ponniah and Seshadari 1953), which are reported to possess anti-helicobacter (Ustun et al. 2006) and anti-ulcerogenic (Sannomiya et al. 2005) properties.

The anti-ulcerogenic activity of Punica includes various mechanisms like cytoprotection and anti-secretory activity as evidenced from data. Since the herbal drugs augment the defensives factors and are reliable and safe, Punica may be considered for use alone or in combination with other anti-ulcer drugs.

Conclusion

On the basis of data generated with AMP, it can be con-cluded that it possesses very good anti-ulcer potential against experimentally-induced gastric ulcer models. At present, it is still unknown whether the entire aqueous methanolic extract of Punica is responsible for its activity or any single constituent. Further studies on isolation and characterization of constituents are under progress in our laboratory.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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protective effects of punica granatum in experimentally-induced gastric...

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