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��ࡱ�>��	�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������[�	���bjbj����	4�ΐΐFN����������������1��:				*0,0,0,0,0,0,0$�3�q66P0�P0		�0�#�#�#�
		*0�#*0�#�#��-h�.	����P�Af�!��!N.0�001&.��6�"��6 �.�6�.P�#P0P0�#1���������������������������������������������������������������������6�	�:	`Healing Potential of Citrullus Lanatus in Acetic Acid Induced Ulcerated Rats
Sharma Swapnil* , Vivek Dave, Sachdev Yadav 
Department of Pharmacy, Banasthali University
Banasthali-304022, Rajasthan (India)
Email*: swapnilsharma1978@gmail.com



Abstract
The present study was carried out to evaluate the healing potential of aqueous extract of Citrullus lanatus (250 mg/kg, 500 mg/kg and 1000 mg/kg p.o.) on acetic acid induced ulcers in rats. Rats were divided in 5 groups of 6 animals each, first group as control, second group served as standard (Carbenoloxone sodium 100mg/kg of bw) while group 3, 4 and 5 were treated with fruit pulp aqueous fruit pulp extract of Citrullus lanatus at doses of 250, 500 and 1000 mg/kg body weight (b.w.), per os respectively, after the induct ion of ulcer. All the three doses of Citrullus lanatus showed reduction in ulcer lesion index as compared to vehicle treated group, providing evidence for ulcer healing capacity. The post treatment with CLE resulted in significant increase in hexoses, sialic acid, enzymic antioxidants i.e. super oxide dismutase (SOD), catalase (CAT), with a significant decrease LPO levels. Histological studies also confirmed the gastroprotective activity of CSE. The presence of the flavonoids and polyphenols may be responsible for the gastro-healer effect of CSE and proved it as potent gastroprotective agent in therapy of ulcer. 
Keywords: Citrullus lanatus; super oxide dismutase; catalase

Introduction
Citrullus lanatus (Watermelon, family Cucurbitaceae) is native to India. It is found in forest lands, riversides, and wasteland, and also gets cultivated on a small scale. It is an excellent source of the arginine, vitamin A, B & C, carotenoids, lycopenes, carbohydrate, sodium, magnesium, potassium, and water [1]. The main constituents present in the fruit are especially from its rind, which is rich in alkaloids, flavonoids, polyphenols, glycosides, steroids and tannins [2].  In Ayurveda, the plant is considered beneficial as diuretic, vermifuge, demulcent, hepatic congestion & intestinal catarrah. Traditionally Citrullus lanatus is in use as energy source, to lower high blood pressure, to prevent erectile dysfunction, as antioxidant and used to treat enlarged liver and jaundice [3]. The exhaustive literature survey revealed that the potential of C. lanatus fruit in gastro-intestinal tract ailment has not been exploited.  An effort is therefore made to evaluate scientifically the healing potential of CLE in gastric ulcers induced in rats.

Materials & methods
Experimental
Fresh Citrullus lanatus (Watermelon) was purchased from the market of Jaipur (Rajasthan, India) and botanical authentification was carried out at the Department of Botany, University of Rajasthan (voucher specimen no. RUBL20694). Fresh fruit pulp was homogenized and dried under shade and thus dark greenish brown powder mass obtained was powdered (yield- 39%). The dried Citrullus lanatus fruit pulp was reconstituted in water (CLE) and the extract so obtained was subjected to qualitative phytochemical analysis and assessment of pharmacological activity.
Animals
Healthy Wistar rats of either sex weighing between 180-250 gm were used in this study. Animals were maintained at 25�20C and kept in well ventilated animal house under natural photoperiodic condition in polypropylene cages with paddy husk as bedding with free access to food and water ad libitum. The experimental protocol described in present study was approved by Institutional animal ethical committee (IAEC) of Pinnacle Biomedical Research Institute, Bhopal (Reg No. 1283/C/09/CPCSEA). 
Acute toxicity studies
Different doses of the CLE were administered orally to six groups of rats each containing 6 rats. The animals received 250, 500, 1000, 1500 and 3,000, 6000 mg/kg body weight orally. They were observed for 24 h after which the number of dead rats was recorded. The method of Karber was employed in the determination of acute oral median lethal dose (LD50). Similarly subacute toxicity studies were performed using 250, 500, 1000 mg/kg body weight orally thrice a day for four weeks on rats of both sexes in three different groups. Effects were observed on physiological (body weight, food and water intake), organ weight (liver, kidney, adrenals and testis), haematological and biochemical liver function (total bilirubin, SGOT, SGPT, alkaline phosphatase, total protein and albumin) and renal function (blood urea and creatinine) tests. For further insight histological studies of liver, kidney, testis and ovaries was performed for any gross or microscopic changes [4-5].
Acetic acid-induced ulcers
The rats were anaesthetized with pentobarbitone (35 mg/kg, i.p.). The abdomen was opened and the stomach was visualized. A cylindrical glass tube of 6 mm in diameter was tightly placed upon the anterior serosal surface of the glandular portion of stomach 1 cm away from the pyloric end. A total of 50% acetic acid (0.06 ml/ animal) was instilled into the tube and allowed to remain 60 s on the gastric wall. After removal of the acid solution, the abdomen was closed in two layers and animals were caged and fed normally. CSE was given in the doses of 250,500 & 1000 mg/kg orally, thrice daily at 8:00 and 14:00 h, 22:00 h for five days, after the application of acetic acid and continued up to 5 days after induction of the ulcer. The animals were then sacrificed after 18 h of the last dose of drug either on day 6 of experiment to assess the ulcer size and healing. Ulcer index was calculated based upon the product of length and width (mm2/rat) of ulcers [6].

Treatment protocol
CSE was administered in the doses of 250, 500, and 1000 mg/kg body weight orally for five days for three times in a day before ulcer induction. Carbenoloxone sodium (100 mg/kg) was used as the standard drug. The doses were administered orally to different experimental groups thrice daily at 8:00 and 14:00 h, 22:00 h respectively, for 5 days after induction of ulcer. Vehicle treated group received suspension of 1% CMC in distilled water.
Physical parameters- cytoprotection studies
Ulcer Index         
             Ulcer healing was observed via measurement severity of lesion. Following   arbitrary  scoring system was used to grade the incidence and severty of lesion

                         P<.05
0 = Normal, 1 = Red colouration, 2 = Spot ulcers, 3 = Haemorrhagic streaks, 4 = Ulcers > 3 but < 5 and 5 = Ulcers > 5
The Mean Ulcer Index UI will be calculated using following formula:
                                   UI = UN+US+UP X 10-1
Where,
	UN	=	Average of number of ulcer per animal.
         				US	=	Average of severity score.
          			UP	=	Percentage of animals with ulcers
The ulcer index was scored for both the models following the method of Hollander et al [7-8].
Histopathological studies
            Gastric tissue samples were fixed in neutral buffered formalin for 24 h, then tissues were fixed in 10% buffered formalin. The processed tissues were embedded in paraffin blocks and sections about 5 �M thick were cut using an microtome. These sections were stained with haematoxylin and eosin. The slides were examined microscopically for pathomorphological changes such as congestion, hemorrhage, edema, and erosions using an arbitrary scale for severity assessment of these changes.
Biochemical parameters 
Estimation of Total Hexoses
The dissolved mucosubstances in gastric juice were estimated in the alcoholic precipitate obtained by adding 1 ml of gastric juice to 9 ml of 90% alcohol, the mixture was kept for 10 min, and the supernatant was discarded. The precipitate separated was dissolved in 0.5 ml of 0.1N sodium hydroxide. To this 1.8 ml of 6N, HCl was added. This mixture was hydrolyzed in the boiling water bath for 2 hrs. The hydrolysate was neutralized by 5N sodium hydroxide using phenolphthalin as indicator and the volume was made up to 4.5 ml with distilled water and used for the estimation of total hexoses. The method adopted is based on the determination of the hexose component of gastric mucosal glycoproteins. It depends on the reaction of carbohydrate in concentrated sulphuric acid with orcinol (5-methyl orcinol) to give a coloured product, measured colorimetrically. In detail, 0.4 ml of 1:20 diluted juice was added to an equal volume of 3.4 ml of the orcinol reagent (1.6%) and 4 ml of 60% sulphuric acid. The mixture was boiled in a water bath for 10 min and then cooled on ice. The optical density was measured at 425 nm [9]. 
Estimation of Sialic acid
To 0.5 ml of the hydrolysate in 0.1N sulphuric acid, 0.2 ml of sodium periodate was added and mixed thoroughly by shaking. A time of 20 min was allowed to elapse before addition of 1 ml of sodium arsinate solution mixture. The developed brown colour was disappeared by shaking. Then 3 ml of thiobarbituric acid was added and while shaking 4.5 ml of cyclohexanone was added, till the entire colour was taken up by the cyclohexanone supernatant. The resulting mixture was centrifuged to get a clear pink layer of cyclohexanone. The supernatant was pipette out and intensity of colour was measured using spectrophotometer at 550 nm. The sialic content of the sample was determined from the standard curve of sialic acid and has been expressed in �g/ml of gastric juice [10].
Estimation of free radical generation
The fundic part of the stomach was homogenised (5%) in ice cold 0.9% saline with a glass homogeniser for 30 sec. The homogenate was then centrifuged at 800 g for 10 min followed by centrifugation of the supernatant at 12,000 g for 15 min and the obtained fraction was used for the following estimations.
Lipid peroxidase (LPO)
LPO product malondialdehyde (MDA) was estimated using 1,1,3,3-tetraethoxypropane as the standard and is expressed as nmol/mg protein. The glandular portion of the gastric mucosa was homogenized with cold 0.15 M Tris-HCl (pH 7.4) to give a 10% (w/v) homogenate. After 10 min, 0.2 ml of tissue homogenate, 0.2 ml of 8.1% sodium dodecyl sulphate (SDS), 1.5 ml of 20% acetic acid and 1.5 ml of 8% TBA were added. The volume of the mixture was made up to 4 ml with distilled water and then heated at 95�C on a water bath for 60 min using glass balls as condenser. After incubation the tubes were cooled to room temperature and final volume was made to 5 ml in each tube. 5.0 ml of butanol: pyridine (15:1) mixture was added and the contents were vortexed thoroughly for 2 min. After centrifugation at 3000 rpm for 10 min, the upper organic layer was taken and its OD read at 532 nm against the blank [11-12].
Superoxide dismutase (SOD) activity
SOD was estimated by following the method of Kakkar et al., (1984). The inhibition of reduction of nitrobluetetrazolium (NBT) to blue coloured Formosan in presence of phenazine methylsulphate (PMS) and NADH was measured at 560  nm using n-butanol as blank. One unit of enzyme activity was defined as the amount of enzyme that inhibits rate of reaction by 50% in one min under the defined assay conditions and the results have been expressed as units (U) of SOD activity/mg protein [13].
Catalase (CAT) activity
100�l of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0). Reaction was started by the addition of 1.0 ml of freshly prepared 30 mM H2O2. Decrease in absorbance was read at 240 nm for 3min at interval of 30 sec. The activity was calculated using extinction coefficient of H2O2 0.041micromole/cm2. Results were expressed as micromole of H2O2 utilized/min/gm tissue. The rate of Decomposition of H2O2 was measured spectrophotometrically from changes in absorbance at 240 nm [14]
Statistical Analysis
Results were expressed as the mean � S.E.M. Statistical significance was determined by one-way analysis of variance followed by Dunnett�s test, with the level of significance set at P < 0.05.
Results
The results of the initial trials carried out with different dosages of Citrullus lanatus extract (250, 500, 1000 mg/kg, p.o. /day) thrice a day for five days showed a significant protection against the experimental ulcers induced acetic acid and was comparable to potent healer carbenoloxone sodium. From the Table 2 it is evident that a dosage of 1000 mg/kg, p.o./day of Citrullus lanatus markedly inhibited the LPO level, the number of lesions along with a elevation  in the hexoses & sialic acid and antioxidant enzyme levels SOD & catalase, re�ecting a positive effect of Citrullus lanatus on acetic acid induced gastric ulcer. In Table 1, the 50% acetic acid (0.06 ml/ animal) caused a signi�cant (p < 0.05) increase in the number of lesions in the gastric mucosa. The gastro-protective effect of aqueous extract of Citrullus lanatus confirmed by a dose dependent healing in acetic acid induced ulceration in albino rats. The study on effects of aqueous extract of Citrullus lanatus indicate that the significantly (p<0.005) reduces the ulcer index of gastric secretion and also has activity against gastric ulcers in albino rats.

Discussion 
Multiplicity of factors responsible for peptic ulcer makes difficult to pinpoint its exact etiology. Nevertheless, it is clear that it results from an imbalance between offensive and defensive factors. Naturally occurring substances have been found to possess anti ulcerogeinc activity by lowering offensive factors of augmenting defensive factors for ulcerogenesis [15]. The efficacy of CLE as healer anti ulcerogeinc could be due to its various actions on offensive and defensive factors. Hence further investigation carried on offensive and defensive factors in the gastric juice and mucosa. CLE The extract protected the stomach against acetic acid induced necrotic damage and its effect was comparable to than carbexoloxone sodium, a potent healer in ulcer therapy. The number of lesions present on the gastric mucosa are indicative of the ulcer severity [16]. A signi�cant reduction in the number of lesions i.e. ulcer index was observed after five days of treatment  Citrullus lanatus groups may be due to its healing properties.
Several mechanisms have been suggested for the effect of antiulcerogenic compounds, including increasing the gastric hexose & hexosamine level and enhancing the strength of the gastric barrier either physically or by blocking the H+, K+�ATPase pump. Probably, the CLE might exert their activities by one or more of these proposed mechanisms. Recently, studies have implicated the generation of oxygen derived free radicals and lipid peroxidation as one of the most important mechanisms involved in the pathogenesis of gastric ulcer. Antioxidants are known to inhibit lipid peroxidation and scavenge free radicals. Here the Citrullus lanatus markedly inhibited the LPO level, the number of lesions along with an elevation in the hexoses & sialic acid and antioxidant enzyme levels SOD & catalase level. Mucin is viscous glycoprotein with physiochemical properties producing relatively resistant acid barrier [18-19]. It makes up the major part of the mucus, an important pre-epithelial factor that acts as a first line of defence against ulcerogens.19 Increase in mucin was due to significant increase in individual mucopolysacharide like sialic acid and total hexoses leading to significant increase in total carbohydrates that is responsible for gastro-healing  potential of CLE. Since Citrullus lanatus aqueous extract significantly enhances gastric tissue SOD & catalase concentration, it is conceivable that it is endowed with antioxidant properties accounting for its gastrohealing potential.

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Table 1.  Effect of dried fruit pulp of Citrullus lanatus (CLE) on acetic acid-induced      gastric ulcers  in rats 
S. No.GroupUlcer Index% Protect1Control8.00 � 0.442Std.0.10 �0.09*68.423CLE 250'*+12MN\]^z������������ٱ��xfT�T?�(h�@Rh�@R5�B*CJOJQJaJph"h�|`5�B*CJOJQJaJph"hVC5�B*CJOJQJaJph"hxX5�B*CJOJQJaJph"h��5�B*CJOJQJaJph(h�|`h�|`5�B*CJOJQJaJph"h�`05�B*CJOJQJaJph+hi�h
5�6�B*CJOJQJaJph(h�|`h
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la�6	yt�X��T5.98 �  0.07*14.474CLE 5003.14  � 0.43*34.25CLE 10001.18  � 0.05*48.68         a(mean �S.E.M., n = 6 in each group)
     * Significant variation as compared to Indomethacin treatment (P<0.05) 





Table 2: Effect of post-treatment of dried fruit pulp of Citrullus lanatus (CLE) on LPO, SOD and CAT level  in rats
S. No.GroupHexosesSialic acid1Control101.02  �  12.2621.64  � 4.162Std.178.35 �  7.65*42.80 � 3.63*3CLE 250113.12  �  6.53*28.41 � 4.18*4CLE 500180.76 �  4.32*32.40  � 3.27*5CLE 1000232.67 �  3.26*37.22  � 4.00*         a(mean �S.E.M., n = 6 in each group)
     * Significant variation as compared to Indomethacin treatment (P<0.05) 








Table 3: Effect of post-treatment of dried fruit pulp of Citrullus lanatus (CLE) on LPO, SOD and CAT level in rats

S. No.GroupSODLPOCATALASE1Control14.0 �  0.670.643 �  0.01210.27  � 0.162Std.87.22 �  0.45*0.275 �  0.017*59.13 �  0.19*3CLE 25026.36 �  0.43*0.576 �  0.023*18.86 �  0.28*4CLE 5002.20 �  0.54*0.449 �  0.018*32.16 �  0.14*5CLE 100077.14 �  0.76*0.309 �  0.01*  0.21*         a(mean �S.E.M., n = 6 in each group)
     * Significant variation as compared to Indomethacin treatment (P<0.05) 






















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