Journal of Ethnopharmacology, 29 (1990) 25 34 Elsevier Scientific Publishers Ireland Ltd.

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EVALUATION OF TURMERIC (CURCUMA LONGA) FOR GASTRIC AND DUODENAL ANTIULCER ACTIVITY IN RATS

S. RAFATULLAH, M. TARIQ, M.A. AL-YAHYA, J.S. MOSSA and A.M. AGEEL Medicinal, Aromatic and Poisonous Plants Research Center and Departments of Pharmacology and Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2b57, Riyadh-1H51 (Saudi Arabia) (Accepted October 18,1989)

Summary An ethanol extract of turmeric was studied in rats for its ability to inhibit gastric secretion and to protect gastroduodenal mucosa against the injuries caused by pyloric ligation, hypothermic-restraint stress, indomethacin, reserpine and cysteamine administration and cystodestructive agents including 80% ethanol, 0.6 M HC1, 0.2 M NaOH and 25% NaCl. An oral dose of 500 mg/kg of the extract produced significant anti-ulcerogenic activity in rats subjected to hypothermic-restraint stress, pyloruic ligation and indomethacin and reserpine administration. The extract had a highly significant protective effect against cystodestructive agents. The reduction in the intensity of ulceration of cysteamine-induced duodenal ulcers was not found to be statistically significant. Turmeric extract not only increased the gastric wall mucus significantly but also restored the non-protein sulfhydryl (NP-SH) content in the glandular stomachs of the rats.

Introduction Upper gastrointestinal bleeding can be a life-threatening complication in patients with gastric mucosal injury (Smith and Kvietys, 1988). In the recent years a wide-spread search has been launched to identify new anti-ulcer drugs from synthetic and natural resources. A large number of dietary nutrients, spices and condiments, namely banana (Best et al., 1984), ginger (Al-Yahya et al., 1989), cinnamon (Akira et al., 1986), a-tocopherol (Tariq, 1988) and selenium (Parmar et al., 1988) have been shown to possess significant antisecretory and gastroprotective activity. Although the modes of action of these agents are far from clear, substances like a-tocopherol and
Correspondence to: M. Tariq. 0378-8741/$03.85 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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selenium have been claimed to protect gastric mucosa by scavenging free radicals (Tariq, 1988; Szabo, 1989). Many plants are known to possess ingredients with antioxidative properties. Tanizawa et al. (1984) have isolated antioxidative compounds from green tea leaves. A potent antioxidative compound was also isolated by Toda et al. (1985a) from Plantago asiatica. Tariq and Al-Yahya (1983) have reported an a-tocopherol rich fraction isolated from date palm seeds, which was found to have significant activity against endotoxin-induced myocardial damage. Turmeric, Curcuma longa L. (Zingiberaceae), is widely used in the traditional medicine of various countries of the world for the treatment of rheumatism and inflammatory and ulcerative conditions (Mehra et al., 1984, Ageel et al., 1987). Curcuma longa rhizomes have been reported to possess anti-oxidative activity (Toda et al., 1985b). Although much work has been done on turmeric and its compounds in relation to rheumatism and its reputed antihepatotoxic action (Kiso et al., 1983), there are no data available regarding its gastric anti-ulcer potential. Therefore, the present study was undertaken to evaluate an ethanol extract of turmeric for its gastric and duodenal anti-ulcer activity. Materials and methods Animals Inbred Wistar albino rats of either sex of approximately the same age, weighing between 200 230 g, were obtained from the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh. The rats were fed standard chow diet (Silos and Flour Mills Organization Feed Mill, Riyad) and water ad libitum. They were divided into various groups of six to eight animals each. The distribution of the animals into groups, the sequence of the trials and the treatment allotted to each group were randomized. Extract preparation Turmeric rhizomes were procured from the local market of Riyadh, Saudi Arabia and identified in the Taxonomy Division of the Medicinal, Aromatic and Poisonous Plants Research Center of King Saud University, Riyadh. The powdered drug (500 g) was extracted using a percolation method and 96% ethanol. The solvent was then removed at low temperature under reduced pressure and the extracts so prepared were stored in a refrigerator for pharmacological studies. The extract yield was 9.5% (w/w) in terms of starting material. All doses are expressed here in terms of the extract itself. Experimental gastric lesions Unless specified otherwise, the animals were taken off food for 36 h with access to water ad libitum before subjecting them to one of the following procedures.

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Pyloric ligation. The pylorus was ligated according to the method of Shay et al. (1945) under light ether anaesthesia with care taken not to cause bleeding or to occlude blood vessels. Six hours after ligation, the animals were killed by an overdose of ether, the stomachs removed, contents collected, measured for volume, centrifuged and subjected to analysis for titratable acidity against 0.1 N NaOH to pH 8 using a pH meter. Total acid output was calculated. Each stomach was examined for lesions in the forestomach portion and indexed according to severity. Hypothermic-restraint stress-induced ulcers. The method of Levine (1971) was followed with slight modification. The animals were taken off food for 24 h with access to water ad libitum and 1 h after receiving the corresponding treatments, they were placed in a metallic restraint cage inside a refrigerator (24C) for 2 h. They were killed at the end of this period. Indomethacin-induced ulcers. Ulcers were induced according to the method of Bhargava et al. (1973). Indomethacin was suspended in 1% carboxymethycellulose in water and administered orally at a dose of 30 mg/kg (5 ml/kg). Six hours after indomethacin administration, the animals were killed. Reserpine-induced ulcers. The method of Gupta et al. (1974) was followed. Reserpine (CIBA) was administered in a dose of 5 mg/kg i.m. (5 ml/kg) and the animals were killed 24 h later. Cytoprotection studies. The method of Robert (1979) was followed. The following necrotizing agents were administered orally to male rats in a volume of 1 ml: 80% ethanol, 0.6 M HC1, 25% NaCl and 0.2 M NaOH. Turmeric extract (500 mg/kg; 5 ml/kg) or the vehicle alone (normal saline) was given i.g., 30 min before the necrotizing agents. One hour after the administration of necrotizing agents, the animals were sacrificed and each was examined for gastric lesions. Experimental duodenal lesions The method described by Szabo (1978) was followed. Female wistar rats, weighing 180 200 g were used. Food and water were available ad libitum throughout the study. Duodenal ulcers were induced by two oral administrations of cysteamine hydrochloride (300 mg/kg, p.o.) in aqueous solution (5 ml/ kg) with an interval of 4 h. All the animals were killed 48 h after the first dose of cysteamine. Gastric wall mucus determination Gastric wall mucus was determined according to the modified procedure of Come et< al. (1974). The glandular segments from stomachs which had been opened along their greater curvature were removed and weighed. 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 pH 5). After immersion for 2 h, excess dye was removed by two successive rinses with 10 ml of 0.25 M sucrose, first for 15 and then for 45 min. Dye complexed with the gastric wall mucus was extracted with 10 ml of 0.5 M magnesium

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chloride by shaking intermittently for 1 min at 30 min intervals for 2 h. Four millilitres of blue extract were then shaken vigorously with an equal volume of diethyl ether. The resulting emulsion was centrifuged at 3600 rev./min for 10 min and the absorbance of the aqueous layer was recorded at 580 nm. The quantity of Alcian blue extracted per gram of net glandular tissue was then calculated. Estimation of non-protein sulfhydryl groups (NP-SH) The animals were fasted for 36 h before the oral administration of 500 mg/ kg turmeric extract (5 ml/kg). Thirty minutes later, the rats were treated with 80% ethanol. One hour after the ethanol, the animals were killed and their stomachs removed. Gastric mucosal NP-SH was measured according to the method of Sedlak and Lindsay (1968). The glandular stomach was removed and homogenized in ice-cold 0.02 M EDTA. Aliquots of 5.0 ml of the homogenates were mixed in 15-ml test tubes with 4.0 ml of distilled water and 1.0 ml of 50% trichloroacetic acid. The tubes were shaken intermittently for 10 15 min and centrifuged at 3000 X g. Two millilitres of supernatants were mixed with 4.0 ml of 0.4 M Tris buffer (pH 8.9), 0.1 ml of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added and the sample shaken. The absorbance was read within 5 min of addition of DTNB at 412 nm against a reagent blank with no homogenate. General procedures The ethanol extract of turmeric (100 mg/ml) was given at a dose of 500 mg/kg p.o. 30 min before the administration of an ulcerogenic agent (twice in case of reserpine and cysteamine) and at the beginning of stress procedures. In Shay rats it was administered immediately after pylorus ligation. Control animals received an equivalent volume (5 ml/kg) of the normal saline vehicle. The animals were killed at the end of specified periods using anaesthetic ether and the stomach and duodenum were excised. The duodenum was opened along its anti-mesenteric side and the stomach along the greater curvature, their contents were rinsed off with saline and the linings examined with a 6.4 X binocular magnifier. Lesions were assessed by two observers unaware of the experimental protocols. Gastric lesions induced by all the procedures used in this study were multiple in each stomach. They were evaluated singly according to their dimensions and severity, and scored using a scale of 0 (no visible ulcers) to 10 (deep lesions with a diameter greater than 8 mm in each stomach). The scores for each single lesion were then summed so that the total score per stomach could exceed the value of 10 (Tariq et al., 1985). The duodenal ulcers were scored for intensity, using a scale of 0 to 3, where 0 = no ulcer, 1 = superficial mucosal erosion, 2 = deep ulcer or transmural necrosis, 3 = perforated or penetrated (into the pancreas or liver) ulcer (Szabo, 1978). The ulcer index is the sum of the arithmetic mean of the intensity for a group and the ratio of positive/total multiplied by 2, e.g. 2.1 + (9/10 X 2).

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The results refer to the average lesion score S.E.M. Statistical analysis of the severity of gastric ulcers was done by Student's t-test. Results As shown in Table 1, pyloric ligation induced ulcers and turmeric extract led to a significant decrease in the ulcer index and total acidity of the stomach contents at the end of the 6 h period. The ulcers were mainly in the forestomach and few haemorrhagic spots were found in the glandular stomach. Administration of indomethacin or reserpine, and subjecting the animals to cold and restraint resulted in the production of gastric mucosal damage mainly in the glandular segment of the stomachs. The majority of these lesions were gastric erosions. Pretreatment with turmeric extract administered orally was effective in reducing the intensity of ulceration in indomethacin, reserpine and restraint stressed groups (Table 2). Lowered gastric wall mucus was observed in the animals subjected to hypothermic-restraint stress, but this could be reversed by treatment with turmeric extract (Table 3). Lesions induced by the various necrotizing agents were grouped in varying sized patches, usually parallel to the major axis of the stomach. Treatment with turmeric extract significantly reduced the severity of these lesions (Table 4). Administration of duodenal ulcerogen cysteamine hydrochloride caused mortality in 12.5% of the rats in 24 h. Rats which died had perforated duodenal ulcers. Cysteamine treatment produced duodenal ulcers in 90% of all the rats. Usually two ulcers were produced close to the pylorus, the larger on the anterior and the smaller on the posterior wall of the duodenum. They were elongated, extending longitudinally down the duodenum and could easily be measured. Turmeric treatment reduced the average lesion score, but this change was statistically not significant (Table 2).
TABLE 1 EFFECT OF AN ETHANOL EXTRACT OF TURMERIC ON THE VOLUME OF GASTRIC SECRETIONS, ACIDITY AND THE DEGREE OF ULCERATION IN 6-H PYLORUS LIGATED (SHAY) RATS

Treatment

Dose (mg/kg, i.p.)

Mean S.E.M. Volume of gastric secretion (ml) 8.66 0.72 4.58 1.01* Titratable acidity (mEq/1) Total acid output Ulcer index

Control Turmeric

6 6

500

70.0 2.7 43.9 8.0*

606 52 229 72**

1.00 0.36 0.33 0.21**

Significance relative to control data: *P < 0.05, **P < 0.01.

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TABLE 2 EFFECT OF AN ETHANOL EXTRACT OF TURMERIC ON EXPERIMENTALLY-INDUCED GASTRIC AND DUODENAL ULCERS IN RATS Treatment N Oral dose (mg/kg) Hypothermic-restraint stress Control 6 Turmeric 6 Indomethacin Control 6 Turmeric 6 Reserpine Control 6 Turmeric 6 Cysteamine Control 7 Turmeric 7 500 X 1 500 X 1 Lesion score (Mean S.E.M.) 26.5 2.3 17.2 + 2.4* 35.2 23.3 37.0 18.0 1.85 1.00

1.6 3.1*** 2.0 3.1*** 0.26 0.37

500 X 2 500 X 2

Significance relative to respective control data: *P < 0.05, **P < 0.01. ***P < 0.001.

TABLE 3 MEAN (S.E.M.) EFFECT OF ETHANOLIC EXTRACT OF TURMERIC ON GASTRIC W A L L MUCUS IN RATS WITH HYPOTHERMIC-RESISTANT STRESS Group N Oral dose (mg/kg) Gastric wall mucus ing Alcian blue/g wet glandular tissue) 305 196 385 P

Normal (unstressed) Control (stressed) Turmeric (stressed)

7 7 7

500

24* 9 31*

< 0.001

< 0.001

TABLE 4 EFFECT OF AN ETHANOL EXTRACT OF TURMERMIC ON THE GASTRIC LESIONS INDUCED BY VARIOUS NECROTIZING AGENTS Procedure N Ulcer index (mean S.E.M.) Control 80% Ethanol 0.6 M HC1 25% NaCl 0.2 M NaOH 6 6 6 6 6.83 + 0.40 6.83 0.79 6.50 + 0.76 6.55 0.80 Turmeric 500 mg/kg, p.o. 4.00 1.50 1.66 2.00

0.40* 0.50* 0.42* 0.36*

Significance relative to respective controls: *P < 0.001.

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TABLE 5 MEAN ( S.E.M.) EFFECT OF AN ETHANOL EXTRACT OF TURMERIC ON NON-PROTEIN SULFHYDRYL CONTENT IN THE GLANDULAR STOMACHS OF RATS TREATED WITH 80% ETHANOL

Treatment

Oral dose (mg/kg)

Non-protein sulfhydryl content (/imol/g of tissue) 3.15 0.18* 2.89 0.23* 1.42 0.06* 2.27 0.12*

Normal Turmeric 80% Ethanol (1.0 ml) Turmeric + 80% ethanol (1.0 ml)

7 7 7 6

500

500

The gastric mucosal NP-SH content was significantly decreased following the administration of 80% ethanol. Treatment with the turmeric extract significantly prevented this reduction (Table 5). Discussion The results of this study clearly indicate that the oral administration of an ethanol extract of turmeric produced significant anti-ulcer and cytoprotective effects in rats. Hypothermic-restraint stress ulcers have been widely used experimentally for the evaluation of anti-ulcer activity in rats because of data reproducibility (Murakami et al., 1985). Disturbances of gastric mucosal microcirculation (Guth, 1972), alteration of gastric secretion (Kitagawa et al., 1979) and abnormal gastric motility (Watanabe, 1966) have been considered to be the pathogenic mechanisms responsible for stress-induced gastric mucosal lesions and gastric mucus depletion (Koo et al., 1986). The increase in gastric acid secretion is considered to be an important factor in the genesis of stress ulcer and is often termed as the "aggressive factor" (Goa and Monk, 1987). The antisecretory activity of turmeric as observed in our Shay rat model might be important in protecting gastric mucosa against stress-induced ulceration. Turmeric has been shown to possess highly significant antioxidant activity and Toda et al. (1985b) have shown that the methanol extract of turmeric possesses better anti-oxidant properties than a-tocopherol. Young et al. (1976) found that a-tocopherol can mitigate stress-induced ischemia in tissues. Our findings are in agreement with earlier authors who reported significant anti-ulcer activity for a number of other anti-oxidants including superoxide dimutase, vitamin E, selenium and AT-acetylcysteine (Javor et al., 1986; Parmar et al., 1988; Tariq, 1988). The ability of turmeric extract to produce a significant reduction of the gastric mucosal damage induced by indomethacin

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and reserpine further confirms its potent anti-ulcer activity. Of these, indomethacin is a potent prostaglandin biosynthesis inhibitor (Whittle, 1981). There is mounting evidence that an increase of certain endogenous prostaglandins can enhance gastric mucosal resistance against ulcerogenic agents (Konturek et al., 1984; Wallace and Whittle, 1985). Many of these antioxidants have been shown to stimulate the synthesis of prostaglandins by inhibiting lipoxygenase (Gwebu et al., 1980; Sok et al., 1980; Reinton et al., 1981). The severity of duodenal ulcers, though reduced, was not statistically significant after oral pretreatment with turmeric extract. Cysteamine-induced duodenal ulcers are considered to be due to a long-lasting hypersecretion of gastric acid (Szabo et al., 1977; Kirkegaard et al., 1980). It is possible that higher and more frequent dosing with turmeric extract might reveal some duodenal anti-ulcer activity in this experimental model. Even the standard H receptor antagonist cimetidine has been found to be less effective in rats against cysteamine-induced duodenal ulceration (Szabo et al., 1979). Turmeric extract significantly protected gastric mucosa againts several known necrotizing agents, namely 80% ethanol, 0.6 M HC1, 0.2 M NaOH and 25% NaCl. These results support the findings of Kiso et al. (1983), who reported a protective effect of turmeric against carbon tetrachloride-induced hepatocellular injury. The extract also inhibited ethanol-induced depletion of the non-protein sulfhydryl (NP-SH) content of the glandular mucosa of the stomach. Glutathione (GSH) has been reported to play an important role in the process of cellular injury (Robert et al., 1983). Szabo et al. (1981) found a significant decrease in NP-SH following necrotizing agents in ulcerative doses; therefore, the replenishment of sulfhydryl levels in gastric mucosa by turmeric extract may contribute to its anti-ulcer activity. Our findings show that the ethanolic extract of turmeric has significant anti-ulcer, antisecretory and cytoprotective activity in rats.
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Acknowledgement The authors are thankful to the King Abdulaziz City for Science and Technology (KACST) for financial support. References
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