INTRODUCTION:

Gastric ulcers arise due to various factors ¹. Even though the etiology of gastric ulcers is still debated, it is accepted that ulcers are caused due to net imbalances in mucosal offensive and defensive factors ². Ulcer therapy is now mainly focused on limiting the deleterious effects of offensive acid secretion, but search for new safer alternative drugs have rekindled the interest in cytoprotective drugs, which protect the gastric mucosa from damaging agents without influencing acid secretion or neutralizing intragastric acidity ³. Although few drugs like sucralfate and prostaglandin analogs, i.e. misoprostol are recognized as cytoprotective agents ⁴, many natural drugs have been reported to possess this activity ^{5 - 7}. Change in gastric emptying has been reported to be responsible for genesis of ulcers ⁸. Free radical shave been reported to be responsible for many ailments including gastro duodenal ulcers ⁹. Azima tetracantha Lam (family Salvadoraceae) is a well-known medicinal plant whose root has been advocated for various ailments such as dropsy and rheumatism.

Azima tetracantha leaves possess stimulant, expectorant and antispasmodic activities ¹⁰; analgesic effect ¹¹ and anti-inflammatory effect ¹². The present study incorporates the evaluation of ulcer protective effects of ethanolic extract of dried leaves of Azima tetracantha by estimating various biochemical parameters of ulcer-induced models.

MATERIALS AND METHODS:

Plant collection and Extraction:

The leaves of Azima tetracantha was collected from Chennai district, Tamil Nadu, India in the month of July 2008. Dr. P. Jayaraman, (Director) Plant Anatomy Research Centre, Chennai, confirmed the identity. A voucher specimen was deposited in the Department of Pharmacognosy at Madras Medical College, Chennai. 500 grams of dried leaves of Azima tetracantha was powdered and then soaked in 1500 mL of ethanol overnight. After filtration, the residue obtained was again resuspended in equal volume of ethanol for 48 h and filtered again. The above two filtrates were mixed and the solvent was evaporated in a rotovac evaporator at 40°- 50°C under reduced pressure. A 16% semisolid dark material obtained was stored at 0-4°C until used. A known volume of the residual extract was suspended in 0.5 % Carboxy methylcellulose (CMC) for animal administration.

Table1: Effect of EEAT on pH, gastric volume, free acidity and total acidity

Treatment Groups	pH	Gastric volume (mL/100g)	free acidity (m Eq/L/100g)	Total acidity (m q/L/100g)
Control	4.13±0.147	1.55±0.1335	58.17±0.2591	44.62±0.4003
APL ^**	1.333±0.08^a**	5.6±0.1065^{a ***}	66.95±0.5396^{a **}	78.53±0.2028^a
EEAT (200 mg/kg)	3.45±0.11^b**	4.383±0.3311^b**	54.13±0.6766^b**	56.2±0.1506^b**
EEAT (400 mg/kg)	3.8±0.97^b**	3.283±0.1493^b**	44.07±0.3853^b**	51.98±0.400^b**
Ranitidine (50 mg/kg)	4.2±0.147^b**	2.783±0.1537^b**	40.8±0.2206^b**	7.08±0.2469^b**

Data are mean ± SEM., n=6, Statistical significance * p<0.05, **p<0.01, ***p<0.001, a - Group I vs. Group II; b - Group II vs. Groups III, IV and V

Table 2: Effect of EEAT on gastric juice mucoprotein (µg/mL) in ALP rats

Treatment Groups	Protein	Total hexoses	Hexosamine	Fucose	Sialic acid
Control	245.3±3.084	458± 6.172	225.3±6.381	104.7±1.8	79.33±1.282
APL	438.3±49.32^{a ***}	212.5±6.25^{a ***}	119.8±4.377^{a ***}	6.9±1.065^{a ***}	23.0±1.653^a***
EEAT (200 mg/kg)	369.8±7.507^b**	358.8±9.13^b**	176.3±6.561^b**	88.5±0.7638^b***	52.5±1.522^b**
EEAT (400 mg/kg)	345.5±11.03^b**	07.2±6.61^b**	186.2±5.406^b**	93.17±1.167^b***	61.17±0.945^b**
Ranitidine (50 mg/kg)	305±5.562^b**	438.2±6.8^b**	190.8±5.076^b**	126.2±1.701^b***	69.67±0.843^b**

Data are mean ± SEM., n=6, Statistical significance * p<0.05, **p<0.01, ***p<0.001, a - Group I vs. Group II; b - Group II vs. Groups III, IV and V

Experimental Animals:

In bred Wistar rats of either sex obtained from Madras Medical College (Chennai, India) weighing 200-250g were used for the study. They were kept in the departmental animal house at 26 ± 2°C and relative humidity 45-55%, light and dark cycles of 12 and 12 h, respectively for 1 week before and during the experiments. Animals were provided with standard rodent pellet diet (Hindustan Lever) and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC) constituted under CPCESA (Ref No: 10/243 - IAEC 2008)

Table 3: Effect of EEAT on Ulcer scores

Treatment Groups	Aspirin and Pylorus ligation	Cold Restraint Stress
Control	0.10±0.035	0±0
Ulcer induced^a***	2.38±0.0640^a***	25.50±0.5627
EEAT (200 mg/kg)	2.25±0.2230^b*	22.83±0.3073^b*
EEAT (400 mg/kg)	1.4±0.210^b**	21.33±0.2108^b*
Ranitidine (50 mg /kg)	1.03±0.049^b***	-----
Omeprazole (10 mg/kg)^b**	------	16.67±0.0201

Data are mean ± SEM., n=6, Statistical significance * p<0.05, **p<0.01, ***p<0.001, a - Group I vs. Group II; b - Group II vs. Groups III, IV and V

Acute toxicity studies:

Albino mice weighing 20-25 g selected by random sampling technique were used in the study. Acute oral toxicity was performed ¹³. The animals were fasted overnight, provided only water after which extract was administered to the groups orally at the dose level of 5 mg / kg body weight by gastric intubation and the groups were observed for 14 days. If mortality was observed in 2 or 3 animals (out of 6), then the dose administered was assigned as a toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher doses such as 50, 300 and 2000 mg / kg body weight. The animals were observed for toxic symptoms such as behavioral changes, locomotion, convulsions and mortality for 72 h.

Aspirin plus pylorus ligation method (APL):

The animals were placed in cages with grating floor to avoid coprophagy and divided into five groups viz, Group I receiving 0.5 % Carboxy methyl cellulose (CMC) served as vehicle control, group II received Aspirin 200 mg / kg p.o suspended in 0.5 % CMC. Groups III and IV received the ethanolic extract of Azima tetracantha (EEAT) at doses of 200 mg / kg and 400 mg/ kg respectively by oral route. Group V received Ranitidine 50 mg / kg orally serving as standard drug control for Aspirin plus pylorus ligation (APL) models. All the extracts and reference drug were suspended in 0.5 % CMC for animal administration. Aspirin was administered once daily for 5 days. Ranitidine and the extracts were administered 30 min before each aspirin administration. On day 6 after last dose, the rats were kept for 18 h fasting. Pylorus ligation was done following the method as described ¹⁴. The animals were deprived of water during the postoperative period. After 4 h, stomachs were dissected out and contents were collected in tubes for estimation of biochemical parameters. Ulcers were scored as described ^15.

Cold restraint stress induced ulcers (CRS):

The animals were placed in cages with grating floor to avoid coprophagy and divided into five groups viz, Group I receiving 0.5 % Carboxy methylcellulose (CMC) served as vehicle control, and group II received Cold Restraint Stress (CRS). Groups III and IV received the extract of Azima tetracantha (EEAT) at doses of 200 mg / kg and 400 mg/ kg respectively by oral route. Group V received Omeprazole 10 mg/kg and served as drug control. On the day 6, for 18 h fasted rats, CRS was given by strapping the rats on a wooden plank and keeping them immobile for 2 h at 4-6 °C. The animals were then sacrificed by cervical dislocation. The stomach was taken out and cut open along the greater curvature, ulcers were scored ¹⁶, and the tissues were subjected to estimate biochemical parameters.

Biochemical Estimation:

In APL induced ulcer models the following were estimated by procedures described, gastric volume ¹⁶, pH of gastric content ¹⁸ free acidity and total acidity ¹⁹, Dissolved muco substances such as total protein ²⁰, hesoses, hexosamine, fucose, sialic acid ²¹were estimated in 90 % alcoholic precipitate of gastric juice and expressed as µg/ml. In CRS induced ulcer model Lipid peroxidases, LPO ²² expressed as nmol/mg protein, Superoxide dismutase, SOD ²³ expressed as activity/mg and Catalase, CAT ²⁴expressed as units (U) of CAT activity/mg protein were estimated.

Statistical Analysis:

The data are expressed as mean ± SEM. Statistical comparisons were performed by one-way ANOVA followed by Dunnet’s‘t’ test. The results were considered statistically significant if the p-values were less than 0.05.

RESULTS:

EEAT did not produce any toxic symptoms or mortality up to the dose level of 2000 mg/kg body weight and hence the drug was considered safe for pharmacological screening. According to OECD-423 guidelines for acute oral toxicity, the LD₅₀ dose of 2000 mg/kg and above is categorized as unclassified.

Estimation of acid secretory parameters such as pH, gastric volume, free acidity and total acidity was increased significantly in the aspirin administered group. Administration of EEAT exhibited a significant (p < 0.01) reduction in all the parameters and the results were comparable with the standard drug Ranitidine 50 mg/kg (Table 1). Determination of the concentrations of various muco proteins such as total protein, total hexoses, hexosamine, fucose and sialic acid revealed a decrease in ulcer induced group. The extract at 400 mg/kg increased the level of the muco proteins significantly and comparably with the standard drug (p <0. 01) (Table 2). The ulcer scores obtained in ulcer induced group of APL and CRS exhibited an increased score. Administration of the extract exhibited a moderate decrease in the ulcer scores in a dose dependent manner in CRS models and a significant decrease in APL models (p < 0.05 and p < 0.01). The standard drug Omeprazole 10 mg/kg decreased the ulcer score significantly in CRS model (P < 0.01) (Table 3). Effect of EEAT on the levels of SOD, LPO and CAT were satisfactory. The stress-induced group had increased level of SOD, LPO and a decreased level of CAT than control group, which was restored by the administration of EEAT in a dose dependent manner. The results were significant and comparable with the standard drug Omeprazole 10 mg/kg (p < 0.01) (Table 4).

The histopathological sections of the drug treated group in ALP and CRS had shown a reduction in ulcer focus and a hyperplastic gastric mucosa with regenerating mucosal epithelium (Fig 1 and 2).

DISCUSSION:

EEAT showed significant dose-dependent ulcer protective effect against cold restraint stress and aspirin plus pylorus ligation induced gastric ulcers. The anomality in ulcer protective effect may be due to variability of factors affecting ulcerogenesis in different models. Ulcers are caused due to imbalances between offensive and defensive mucosal factors ² and hence the effects of EEAT can be explained based on these factors. Mucin is a viscous glycoprotein with physiochemical properties producing relatively resistant acid barrier ²⁵. It makes up the major part of the mucus, an important pre-epithelial factor that acts as a first line of defence against ulcerogens ²⁶. Increase in mucin can be due to increased levels of individual mucopolysaccharide like sialic acid and total hexoses. The increase in mucosal defence may also be due to decrease in cell exfoliation ²⁷. Hence, the protection afforded by EEAT in APL induced ulcers may be predominantly due to strengthening mucosal defense ². The ability of EEAT to protect stomach against ulcerogens by neutralizing intra gastric acidity can as well lead it to classify as a cytoprotective agent ³ Prostaglandins have often been quoted as a model cytoprotective agent, although this has been disputed ⁴. Aspirin, an irreversible COX inhibitor decreases prostaglandin synthesis and reported to produce ulcers both by local and systemic effects. Aspirin has direct irritant effect by increasing the H⁺ion transport. On the mucosal epithelial factors, it decreases mucin, surface-active phospholipids bicarbonate secretion mucosal proliferation and on the microvasculature produced damage by formation of free radicals ²⁸. To ascertain this effect, the activity of EEAT was studied on aspirin plus pylorus ligation model, where aspirin is known to further aggravate mucosal damage caused by pylorus ligation ²⁶. It was found that EEAT was effective in this model, suggesting that the activity of EEAT may also involve other defensive factors apart from PG synthesis.

The role of the free radicals in gastric ulcerations is well-documented ²⁹. EEAT significantly reduced lipid peroxidation in rat gastric mucosa. Azima tetracantha has been reported to possess antioxidant activity ³⁰SOD scavenges the super oxide radical O₂^- , one of the reactive oxygen species (ROS) responsible for lipid

peroxidation ³¹. This reaction leads to increase in generation of producing more oxidative damage ³². CAT and other peroxidases further reduce H₂O₂^- . Hence, the anti oxidant activity in gastric mucosal homogenates observed from the decrease in the LPO may be due to increase in SOD and CAT levels. Stress induced ulceration involves damage by ROS apart from acid and pepsin related factors ³³ during stress LPO and SOD were significantly increased and CAT level was significantly decreased. The increase in SOD was due to increased ROS generation during mucosal damage. This led to increased generation of H₂O₂^-and its accumulation due to decreased CAT level. Inactivation of gastric peroximes during stress ³⁴ may also aggravate the mucosal damage. This evidently caused increased lipid peroxidation and mucosal damage as seen from the increase in ulcer scores in comparison to the control group. EEAT effectively alleviated stress induced ulcers with marked decrease in LPO suggesting decrease in oxidative damage. This may be due to restoration of balance between free radical scavenging enzymes SOD and CAT in the gastric mucosa, effectively counteracting the free radicals generated by cascade of reactions as described earlier. Thus the ulcer protective activity of EEAT may also be due to its anti oxidant effect.

Hence, the gastro duodenal ulcer protecting effect of EEAT may be due to its predominant effect on the mucosal defensive factors rather than offensive factors. Further conclusion on the precise mechanism of the action can be arrived after studies on its CNS effect and on the mucosal defensive factors like Nitric oxide, cAMP etc.

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Received on 15.05.2008 Modified on 10.07.2008

Research J. Pharm. and Tech.2 (2): April.-June.2009,;Page 344-348

Treatment Groups	LPO (MDA, nmol/g wet tissue)	SOD (Units/g wet tissue)	CAT (Units/g wet tissue)
Control	132.2±1.641	84.17±1.797	51.5±2.363
Restraint stress	265.5±1.544^a***	217.2±1.249^a***	15.83±1.078^a***
EEAT (200 mg/kg)	90.5±1.607^b**	155.2±1.989^b**	29.17±2.088^b**
EEAT (400 mg/kg)	52.5±1.945^b***	105.2±2.574^b**	39.67±1.994^b**
Omeprazole (10 mg/kg)	67.0±1.461^b***	93.67±1.202^b**	44.0±2.1600^b**