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Hepatoprotective and Antioxidant Activity of Ethanol Extracts of Dipterocarpus turbinatus (Dipterocarpaceae) from Tripura

Ipomoea reniformis Chaos is claimed in Indian traditional medical practice to be useful in the treatment of epilepsy and neurological disorders. In the present study, pretreatment effect of methanolic extract of Ipomoea reniformis on epilepsy and psychosis was evaluated in rodents using standard procedures. Besides evaluating epileptic and behavioral parameters, neurotransmitters such as Gamma-Amino Butyric Acid (GABA) in epilepsy and in psychosis dopamine, noradrenaline and serotonin contents in the rodent brain were estimated. The extract pretreatment reduced maximal electro shock; Isoniazid (INH) and Pentylenetetrazole (PTZ) induced seizures and also significantly inhibited the attenuation of brain GABA levels by INH and PTZ in mice. These results suggested that the observed beneficial effect in epilepsy may be by enhancing the GABAergic system. The test drug also inhibited the apomorphine induced climbing and stereotyped behavior and showed significantly reduced levels of brain dopamine, noradrenaline and serotonin which may be due to blocking of central dopaminergic, noradrenergic and serotonergic pathways or by enhancing the GABAergic system. The results obtained in present study suggest that the title plant possesses antiepileptic and antipsychotic activities in rodents.


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].
The plant contains various neuroprotective chemical constituents such as caffeic, p-coumaric, ferulic and sinapic acid esters. Petroleum ether extract contains fats and fixed oil while aqueous extract contains amino acids, tannins (condensed and pseudo tannins) and starch [5]. IR has been reported to possess various pharmacological actions, mainly antidiabetic [6], antiinflammatory [7], nephroprotective [8], antibacterial [9], antioxidant and antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological

Introduction
Liver is vital organ in human body that perform detoxification of exogenous xenobiotics, infection and regulate various biochemical enzymes for metabolisms thus help to fight against disease 1 . Liver disease is a worldwide problem which is caused by various agents like alcohol, drugs and chemicals 2 . In spite of phenomenal growth of modern medicine there are no synthetic drugs available for the treatment of hepatic disorder. However herbal formulation claimed to have beneficial effect on liver disorder 3 . Preliminary phyto chemical analysis of the extracts shows presence of phenol and flavonoids 4 which is known to be hepatoprotective and anti oxidant activity. Free radicals like ROS and NOS due to the special characters can initiate the lipid peroxidation, break nucleic acid strands and oxidize virtually all molecules in the biological membrane resulting in the injury of liver. Thus anti oxidant effects drug can prevent liver's oxidative damage 5 .
Dipterocarpus turbinatus is a large woody plant height of 100-120 ft. and a girth of 8-15 ft. It is found in the tropical forests of Tripura, Assam and Andaman. D. turbinatus is used traditionally as anti diarrhoeal, astringent, wound healer, antiulcer, burns, tuberculoid leprosy [6][7][8] . In Ayurveda, Asanadi gana was one of the ingredients out of 23 plants which were used in the diabetic treatment 9 . Tribal people of Tripura use D. turbinatus plant and plant derived product for various purposes. This drug possess anti oxidant activity 10 , aqueous extracts of the plant used for reducing gum and tooth ache 11 . It shows cyto-toxic activity 12 and other species of the plants also shows anti cancer properities 13 . In this work we have done the hepatoprotective and anti oxidant activity of ethnolic extract of D. turbinatus bark by in vivo and in vitro method respectively.
Barks were procured from wild source from the forest of Tripura. The plant material was authentified by Prof P. Jayaraman, M.Sc., Ph.D., Director PARC., Chennai. With reference number (PARC/2012/1277). The specimen was preserved at the herbarium of the institute.

Chemical and Reagent
Paracetamol and Silymarin were procured from the Micro Lab Ltd., India. Standard biochemical kits {Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Serum Alkaline Phosphate (ALP), Bilirubin, Protein kits} were procured form Agappe diagnostics Ltd, Kerala, India. DPPH was procured from Hi Media Mumbai. All other reagents for the study were procured from SD fine lab, Mumbai, India. Remi research centrifuge was used for centrifugation purpose. Mispa excel semi autoanalyzer made of Agappe diagnostic Ltd., Kerala, India and double beam UV-Visible spectrophotometer of Lab India, were used for various analyses.
Coarsely powdered dried bark of 500 g was extracted up to exhaustion with petroleum ether, chloroform, and ethanol (70%) using soxhlet apparatus. Ethanol extracts (18% w/w) thus obtained was dried under controlled temperature and was selected for the study.

Experimental Animals
The animals (Wistar albino rats for hepatoprotective activity and albino Mice for acute toxicity) were kept at Standard environmental conditions for 7 days before the experiments. All the animals were maintained under strict hygienic conditions with normal feed and water. The entire animals were utilized for the studies and used according to the protocol approved by (No. IAEC/CESCOP/2016-07) Institutional Animal Ethics Committee. For each hepatoprotective activity 5 groups were made for both models (paracetamol and carbon tetrachloride) containing 6 animals. Group I serves as vehicle control, group II contains hepatotoxin, group III contains standard (Silymarin), group IV and V serve as drugs treated (200mg/kg & 400mg/kg of EEDT) group.
Acute toxicity for EEDT was determined by the method adapted by CPCSEA, Government of India, -acute toxic class method (OCED Guideline no. 423, Annexure-2d) in albino mice. The mortality was observed after oral administration of 2000mg/kg b.w of test sample 14 . Common side effects like mild diarrhea, weight loss and depression of treated groups of animals were recorded within the one week of observation 15 .
Group I (vehicle control) was given 2% acacia suspension in normal saline, group II received paracetamol (3gm/kg) 16 as a single dose at 0hr followed by vehicle for 1hr, 24hr, 48hr. Group III received paracetamol (3gm/kg/p.o) as a single dose at 0hr followed by Silymarin (50mg/kg) for 1hr, 24hr, 48hr. Group IV and V received paracetamol 3gm/kg at 0hr followed by drug extracts 200mg/kg and 400mg/kg at 1hr, 24hr, 48hr. At 72 nd hr, blood was collected from animals and biochemical parameters were evaluated related to liver disorder to check the therapeutic effects of the drug extracts 17 . All the dosage has administrated by oral route.

Carbon Tetra Chloride Induced Hepatotoxicity
Group I serves as (-ve) control and were received 1 ml 2% acacia solution for 5 days and olive oil 2ml/kg on 2 nd and 3 rd day. Group  Assessments of liver function were done by collecting blood from retro orbital puncture. Blood was centrifuged at 4000 rpm for 15min and serum was collected. Various biochemical parameters like SGOT, SGPT, serum ALP, total cholesterol, total bilirubin and total protein were evaluated using biochemical kits 20 .
The animals were sacrificed and liver were collected and washed with normal saline. Isolated liver were stored in 40% formalin solution and histopathology was done to evaluate the details change hepatic cell structure in each group microscopically for both the models.

DPPH (2, 2-diphenyl-1-picrylhydrazyl)
Radical Scavenging Activity DPPH scavenging activity was measured according to the method of Sanjay Rai et al. 21 with some modifications. The free radical-scavenging activities of all samples were estimated in terms of hydrogen donating or radical scavenging ability. A Solution of 0.1 mM DPPH was prepared in ethanol. 1 ml of this solution was added to 3 ml of all the extracts in water at different concentrations (1-10μg/ml). Thirty minutes later the absorbance was measured at 517nm. Ascorbic acid is used as standard antioxidant. The results expressed as IC 50 or inhibitory concentration 50 value.

Reducing Ability
Reducing power of EEDT was determined based on the ability of anti oxidant to form complex with potassium ferricyanide, TCA and FeCl 3 . Add different concentration of sample(s) in 1 ml of distilled water in order to get 1μg, 2μg, 4μg, 8μg and 10μg/ml concentration. A test tube also makes as control (without any test sample). Add phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide (2.5ml, 1%) to the sample. The mixture was incubated at 50μC for 20 min. Portions (2.5 ml) of trichloroacetic acid (10%) added to the mixture and centrifuged at 3000 rpm for 10 minutes. The upper layer of the mixture solution (2.5 ml) was mixed with distilled water (2.5 ml) and 0.1% ferric chloride (0.5 ml). The absorbance (OD) was measured at 700 nm 22 by using Ascorbic as standard. The % reducing power was calculated by using by conventional formula.

Superoxide Anion Scavenging Activity
The Nitric oxide scavenging activity of extracts was determined by using Griess reagent (1% sulphonilamide, 2% phosphoric acid and 1% naphthyl ethylene diamine di hydrochloride). Reaction mixture containing 3 ml of sodium nitroprusside (10 M.mol) in phosphate buffer and test extracts in different concentrations (1μg, 2μg, 4μg, 8μg and 10μg/ml) were incubated at 25°C for 150 minutes. Control was prepared by omitting the sample. After incubation, 0.5 ml of Griess reagent was added and the absorbance was measured at 546nm using UVvisible spectrophotometer 24 . Percentage inhibition was calculated and the activity was expressed as an inhibition concentration 50 (IC 50 ).

Statistical Analysis
Data were expressed as Mean values ± SEM. The differences between the groups were analyzed by one way ANOVA and compare with toxic group. A value *p< 0.05 was considered as statistically significant.

Effects of Ethanol Extracts of D. turbinatus on Various Biochemical Parameters Associated with Liver Toxicity
Animal treated with 200mg/kg and 400mg/kg P.O of ethanol extracts from D. turbinatus barks exhibit a significant (*p<0.05) reduction in various biochemical parameters. In paracetamol induced and carbon tetra chloride induced groups, there was an increase in the level of SGOT, SGPT, ALP, serum bilirubin and decrease in the level of total proteins. Treatment with the extracts 200mg/kg and 400mg/kg P.O take down to normal in the level of SGOT, SGPT, ALP, Cholesterol and serum bilirubin and increase the level of total proteins. The results were explained in Table 1 and Table 2. D. turbinatus bark extracts showed the degree of protection with the higher dose. Histology of liver was done for all groups to find out change in liver architecture; like central vein, cytoplasm, nature of polygonal hepatocytes with their round nucleus and cytoplasm. Blood cell infiltration in hepatic vein, sinusoidal area and aggregation of Kuffer cells surrounding the central veins was also observed. Figure 1(a) and 2(a) shows the histopathology of the liver of untreated animals with normal hepatic vein, with less sinusoidal space polygonal hepatocytes without any ballooning. Figure 1(b) intoxicated with paracetamol shows damage in hepatic cell enlargement sinusoidal space and with infiltration of blood cell in hepatic vein, where as Figure 2(b) intoxicated with CCl 4 shows so much damage in the cellular system in liver cell. In both Figure 1(c) and 1(d) standard drug treated animal able to normalize the hepatic cell with less sinusoidal space, polygonal shape of kuffer cell, and hepatic vein with less blood cell in filtration. Figure 1(d) and 1(e) shows test drug treated with 200mg/kg and 400mg/kg, in paracetamol induced hepatotoxic animals liver, where we can see improvement of hepatocyte, sinusoidal space and hepatic vein as compared to the toxic (Paracetamol treated group) group. Figure 2

Anti Oxidant Activity
D. turbinatus bark extracts posses a significant anti oxidant activity. Table 3 illustrates the results of anti oxidant activity by various in vitro anti oxidant methods. Preliminary phyto chemical analysis of the extracts shows presence of phenol and flavonoids 4 which is known to be hepatoprotective and anti oxidant activity. Anti oxidant capacity of the plant extracts may be helpful in the regeneration of hepatic cells from their oxidative damage and protect the liver cell from toxicants. Paracetamol is widely used as anti pyretic and analgesic. The metabolite of paracetamol N-acetyl-pbenzoquinonimine breaks sulphydryl group of proteins and causes liver damage 25,26 . CCl 4 has been widely used as an indicator of the liver protective activity of drug 27 . CCl 4 gets metabolised in liver and release CCl 3 radicals, a free radical that binds to lipoproteins and leads to peroxidation of lipids of endoplasmic reticulum 28 . CCl 4 causes liver injury that is characterized by centrilobular necrosis that is followed by hepatic fibrosis 29 . In our study we performed paracetamol and carbon tetra chloride induced hepatotoxicity. In paracetamol induced hepatotoxicity, toxicant (Paracetamol) was given at 0 hr and followed by ethanol extract. In CCl 4 induced hepatotoxicity toxicant CCl 4 was administrated (at 2 nd and 3 rd day) along with ethanol extract from the bark of D. turbintaus in dose dependent manner for 0 to 5 days, once/day. The ethanolic extract from the barks of D. turbinatus able to decrease the elevated biochemical parameters like SGOT, SGPT, ALP, cholesterol, bilirubin level in both carbon tetra chloride and paracetamol induced hepatotoxicity in rats and also significantly increased the total protein in the blood of treated animals with test drugs and silymarin. These findings suggest that ethanolic extract of D. turbinatus is effective in bringing the functional improvement of liver. Protection in hepatocyte by EEDT at 200mg/kg and 400mg/kg also was confirmed by histopathological studies of the liver for various groups animal. Anti oxidant activity shows that the drug extract has beneficial anti oxidant property with significant IC 50 values with the reference standard of ascorbic acid. Treatment with EEDT brought down the elevated levels of various biochemical parameters related to hepatic function and also restores the damage of liver cell. An overall result is concluded in these studies that the drug can be a therapeutic agent in liver damage in future. Hepatoprotective effect of EEDT may be due to its anti oxidant activity and ability to scavenging the free radicals which may be due to the presence of phenol and flavonoids in the extracts. However further studies are need to confirm involvement of cytochrome p450 enzyme inhibition to check the proper mechanism of liver protection from toxicity.  Values are expressed as the mean SEM with p<0.01 compared to positive control group. Symbol represent *p<0.05., **p<.0.01., ***p< 0.001.