Gastroprotective Effect of Tectona Grandis in Experimental animals Anupama a suralkar

Gastroprotective Effect of Tectona Grandis in Experimental animals
Anupama A Suralkar^*, Avinash D Deshpande, Abhijeet R Borate, Kishor N Rodge, Rahul D Kamble, Sandeep R Khandeshe

Department of Pharmacology,

Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences & Research,

Pimpri, Pune-411 018.

Name and address of corresponding author*:

Anupama A Suralkar^*,

Department of Pharmacology,

Padm.Dr.D.Y.Patil Institute of

Pharmaceutical Sciences and Research,

Pimpri, Pune-411 018.

E-mail: [e-mail]

Mobile: [phone]

ABSTRACT:

The gastroprotective activity of ethanolic extract of Tectona grandis bark (EtOH-TG) was investigated by using ethanol induced ulcer model in rats. In this model ulcer index, antioxidant parameters like Superoxide dismutase, Catalase, Reduced glutathione, Lipid peroxidation and pH of gastric juice were determined. Administration of EtOH-TG for a period of 15 days at the dose of 100 and 200 mg/kg p.o. /day body weight reduced the chances of the gastric mucosal lesions induced by ethanol administration to the rats. EtOH-TG at the dose of 100 and 200 mg/kg, p.o. /day produced significant (p<0.05 and p<0.01) decrease in ulcer index and significant (p<0.05 and p<0.01) increase in antioxidant parameters and pH of gastric juice when compared to the ethanol control group respectively. Thus the EtOH-TG showed significant gastroprotective activity which may be due to its increase in mucin secretion as well as free radicals scavenging activity in the stomach which reduces the gastric ulcer production. Thus the results obtained indicates that ethanolic extract of Tectona grandis may prove to be useful in treatment of gastric ulcer this may be due to its significant antiulcer and cytoprotective effect on gastric mucosa.

Key words: Gastroprotective, EtOH-TG, ethanol induced ulcer, ulcer index.

INTRODUCTION:

Gastric ulcer is a very common global problem today. The pathophysiology of these ulcers involves an imbalance between offensive (acid, pepsin, and Helicobacter pylori) and defensive factors (mucin, prostaglandin, bicarbonate, nitric oxide and growth factors). Today, there are two main approaches for treating peptic ulcer. The first deals with reducing the production of gastric acid and the second with re-enforcing gastric mucosal protection. ⁽¹⁾ Modern approach includes proton pump inhibitors, histamine receptor blockers, drugs affecting the mucosal barrier and prostaglandin analog but clinical evaluations of these drugs have shown incidences of relapse, side effects and drug interactions. ⁽²⁾ For examples, H₂- receptor antagonists like cimetidine may cause gynecomasia in men and galactorrhea in women.⁽³⁾ while proton-pump inhibitors like omeprazole and lansoprazol can cause nausea, abdominal pain, constipation and diarrhea.⁽⁴⁾ These therapeutic limitations have provided incentive anti-ulcer drugs. Due to those side effects, there is a need to find new anti-ulcerogenic compound(s) with potentially less or no side effects and medicinal plants have always been the main sources of new drugs candidates for the treatment of gastric ulcer. ^{(5, 6)}

Tectona grandis Linn. (Verbenaceae) is a large deciduous tree. Branchlets are quadrangular, channeled and stellately tomentose. The tree is growing in higher situations, native to central India, Konkan, Western Deccan peninsula, South India and Burma. It is commonly known as sagwan (Hindi), saka (Sanskrit) and teak tree (English). Teak is a hardwood species of worldwide reputation. Root contains lapachol, tectol, tectoquinone, β-sitosterol and a diterpene, tectograndinol. Bark is useful in bronchitis, hyperacidity, vitiated conditions of pitta; dysentery, burning sensation, diabetes, leprosy and skin diseases. ⁽⁷⁾

T. grandis has been investigated for its nitric oxide scavenging activity and various phytochemicals present in plants have been reported to possess great potential in treatment of various diseases, many of them have been shown to be very useful in wound care i.e. promoting the rate of wound healing, bringing about decrease in pain, discomfort, and scarring ⁽⁸⁾ and naphthoquinones like juglone, lapachol and deoxylapachol reported in teak also possess antimicrobial activity. ⁽⁹⁾ T. grandis contains tannin, which is used as an anti-inflammatory agent and is also used topically for the treatment of burns. ⁽⁷⁾

Traditionally this plant is used as anti-inflammatory, anti-bacterial, cytotoxic, anti-anemic, anti-ulcer, anti-viral, wound healing. Lapachol is the main chemical constituent which is reported in this plant. ⁽¹⁰⁾ Thus, the present study involves the gastroprotective activity of the ethanolic extract of T. grandis bark in ethanol induced ulcer model in rats.

MATERIAL AND METHODS:

Plant material and preparation of extract

Fresh bark of Tectona grandis Linn. (Verbenaceae) was collected from Nanded, Maharashtra, India. The specimen was authenticated at Botanical Survey of India (BSI), Pune. The bark was washed with distilled water and shed dried and latter powdered. This powder was then defatted with petroleum ether and then macerated with ethanol for 72 h with occasional shaking. It was then filtered and the solvent was evaporated under vacuum ⁽¹¹⁾. The yield of ethanolic extract of bark of Tectona grandis (EtOH-TG) was 3.1% (w/w).

Experimental animals

Wistar albino rats weighing 150-200g were housed in standard cages at room temperature 22 ± 2 °C and 50±5% relative humidity, under a light/dark cycle of 10/12 h, for 1 week before the experiments. Animals were provided with standard rodent pellet diet (Amrut, India), and water ad libitum. The animals were deprived of food for 24 hrs before experimentation, but had free access to drinking water. All experiments were performed in the morning. Experimental protocols were approved by our Institutional Ethical Committee which follows guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) and complies with international norms of INSA (Indian National Science Academy).

Acute oral toxicity study and selection of doses

A safe oral dose of EtOH-TG was determined through the acute oral toxic test in rats as described by the Organization of Economic Co-Operation and Development (OECD) as per 423 guidelines. The EtOH-TG, at different doses up to 2000 mg/kg, was prepared by dissolving the extract in distilled water and the concentration was adjusted in such a way that it did not exceed 1 ml/100 g of the rat. The extract was then administered by oral route and animals were observed for behavioural changes, any toxicity and mortality up to 48 h. Two different doses (100 and 200 mg/kg, p.o) of EtOH-TG were later chosen for this study based on the acute toxicity testing.

Gastric Ulcer Induction in Rats: ⁽¹²⁾

One ml of 80% ethanol was used orally to induce gastric ulcer. The animals were divided into four groups (n=6). Group-I represented the normal control group, which received 5 ml/kg body weight of vehicle (1% gum acacia, p.o. /day). Group-II received ethanol (80%, 1.0 ml) and served as ethanol control. Group-III received Omeprazole (20 mg/kg p.o. /day) one hour before ethanol (80%, 1.0 ml) administration and served as standard. Group-IV and Group-V received EtOH-TG at the dose of 100 and 200 mg/kg, p.o/day) respectively of one hour before ethanol (80%, 1.0 ml) administration and served as test.

EtOH-TG was administered by oral intubation at a dose of 100 and 200 mg/kg, p.o/day) and was given for a period of 15 days. After the experimental period of 15 days, prior to sacrifice, animals were fasted for 24 h. Group-II to Group-V animals received 1.0 ml of 80% ethanol orally. One hour after the ethanol administration the animals were sacrificed by cervical dislocation and the abdomen was incised and irrigated with normal saline. Subsequently, the stomach was incised along the greater curvature and its mucosal erosion was determined randomly by measuring the area of the lesions. The following arbitrary scoring system was used to grade the incidence and severity of lesions, ⁽¹³⁾

0 = No ulcer, 1 = Superficial ulcer, 2 = Deep ulcers, 3 = Perforations

The Mean Ulcer Index UI was calculated using following formula:

UI =UN+US+UP X 10-1

Where UI=Ulcer Index, UN = Average of number of ulcer per animal, UN = Average of severity score, UP = Percentage of animals with ulcers.

Effect on antioxidant enzyme parameters:

The stomach was weighed and then further processed for antioxidant enzymes, in which the stomach was homogenized in chilled Tris buffer (10 mM, pH 7.4) at a concentration of 10% (w/v). The homogenates were centrifuged at 10,000 x g at 0^oC for 20 min using Remi C-24 high speed cooling centrifuge. The clear supernatant was used for the assays of lipid peroxidation (MDA content), endogenous antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT), and reduced glutathione (GSH)].

Biochemical Estimations

Superoxide dismutase was determined by the method of Mishra and Fridovich. ⁽¹⁴⁾Catalase was estimated by the method of Hugo Aebi as given by Colowick et al.⁽¹⁵⁾ Reduced glutathione was determined by the method of Moron et al.⁽¹⁶⁾ Lipid peroxidation or malondialdehyde formation was estimated by the method of Slater and Sawyer. ⁽¹⁷⁾

STATISTICAL ANALYSIS:

The values expressed as mean ± SEM from 6 animals. The results were subjected to statistical analysis by using one way ANOVA followed by Dunnett’s test to verify the significant difference if any among the groups. P<0.05*, P<0.01**and P<0.001*** were considered significant.

RESULTS:

Study of Anti-Ulcer and Antioxidant Activity Using Ethanol-Induced Ulcer Method

Administration of ethanol produced significant ulcers in the ethanol control group. There was a significant (p<0.05 and p<0.01) reduction in ulcer index at the selected dose of 100 and 200 mg/kg, p.o/day of EtOH-TG and % protection was found to be 54.17 and 63.01 % respectively which was comparable to standard drug Omeprazole (75.61%).

Ethanol administration was found to increase lipid peroxidation and decrease SOD, catalase, and reduced glutathione in the ethanol control group when compared to normal control rats. Administration of EtOH-TG significantly (p<0.05 and p<0.01) decreased lipid peroxidation and increased the levels of SOD, catalase and reduced glutathione level at the dose of 100 and 200 mg/kg p.o. /day respectively. The results of this work are shown in Table 1 and Table 2.

Effect on Gastric Mucosa of Ethanol Treated Rats

Histologically, ethanol treated rats showed ulcerated mucosa with marked mucosal hemorrhage and destruction of glandular elements and these were characterized by multiple red bands (patches). Pretreatment with EtOH-TG showed significant (p<0.05 and p<0.01) protection against ethanol induced gastric mucosal damage when compared to ethanol control group. Animals in ethanol control group (Fig.1) showed total mucosal ulceration, hemorrhage and segmental mucosal necrosis of gastric epithelium when compared to the animals treated with Omeprazole (standard). In animals treated with 100 and 200 mg/kg p.o. /day of EtOH-TG (test), EtOH-TG at the dose of 200 mg/kg p.o. /day the gastric mucosal epithelium showed maintenance of normal architecture than 100 mg/kg dose of EtOH-TG when compared to ethanol control group.

DISCUSSION AND CONCLUSION:

It is generally accepted that gastric ulcer results from an imbalance between aggressive factors and the maintenance of the mucosal integrity through the endogenous defense mechanism. To regain the balance, different therapeutic agents including herbal preparations are used to inhibit the gastric acid secretion or to boost the mucosal defense mechanism by increasing mucus production. Experimental studies have demonstrated that oxygen-generated free radicals (ROS) and lipid peroxidation are involved in the pathogenesis of acute gastric lesions induced by ethanol, nonsteroidal anti-inflammatory drugs or Helicobacter pylori. Absolute ethanol method of inducing gastric lesions is rapid and convenient way of screening plant extracts for anti-ulcer potency and cytoprotection in macroscopically and microscopically visible lesions. Disturbances in gastric secretion, damage to gastric mucosa, alterations in permeability, gastric mucus depletion and free-radical production are reported to be the pathogenic effects of ethanol. Absolute ethanol is highly corrosive to the gastric mucosa and it causes superficial necrosis of gastric mucosa and release of histamine and leukotriene-C4 as tissue-derived mediators. These mediators act on gastric microvasculature and result in destruction of mucosa and submucosa of gastric wall. Extensive experimental evidences indicate that certain substances, through scavenging of free radicals, protect the gastric mucosa. ⁽¹²⁾

In the present study, The EtOH-TG was subjected for phytochemical investigation and acute toxicity studies. In phytochemical evaluation EtOH-TG showed the presence of beta-sitosterol, terpenoids, phenolic compounds, saponins, flavonoids, quinones, glycosides and tannins. In acute toxicity studies, EtOH-TG has shown it to be safe up to a dose of 2000 mg/kg p.o. body weight of rat. Further EtOH-TG was evaluated at a dose of 100 and 200 mg/kg p.o. /day body weight for its potential gastroprotective activity in ethanol induced gastric mucosal ulceration in rats and we found that EtOH-TG exhibited significant gastric mucosal protection in a dose dependent manner against this model, with maximum protection observed at 200 mg/ kg, p.o. /day when treatment is given for 15 days.

From the results obtained, the gastroprotective activity of ethanolic extract of TG bark may be due to its free radicals scavenging activity as well as increase in mucin secretion in the stomach which reduces the chances of gastric ulcer production. Earlier studies have shown that TG exerts anti-ulcer activity in experimental gastric ulcer models but the mechanism of actions through which it mediates gastroprotective effects were not elucidated.

Though we have not studied the active principles responsible for the anti-ulcer activity of ethanolic extract of Tectona grandis, this may be due to the presence of tectoquinone, lapachol, deoxylapachol & flavonoidal compounds such as rutin and quercitin in the extract. Lapachol have been reported to possess significant anti-ulcer activity in various experimental models of gastric and duodenal ulceration. ^{(18, 19)}

CONCLUSION:

Thus the results obtained in the present investigation indicates that ethanolic extract of Tectona grandis may prove to be useful in preventing gastric ulcer owing to its ability to increase the mucin secretion and antioxidant activity in the stomach. Thus our studies establish a significant antiulcer and cytoprotective effect of EtOH-TG. However, further studies are required to establish its exact mode of action and the active principles involved in its anti-ulcer effect.

ACKNOWLEDGEMENT:

Authors are grateful to Dr. A. D. Deshpande, Director Pad. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune, M.H., India, for providing laboratory facilities.

CONFLICT-OF-INTEREST:

The authors declare that they have no conflict of interest.

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Table 1: Effect of Ethanolic Extract of Tectona grandis Bark (EtOH-TG) on ulcer index & % inhibition of ulcers.

Group	Dose (mg/kg)	Total no. of Ulcers	Ulcer Index	% protection
I	Normal Control (Vehicle 5 ml/kg p.o)	-	-	-
II	Ethanol Control (80% Ethanol, 1.0 ml)	20.37±0.98	61.1	-
III	Standard Omeprazole (20 mg/kg p.o)	3±0.46**	14.9	75.61**
IV	EtOH-TG (100 mg/kg, p.o)	7.27±0.71*	28	54.17*
V	EtOH-TG (200 mg/kg, p.o)	5.87±0.53**	22.6	63.01**

EtOH-TG: Ethanolic extract of Tectona grandis bark; Values are expressed as mean ± SEM (n=6). Ethanol control group was compared with normal control group. EtOH-TG treated groups were compared with ethanol control group; *p<0.05; **p<0.01. Values in parenthesis indicate the % reduction in ulcer index in relation to the ethanol control group.

Table 2: Effect of Ethanolic extract of Tectona grandis Bark (EtOH-TG) on antioxidant parameters in stomach of ethanol-treated rats.

Parameters	Normal Control (Vehicle 5 ml/kg p.o)	Ethanol Control (80% Ethanol, 1.0 ml)	Standard Omeprazole (20mg/kg p.o.)	EtOH-TG (100mg/kg, p.o.)	EtOH-TG (200mg/kg, p.o.)
SOD (unit/mg protein)	5.61 ± 0.33	2.23 ±0.03**	8.09 ± 1.25**	4.21± 0.95*	6.39 ± 0.79**
Catalase (μmoles of H2O2 consumed/min mg protein)	8.25 ± 0.27	5.93 ± 0.61*	7.61 ± 1.13**	4.59± 1.25*	6.68 ± 1.55**
Reduced glutathione (μg of GSH/mg protein)	3.25 ± 0.15	0.79 ± 0.17**	4.65 ± 6.25**	1.48± 3.32*	2.26 ± 4.10**
Lipid peroxidation (nmoles of MDA/mg protein)	3.56 ± 0.33	6.75 ± 0.70**	1.43 ± 0.08**	2.14± 0.45*	1.69 ± 0.08**
pH of gastric juice	1.22 ± 0.07	2.6± 0.15	5.50 ± 0.87**	3.72± 0.33*	4.2± 0.18**

EtOH-TG: Ethanolic extract of Tectona grandis bark; Values are expressed as mean ± SEM (n=6). Ethanol control group was compared with normal control group.

EtOH-TG treated groups were compared with ethanol control group; *p<0.05; **p<0.01. Values in parenthesis indicate the % reduction in ulcer index in relation to the ethanol control group.




Ulcerated mucosa



(Normal control) (Ethanol control)





Ulcerated mucosa


Standard, Omeprazole (20 mg/kg) EtOH-TG (100 mg/kg)





Ulcerated mucosa

EtOH-TG (200mg/kg)

Figure 1: Effect of Ethanolic Extract of Tectona grandis Bark (EtOH-TG) on gastric mucosa of stomach.