A POSSIBLE GASTROPROTECTIVE AND IN VITRO ANTI-OXIDANT EFFECT OF HIBISCUS AGAINST EXPERIMENTALLY INDUCED ULCER IN RATS

at doses 200 mg/kg and 400 mg/kg p.o produced significant inhibition of the gastric lesions produced by ethanol. It also significantly (P<0.05) reduced gastric volume, free acidity and ulcer index as compared to positive control and standard drug treated animals. The obtained data suggested that methanolic extract possess potent anti-ulcer activity, which might be due to its anti-secretory activity. The presence of flavanoids in the extract supports its activity. Thus, the present investigation concluded that the methanolic extract of Hibiscus tiliaceus is possessed anti-oxidant and anti-ulcer property.

The anti-ulcer activity of methanolic extract of whole plant Hibiscus tiliaceus (Malvaceae) was investigated in ethanol induced ulcer model in wistar rats. Ulcer index was used as the common parameter to determine the activity. Maceration was the extraction procedure used. Then, the extract was screened for the presence of phytochemical constituents. In-vitro anti-oxidant studies were performed using H2O2 radical scavenging and reducing power assay. Albino rats of either sex weighing between 160-250 gms were divided into five groups of six animals each (n=6). The in-vitro anti-oxidant studies revealed that the inhibitions were statistically significant (p < 0.05). The methanolic extract at doses 200 mg/kg and 400 mg/kg p.o produced significant inhibition of the gastric lesions produced by ethanol. It also significantly (P<0.05) reduced gastric volume, free acidity and ulcer index as compared to positive control and standard drug treated animals. The obtained data suggested that methanolic extract possess potent anti-ulcer activity, which might be due to its anti-secretory activity. The presence of flavanoids in the extract supports its activity. Thus, the present investigation concluded that the methanolic extract of Hibiscus tiliaceus is possessed anti-oxidant and anti-ulcer property.

…………………………………………………………………………………………………….... Introduction:-
Globally, peptic ulcer is serious public health problem that affects about 5-10% of population, with high rate of morbidity and mortality; need to be focused on experimental and clinical investigations (Alkofahi A and Atta, 1999). Usually, peptic ulcers are aggravated by an imbalance between destructive and defensive factors in the stomach (Peskar B M and Maricic, 1998). In the treatment of peptic ulcers, control over the acidic hypersecretion is critical, and the conventional drugs chosen have direct effects on the gastric mucosa (Toma W et al., 2005).. The two main classes of drugs used to treat acid-related disorders include proton pump inhibitors (PPI) that inhibit the hydrogen pump in the parietal cell directly, independently of any membrane receptor stimulation, and histamine type 2 receptor antagonists (H2RAs), which block the histamine receptor on parietal cells thereby reducing hydrogen ion release. Long-term use of H2RAs is associated with the development of undesirable effects such as gynecomastia and galactorrhea as well as alteration of the bacterial flora of the gastrointestinal tract (Borelli F and Izzo A A,  2000).

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There are two types of factors which are responsible for the ulcers. The endogenous destructive factors in the stomach are HCl, pepsin, biliary reflux, lipid peroxidation, and the formation of reactive oxygen species (ROS). The exogenous factors are excessive use of ethanol, indiscriminate use of non steroidal anti-inflammatory drugs (NSAID), stress, smoking, and infection by Helicobacter pylori bacteria. The defensive factors are mucusbicarbonate barrier, mucin secretion, surface phospholipids, prostaglandins (PGs), nitric oxide (NO), mucosal blood flow, cell renewal, growth factors, and antioxidant enzymes (Zhongzhi W et al., 2011). Oxidative stress, present in the process of gastric ulceration, increases the formation of ROS that can disrupt epithelial cell integrity. In addition, ROS accumulates neutrophils in the tissues of the mucosa during gastric ulceration. Studies have shown that proinflammatory cytokines induce the activation of neutrophils and are strong contributors to the of ulcer damage (Melese E, 2011).
The presence of these phytochemical constituents in plants, exert a beneficial effect on health by different actions such as antioxidant, anti-inflammatory, and modulating gene expression. In fact, the intake of antioxidants from the natural source such as fruits and vegetables can provide protection against oxidative stress. Keeping in view of the use of this in ethnomedicine, the present study was taken up to investigate the effect of methanolic extract of whole plant Hibiscus tiliaceus on ethanol induced gastric ulcers in rats to determine its effect on gastric secretion, development of gastric ulcers in rats and also involves in-vitro antioxidant activity.

Collection of plant material:-
The whole plant of Hibiscus tiliaceus was collected from the local market in kukatpally, telangana, India which was identified and authenticated by Prof. Suresh babu, Dept. of botany, Govt. Degree College, Kukatpally.

Preparation of extract: (Dhandapani R and Sabna B, 2008):
The whole plant species were collected and then dried under shade for a period of three weeks. The dried plant material was milled to a fine powder using commercial laboratory blender and were stored in air tight containers until extraction.
Maceration was a simple widely used procedure involves leaving the pulverized plant to soak in a suitable solvent in a closed container. Simple maceration was performed at room temperature by mixing the ground drug with the solvent (drug solvent ratio 1:5 or 1:10), methanol was used as a solvent.

Tests carried out for different phytochemical constituents:
Methanolic extract of Hibiscus tiliaceus (MEHT) was subjected to preliminary phytochemical investigations to identify various phytoconstituents present in whole plant according to the method.

Test for alkaloids:
A small portion of crude extract was dissolved in 5 ml of 1% hydrochloric acid, filtered and tested with Dragendorff's reagent and Mayer's reagent separately. Any precipitate or turbidity with the reagents suggested the presence of alkaloids.

Test for flavonoids:
A few drops of conc. hydrochloric acid and 1-2 magnesium turnings were added to 1 ml of methanolic extract. The presence of flavonoids was indicated by the development of pink or magenta-red colour.

Test for phenols (Ferric chloride test):
A fraction of the extracts was treated with aqueous 5% ferric chloride and observed for formation of deep blue or black color.
Test for amino acids and proteins (1 % ninhydrin solution in acetone): 2 ml of filtrate was treated with 2-5 drops of ninhydrin solution placed in a boiling water bath for 1-2 minutes and observed for the formation of purple color.

Test for carbohydrates (Molisch test):
To a fraction of extract α-naphthol and alcohol was added. It was mixed well and conc. sulphuric acid was added drop by drop by keeping the test tube in inclined position. Violet ring is formed at the junction of two layers which shows the presence of carbohydrates.

Test for saponins (Foam test):
To 2 ml of extract was added 6 ml of water in a test tube. The mixture was shaken vigorously and observed for the formation of persistent foam that confirmed the presence of saponins.

Test for sterols (Liebermann-Burchard test):
2 ml of extract was treated with drops of chloroform, acetic anhydride and conc. H2SO4 and observed for the formation of dark pink or red colour.

Test for tannins (Braymer's test):
2 ml of extract was treated with 10% alcoholic ferric chloride solution and observed for formation of blue or greenish colour solution.

In vitro anti oxidant assay:
Methanolic extract of Hibiscus tiliaceus (MEHT) was tested for in-vitro antioxidant activity using standard methods. Amongst them, two methods were chosen for the present study. The absorbance was measured spectrophotometrically against corresponding blank solution. The percentage inhibition was calculated by the following formula. Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H2O2 can probably react with Fe 2+ , and possibly Cu 2+ ions to form hydroxyl radical and this may be the origin of many of its toxic effects. It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate.

Method:-
A solution of hydrogen peroxide (2 mmol/l) was prepared in phosphate buffer (P H 7.4) Test compounds (10-50 μg/mL) were added to hydrogen peroxide solution (0.6 ml). Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide and compared with ascorbic acid, the reference compound.

Reducing Power Assay (Cabral B, 2016):
This method was based on the principle of increase in the absorbance of the reaction mixtures. Increase in the absorbance indicates an increase in the antioxidant activity. In this method, substances, which have reduction potential, react with potassium ferricyanide (Fe 3+ ) to form potassium ferrocyanide (Fe 2+ ), which then reacts with ferric chloride to form ferric ferrous complex that has an absorption maximum at 700 nm. Potassium ferriccyanide + Ferric Chloride  Potassium ferrocyanide + Ferrous Chloride

Method:-
To 1 ml of test and standard compounds added 2.5 ml of potassium ferricyanide (1 % w/v), 2.5 ml of phosphate buffer (pH 6.6) and incubated at 50 0 C for 30 min. To 2.5 ml of above supernatant liquid, added 2.5 ml of distilled water and 0.5 ml of FeCl3 solution (0.1% w/v).The absorbance of ferric ferrous complex was measured using phosphate buffer pH 6.6 as control at 700 nm using UV-Visible spectrophotometer and estimated the increase in absorbance.

Acute Toxicity Studies of METS (OECD):
Acute toxicity studies were performed according to the OECD 425 guidelines.
Female Wister rats weighing 100-150g were selected and divided into four groups containing three animals in a group. Depending on the mortality or morbidity of animals a few steps may be necessary to judge the toxicity of the test substance. Minimal usage of animals while it allows for acceptable data is the main advantage of this method. were given orally 30 minutes before administration of ethanol (90% v/v, 1 ml/200g) and were sacrificed after 15 minutes. The stomach was dissected out, gastric juice was collected, and its volume was measured. The free and total acidity was also determined. The stomach was opened along the greater curvature, rinsed under a stream of water and pinned flat on a corkboard. Erosions formed on the glandular portion of stomach were counted and each was given a severity rating on 1-3 scale, based on diameter of the ulcer. The overall total diameter of ulcers in one stomach divided by factor 10 was designated as ulcer index (UI).

Evaluation of parameters: Collection of gastricjuice:
The stomach was excised carefully opened along the greater curvature and the gastric contents were removed. The gastric contents were collected in plain tubes and centrifuged at 3000 rpm for 5mins; the volume of the supernatant was expressed as ml/100 gm body weight. The mucosa was flushed with the saline and observed for gastric lesions using a dissecting microscope, ulcer score wasdetermined.

Ulcer scoring:
After sacrificing the rat, stomach was removed and opened along the greater curvature, and washed slowly under running tap water. Then keep it on the glass slide and observed under 10X magnification for ulcer. Score the ulcer as below.

Free acidity and Totalacidity:
Onemlofgastricjuicewaspipettein to a 100 mlconicalflaskandtitratedwith0.01NNaOH using topfers reagent as an indicator. It is Dimethyl-amino-azo-benzene with phenolphthalein, used for the detection and estimation of hydrochloric acid and total acidity in gastric fluids. The end point was to orange color. Noted the volume of NaOH which responded, and continued with titration further till the solution regained its pink colour. Noted the volume of NaOH which corresponded to the free acidity. Acidity (mEq/L/100g) was be expressedas -Acidity = Volume of NAOH × Normality of NAOH/0.1×100

Statistical Analysis:
The statistical analysis is carried out using analysis of variance (ANOVA), followed by dunett's test. P values < 0.05 considered as significant.

Histopathological Studies:
One animal of each group was euthanized at the end of experiment. Gastric tissue samples from each group were fixed in 10% formalin for 24 h. The specimens were then embedded in paraffin, sectioned and stained with hematoxylin and eosin, before being evaluated by light microscopy. Open excised stomach was also observed for histopathological study.

Percentage yield of plant extract:
The yield obtained from the methanolic extract of Hibiscus tiliaceus L. was about 50 g which was obtained from 1000 g.% Yield =Actual yield/Theoretical yield X100% Yield =50/1000 X 100The percentage yield of extract=5%

Preliminary Phytochemical screening:
The results of preliminary phytochemical screening of the methanolic extract of Hibiscus Tiliaceus L. were given in   In the determination of anti-oxidant property of the methanolic extract of Hibiscus tiliaceus using H2O2 assay, the IC50 value of the test extract showed 45±0.16 μg/ml, where as the standard Ascorbic acid (positive control) was found to be 42±0.18 μg/ml, which was commonly used as a standard for anti-oxidant activity. Similarly, in the reducing power assay method, the reducing power of extract was determined and compared to standard used; absorbance with increased concentration significantly increased the reducing power. The test extract exhibited significant (p<0.0001) reducing ability. MEHT reduced the generation of free radicals, showed the absorbance of 1.88 indicated good (p<0.0001) reducing ability power which was comparable to Ascorbic acid.
Biologically active antioxidants may help protect the gastric mucosa against cell damage caused by oxidative stress, as well as enhance the defense systems against degenerative diseases. Studies have associated the administration of antioxidants (synthetic or natural) with protection of the gastric mucosa against necrotic agents, including ethanol and non steroidal anti-inflammatory drugs. The in vitro antioxidant study showed that the methanolic extract of Hibiscus tiliaceus contains classes of bioactive compounds involved in antiulcer processes, and this observation may contribute to the justification of its use in traditional medicine.  Values were showed as the mean ± SEM. *p < 0.05 compared with the control, ethanol and ethanol + standard rats 660 The etiology of peptic ulcer unknown in most of the cases, yet it is generally accepted that it results from imbalance between aggressive factors and the maintenance of mucosal integrity through the endogenous defense mechanisms.
To regain the balance, phytomedicines are used (in experimental animals) to inhibit the gastric acid secretion or to boost the mucosal defense mechanisms by increasing mucous production,stabilizingthesurfaceepithelialcellsorenhancingprostaglandinsynthesis (Mahattanadul S et al., 2009). The present results demonstrated that the methanolic extract of Hibiscus tiliaceus protected the rat gastric mucosa against hemorrhagic lesion produced by aspirin. This was a convenient way of screening plant extracts for anti-ulcer effect and cytoprotection in macroscopically visiblelesions.
Hibiscus tiliaceus L.had significantly protected the gastric mucosa against ethanol challenge as shown by reduced values lesionas compared to control group suggesting its potent cytoprotective effect (Matsumoto T et al., 2002). It showed a significant inhibition of gastric ulcer and also decreased mucosal parameters like acid concentration, gastric volume and increased the pHvalues. Hence, it was suggested that methanolic extract of Hibiscus tiliaceus L. can suppress the gastric damaged induced by aggressive factors. The phytochemical screening of the extract of Hibiscus tiliaceus L. revealed the presence of flavonoids, tannins, glycosides, triterpenes and saponins (Bi W P, 2014). Any of these metabolites may be responsible for the antiulcer activity of hibiscus tiliaceus L. Further, additionally the presence of terpenoids might be responsible for more effect on ulcer inhibition.

Conclusion:-
The test extract showed protection against mucosa against gastric lesions induced by ethanol. This antiulcer effect of MEHT may be due to both reductions in gastric acid secretion and gastric cytoprotection. However, further investigation is to be carried out to elucidate the exact mechanism of anti-ulcer activity through the use of additional experimental models.

Conflicts of interest:
The authors have no conflicts of interest with anyone.