INTRODUCTION:

The liver is the largest solid organ and the largest gland in the human body. It carries out over 500 essential tasks. The liver plays essential roles in the metabolism of fat, protein and carbohydrates. Also, Metabolic detoxification, the liver alters exogenous and endogenous chemicals (e.g. drugs), foreign molecules, and hormones to make them less toxic or less biologically active. Different types of homeostatic mechanisms are affected if liver function is impaired, with potentially serious consequences. About 20,000 deaths occur every year due to liver diseases.

Hepatocellular carcinoma is one of the ten most common tumors in the world. Although viruses are the main cause of liver diseases, also excessive drug therapy, environmental pollution and alcoholic intoxication¹ are not uncommon. CCl4induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl₄ metabolites to cell components and/or peroxidative damage as the cause of injury. Covalent binding of 14C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl₄. Low oxygen partial pressure increased the reductive metabolism of CCl₄ and thus covalent binding. CCl₄ was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. CCl₄ is commonly used for free radical induced liver injury. Liver is not the only target organ of CCl₄ but it also affects several organs of the body such as lungs, hearts, testes, kidneys and brain. It was reported from the investigation carried out on animal models of acute CCl₄ induced liver damage, It is now generally accepted that CCl₄ toxicity results from bioactivation of CCl₄ into trichloromethyl free radical by cytochrome P450 system in liver microsomes² and consequently causes lipid peroxidation of membranes that leads to liver.

CCl₄ is the most recognized chemical substance used in developing models of liver and kidney damage. Therefore, CCl₄-induced liver damage is one of the best ways of inducing damage by xenobiotics and also one of the common methods of screening hepatoprotective or liver treatment drugs. The metabolism of CCl₄ begins with the formation of trichloromethyl and proxy chloromethyl free radicals via the activity of oxygenase system of cytochrome P450 in endoplasmic reticulum. The trichloromethyl radical reacts with various important biological substances such as fatty acids, proteins, lipids, nucleic acids, and amino acids

The lipid peroxidation potency of CCl₄ revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe3+ and cumolhydroperoxide, respectively. CCl₄, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins.^[2] The anti-oxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. The radical scavenger piperonylbutoxide prevented CCl₄-induced lipid peroxidation as well as covalent binding of CCl₄ metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl₃ radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl₃-OO., initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure.

The study plant, Hibiscus surattensis (H. surattensis) is a medicinal plant belongs to Malvaceae family. Hibiscus surattensis is an indigenous scrambling annual commonly known as Wild Sour distributed throughout Africa and Asia. surattensis, the species name, is derived from the Indian trade port, Surat. Leaves and stems are densely pubescent, with silvery hairs visible to the naked eye. Leaf margins are serrated and often appear reddish, suggesting the presence of anthocyanin compounds. Flowers are bisexual, pentamerous. The ability of Hibiscus surattensis to self-pollinate is considered as an evolutionarily advanced trait. Hibiscus surattensis is growing abundantly in Pondicherry and Tamilnadu along the Coromandel Coast³ of India.The reported ethnobotanical properties of H. surattensis span a range of illnesses and have been documented throughout various parts of Africa and India.

The flowers of Hibiscus surattensis were consumed for treatment of hypertension in Nigeria, Stems and leaves were used for the treatment of ureteritis and venereal diseases. In West Africa; Leaves were used for treatment of malarial disease. Crushed leaves used for wound healing, abscess and gonorrhea in Tanzania. Whole plant used for stomach ache in Nigeria. The crude leaf extracts of H. surattensis possesses significant antiinflammatory (mild to moderate), anti-oxidant (moderate), analgesic and anti-diarrheal activities. Preliminary phytochemical tests on Hibiscus surattensis revealed that, the presence of sterols, carbohydrates, phenolic compounds and flavones in dry leaves.

MATERIALS AND METHODS:

Materials:

All the materials used for this experiment were of analytical grade. The chemicals used were manufactured by Sigma Chemical Co. Diagnostic kits for the estimations were manufactured by Ranbaxy Diagnostics Ltd., New Delhi, India. Standard or gastric cannula was used for oral drug administration. Chang liver cell line was purchased from NCCS Pune was maintained in Dulbecco’s modified eagles media (HIMEDIA).

Plant material:

The aerial parts of Hibiscus surattensis were collected from Tirunelveli District, Tamilnadu, India during the month of March 2016. The plant was identified and authenticated by Dr. Chelladurai, Research Officer- Botany, Central Council for Research in Ayurveda and Siddha, Government of India (Ref No: DCP/CH/AN 02)

Collection and garbling:

Fresh leaves were collected from the hill tracts of Tamilnadu in the month of March- April 2016 which was the period when the plant grows the most. The extraneous, undesired substances from the plant material were removed at two stages. At first the rotten leaves, stems etc were removed by hands immediately after collection. The soil was removed by sieving through a net aided by a flow of air from an electric fan before the plant materials⁴ were dried.

Drying and grinding:

The leaves were then subjected for shade dry at temperature not exceeding 50°C. Next, these leaves were grinded into coarse powder with the help of a grinder. The powder were stored in airtight containers and kept in a cool, dark and dry place until extraction was commenced.

Extraction:

The aerial parts of Hibiscus surattensis were collected, shade dried for 3 weeks, powdered mechanically and sieved through No. 20 mesh sieve. About 800g of the powdered aerial part was first defatted with petroleum ether (PEF, 60º - 80ºC) and then consecutively extracted with ethyl acetate (EAEF) and methanol (MEF) by soxhlet extraction (order of increasing polarity). The crude extracts⁵ were concentrated by using rotary vacuum evaporator and dried at room temperature. The extract obtained with each solvent was weighed and the percentage yield was calculated in terms of dried weight of the plant material.

Acute toxicity studies:

The acute toxicity for MEHS was determined⁶ on male Wistar rats, maintained under standard conditions. The animals were fasted overnight prior to the experiment. Fixed dose method of OECD guideline No.423 given by CPCSEA was adopted for toxicity studies, selection and acclimitization of animals. Albino rats of Wistar strains weighing between 180-220gm were produced from animal experimental laboratory, and used throughout the study. They were housed in micro nylon boxes in a control environment at a temperature 25±2⁰C and 12 hrs dark/light cycle with standard laboratory diet and water. The study was conducted after obtaining institutional animal ethical committee clearance. As per the standard practice, the rat were separated based on their gender and quarantined for 15 days before the commencement of the experiment. They were fed on healthy diet and maintained in hygiene environment in our animal house.

Hepatoprotective activity in ccl₄ induced hepatotoxicity:

Treatment Protocol:

The acclimatized animals were divided into 5 groups of each 6 animals of male sex, designated as

Group I:

Serve as normal control and administer olive oil in 10% acacia suspension (1ml/kg, p.o) daily for 5 days with olive oil (1ml/kg, i.p) on days 2 and 3.

Group II:

Serve as CCl₄ control and administer CCl₄, olive oil suspension in 10% acacia (1ml/kg, p.o) daily for 5 days with CCl₄: olive oil (1:1, 2ml/kg, i.p) on days 2 and 3.

Group III:

Treat with reference drug Silymarin (50mg/kg, i.p) along with carbon tetrachloride, olive oil suspension in 10% acacia (1ml/kg, p.o) daily for 5 days with CCl₄: olive oil (1:1, 2ml/kg, i.p) on days 2 and 3, 30 mins. after administration of reference drug.

Group IV:

Treated with methanolic extract of the aerial parts of the plant, Hibiscus surattensis (200mg/kg) along with carbon tetrachloride, olive oil suspension in 10% acacia (1ml/kg, p.o) daily for 5 days with CCl₄: olive oil (1:1, 2 ml/kg, i.p) on days 2 and 3, 30 mins. after administration of test dose.

Group V:

Treated with methanolic extract of the aerial parts of the plant, Hibiscus surattensis (400mg/kg) along with carbon tetrachloride, olive oil suspension in 10% acacia (1ml/kg, p.o) daily for 5 days with CCl₄: olive oil (1:1, 2 ml/kg, i.p) on days 2 and 3, 30 mins. after administration of test dose

In vitro hepatoprotective effect determination by MTT assay^7,8

The cell line was cultured in 25cm² tissue culture flask with DMEM supplemented with 10% FBS, L-glutamine, sodium bicarbonate and antibiotic solution containing: Penicillin (100U/ml), Streptomycin (100µg/ml), and Amphoteracin B (2.5µg/ml). Cultured cell lines were kept at 37ºC in a humidified 5% CO₂ incubator (Galaxy^® 170 Eppendorf, Germany).

The viability of cells were evaluated by direct observation of cells by Inverted phase contrast microscope and followed by MTT assay method.

Cells seeding in 96 well plate:

Two days old confluent monolayer of cells were trypsinized and the cells were suspended in 10% growth medium, 100µl cell suspension (5x10⁴ cells/well) was seeded in 96 well tissue culture plate and incubated at 37ºC in a humidified 5% CO₂ incubator.

Preparation of plant extracts and compound stock:

1 mg of each plant extract or compound was added to 1ml of DMEM and dissolved completely by cyclomixer. After that the extract solution was filtered through 0.22 µm Millipore syringe filter to ensure the sterility. CCl₄ (0.1%) was added to induce toxicity.

Cytotoxicity Evaluation⁹:

After attaining sufficient growth, CCl₄ (0.1%) was added to induce toxicity and incubated for one hour, prepared extracts in 5% DMEM were five times serially diluted by two fold dilution (100µg, 80µg, 40µg, 20µg, and 10µg in 100µl of 5% DMEM) and each concentration of 100µl were added in triplicates to the respective wells and incubated at 37ºC in a humidified 5% CO₂ incubator.

Cytotoxicity Assay by Direct Microscopic observation:

Entire plate^(10-12) was observed at an interval of each 24 hours; up to 72 hours in an inverted phase contrast tissue culture microscope (Labomed TCM-400 with MICAPS^TM HD camera) and microscopic observation were recorded as images. Any detectable changes in the morphology of the cells, such as rounding or shrinking of cells, granulation and vacuolization in the cytoplasm of the cells were considered as indicators of cytotoxicity.

Cytotoxicity Assay by MTT Method^(13,14):

Fifteen mg of MTT (Sigma, M-5655) was reconstituted in 30µl PBS until completely dissolved and sterilized by filter sterilization. After 24 hours of incubation period, the sample content in wells were removed and 3µl of reconstituted MTT solution was added to all test and cell control wells, the plate was gently shaken well, then incubated at 37ºC in a humidified 5% CO₂ incubator for 4 hours. After the incubation period, the supernatant was removed and 100µl of MTT Solubilization Solution (DMSO was added and the wells were mixed gently by pipetting up and down in order to solubilize the formazan crystals. The absorbance values were measured by using microplate reader at a wavelength of 570 nm

The percentage of growth inhibition was calculated using the formula:

RESULTS AND DISCUSSION:

Acute toxicity study:

The LD₅₀ of the extracts when administered orally to mice was found to be 2000 mg/kg according to OECD guidelines 423. During the acute toxicity study, the methanolic extract was administered orally and animals were observed for mortality, changes in the autonomic nervous system, central nervous system and behavioral responses. There was no mortality observed even at 2000mg/kg for the extract. All the animals were found to be normal and there were no gross behavioral changes till the end of the observation period. This observation revealed that the methanolic extract of the aerial parts was found to be very safe up to 2000mg/kg of body weight known as maximum tolerated dose (MTD) by acute toxicity model study as per OECD guidelines 423. Hence from this 1/10th and 1/5th of MTD was selected and the effective doses were fixed as 200 and 400mg/kg for the further pharmacological studies.

Biochemical analysis:

During the period of treatment the rats should be maintained under normal diet and water. After the experimental period the overnight fasted rats will be sacrificed. All treated group animals and one animal of control group will be humanely sacrificed on the 5^th day of the study using CPCSEA recommended euthanasia procedure. (Carbon dioxide inhalation method). Blood samples will be collected and SGOT (AST), SGPT (ALT) estimation will be carried out using commercial kits and it is shown in table no 1. The serum was separated by using refrigerated centrifuge and used for the assay of marker enzymes viz AST, ALT, ALP, TP, TB, LDH. The livers were dissected out immediately, washed with ice-cold saline and 10% homogenates in phosphate buffer solution were prepared. Histopathological assessment of liver damage will be studied.

Statistical analysis:

The Statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Newmann Keul’s multiple range tests. The values are represented as Mean±SEM (table no 1). Probability value of P < 0 .01) Serum Aspartate Transaminase (AST), Alanine Transaminase (ALT), Alkaline phosphatase (ALP), Total bilirubin (TB) and Lactate dehydrogenase and significant decrease in (P< 0.01). Total protein levels were observed in animals treated with CCl4 (Group II) as compared to normal control group(Group I).

Table No 1: Effect of methanolic extract of Hibiscus surattensis on biochemical estimation of SGOT, SGPT, TB, TP, LDH of CCl4 induced toxicity in rats

Group.

No.

TREATMENT

DOSE (mg/Kg)

AST

(IU/mL)

ALT

(IU/mL)

ALP

(IU/mL)

(gm/dl)

(mg/dl)

LDH

(U/L)

Normal control

Olive oil in 10% acacia 1ml/kg

81.70±

3.95

48.65±2.44

63.90±

2.12

6.30±

0.70

0.48±

0.15

212.40±

6.52

Negative control

CCl4: Olive oil

307.22±

8.35*a

324.30±

7.75*a

296.20±

6.30*a

3.40±

0.30*a

1.22±

0.30*a

355.30±

7.20*a

III

Standard control

silymarin 50mg/kg

156.36±

4.25*b

161.65±

4.40*b

195.42±

5.20*b

5.50±

0.60*b

0.60±

0.15*b

249.20±

6.75*b

Treatment control

MEHS 200mg/kg

239.60±

6.62*b

221.70±

5.20*b

266.30±

5.75*b

4.58±

0.42*b

0.90±

0.18*b

283.18±

7.02*b

Treatment control

MEHS 400mg/kg

215.55±

5.68*b

202.50±

5.05*b

249.30±

5.30*b

4.75±

0.48*b

0.82± 0.16*b

261.20±

6.80*b

Evaluation of in vitro hepatic effect by MTT assay:

Methanolic extract of Hibiscus surattensis has significant hepato protective effect on cultured cell line in concentration range between 10µg/ml by using MTT assay. The highest hepatoprotective activity of this extract against cultured cell was found in 100µg/ml concentration by MTT assay method.

Table No 2: In vitro hepato protective effect by MTT assay

HAJ protective study
Samples (µg/ml)	% of viability± SD
Control	100
10	32.90±0.94
20	44.11±0.52
40	59.95±0.70
80	73.26±0.91
100	81.32±0.87

Fig.1: MTT Assay

Histopathological observations:

Orally administered doses of 200 and 400mg/kg of aqueous extract of leaves of Hibiscus surattensis produced significant decrease in AST, ALP, ALT levels and bilirubin. The activity of the extract is found to be dose dependent. The hepatoprotective effect of the drug was further supported by the histopathological examinations of the liver sections which reveal that the normal liver shapes was disturbed by hepatotoxic intoxication [Fig 2]. In the liver sections of the rats treated with Hibiscus surattensis extract and intoxicated with Alcohol the normal cellular shape was retained as compared to silymarin, thereby confirming the protective effect of the extracts of Hibiscus surattensis [Fig 3-6].

Fig No 2: Liver section of normal control rats showing normal liver lobular architecture with well brought out central vein and prominent nucleus and nucleolus.

Fig No 3: Liver of the negative control showing histopathological alteration with normal hepatocytes

Fig no 4: Standard: liver section of rats showing microvesicular central vein with mild fatty change

Fig No 5: liver section of rat showing congested sinusoid which is treated with 200mg extract

Fig No 6: liver extract of rat exract treated with 400mg showing regenerative hepatocytes

CONCLUSION:

In the present study, Methanolic Extract of Hibiscus surattensis exhibit strong hepatoprotective activity, afforded protection CCl₄ induced liver damage. Hepatoprotective activity of Methanolic Extract of Hibiscus surattensis may be due to free radical scavenging activity due to presence of flavonoids, tannins and antioxidants. Additional studies are needed to better understand the mechanism of action of Methanolic Extract of Hibiscus surattensis that is responsible for hepatoprotective activity. The methanolic extract was found to be very safe up to 200mg/kg body weight by acute toxicity model study as per the OECD guidelines 423. The methanol extract showed significant hepatoprotective activities in a dose dependent manner. The hepatoprotective effect of the drug was further supported by the histopathological examinations of the liver sections which reveal that the normal liver shapes was disturbed by hepatotoxic intoxication The methanol extract at the dose of 400mg/kg exhibited significant hepatoprotective activity. This activity can be attributed to the phenolic and flavonoid content in the methanol extract.

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Received on 30.09.2019 Modified on 12.11.2019

Research J. Pharm. and Tech. 2020; 13(10):4635-4640.

DOI: 10.5958/0974-360X.2020.00816.1

Cytotoxicity Assay by MTT Method(13,14):

Fig No 3: Liver of the negative control showing histopathological alteration with normal hepatocytes

Cytotoxicity Assay by MTT Method^(13,14):