INTRODUCTION:

Liver is multifunctional largest organ, serves vital function in human body. The term hepatotoxicity use for liver damage cause by different types of drugs or other xenobiotic compound. Hepatocyes which consume major part of liver cells involve in protein synthesis and storage, carbohydrate transformation, cholesterol bile salt and phospholipid synthesis and detoxicification, excretion of xenobiotics(including drugs).These cells detoxify drugs using different enzyme like enzymes cytochromes P-450, glutathione S-acyltransferases mixed. oxidases, by performing biochemical transformations reactions like oxidation-reduction, hydroxylation, sulfonation and dealkylation. Because of the role of clearance blood and excretion of drugs and exogenous compounds. liver abundantly exposed to the adverse effects of these compounds and is hence prone to hepatotoxicity.

Many drugs have been withdrawal form market just because of DILI (drug induced liver injury) and most of the time severe case of liver dysfunction required liver transplant or some time death. Mechanism of hepatotoxicity can be investigated through mitochondrial dysfunction and DNA damage. This imperial function caused by drug itself or it’s cytochrome P450 mediated metabolites. Resent reports on hepatotoxicity suggest that oxidative stress, microvacular steatosis, imbalance energy storage are major out come of mitochondrial dysfunction.

There is no effective drug available in modern medication that can stimulate liver function or regeneration of liver cell in spite of their adverse effect. Therefore it is necessary to find out some alternative medication for liver damage (1).

In Tradition System of Medicine Chenopodium album Linn. is used as an antianthelmintic, antidiarrhoeal, antiphlogistic, antirheumatic, contraceptive, odontalgic, laxative, cardiotonic, antiscorbutic, blood purifier, spleen enlargement, biliousness, intestinal ulcers, digestive, carminative, aphrodisiac, dyspepsia, flatulence, strangury, seminal weakness, pharyngopathy, splenopathy, hemorrhoids, ophthalamopathy, cardiac disorder and general debility. The pharmacological activity reported so far from this plant are antipruritic and antinociceptive activity, anthelmintic activity and as vaginal contraceptive (2).

The principle aim of present investigation is to identify the hepatoprotective activity of Chenopodium album Linn. in carbon tetrachloride induced hepatotoxicity rats.

MATERIALS AND METHODS:

Extraction of Chenopodium album Linn.:

The leaves plant of Chenopodium album Linn. was dried under shade and then powdered with a mechanical grinder to obtain a coarse powder (500 gm) the fine powder of whole plant was packed in high quality filter paper, which was then subjected to successive extraction in a soxhlet apparatus using 50% ethanol for about 72 hour, solvent was recovered. Extractive yield of Chenopodium album Linn. was 17 %. . After vacuo evaporation the crude extract was dissolved in distilled water freshly as required.

Animals:

Albino rats (Wistar strain) weighing 125 - 150 gm of either sex were used for the present study. The animals were housed in polypropylene cages at control temperature (26 ± 2° C) relative humidity (60 ± 5%) and light. Rats were fed with standard laboratory diet and drinking water was given through drinking bottle throughout the experiment. The animals were maintained as per CPCSEA regulation and cleared by IAEC at Bhupal Nobles’ College of Pharmacy, Udaipur (Rajasthan), India.

Drug Formulation:

The extract of plant fully dissolves in distilled water. The solution of the whole plant extract (300 mg/ml) was freshly prepared in distilled water.

Experimental Induction of Hepatotoxicity:

Liver toxicity was induced in rats by administrating carbon tetrachloride (CCl₄) subcutaneously (sc) in the lower abdomen, in a suspension of liquid paraffin (LP; 1: 2 v/v) at the dose of 1 ml/kg body weight (BW) on alternate days for one week (3).

Experimental Design:

After acclimatization the rats were divided in to 6 groups of 6 rats each in equal number of males and females.

Group 1: Rats served as control and received subcutaneous administration of liquid Paraffin (LP) only 1 ml/ kg on alternate day for one week and vehicle for three weeks orally.

Group 2: Rats were given carbon tetrachloride (CCl₄) subcutaneously (sc) in the lower abdomen, in a suspension of liquid paraffin (1: 2 v/v) at the dose of 1 ml/kg BW on alternate days for a week and vehicle for three weeks orally.

Group 3:Rats were given carbon tetrachloride (CCl₄) sc, in a suspension of liquid paraffin (1: 2 v/v) at the dose of 1 ml/kg BW on alternate days for a week and after one hour Silymarin (50 mg/kg BW/ d)) for three weeks orally.

Group 4-6: Rats were given carbon tetrachloride (CCl₄) sc, in a suspension of liquid paraffin (1: 2 v/v) at the dose of 1 ml/kg BW on alternate days for a week and after one hour Chenopodium album Linn. (200 mg/kg, 400 mg/kg, 600 mg/kg BW/d) respectively for three weeks orally.

Different doses of above mentioned drug, LP and CCl₄ were administered to rats daily “between” 10.00 to 11.00 am.

Biochemical Studies:

The blood was obtained from all animals by puncturing retro-orbital plexus. The blood samples were allowed to clot for 45 min at room temperature. Serum was separated by centrifugation at 2500 rpm for 15 min and utilized for the estimation of various biochemical parameters namely SGOT (4), SGPT (4), SALP (5), serum bilirubin (6) total cholesterol (7) and total protein (8). After collection of blood samples the rats in different groups were sacrificed and their livers were excised immediately and washed in ice cold normal saline, followed by 0.15 M Tris-Hcl (pH 7.4) blotted dry and weighed. A 10%w/v of homogenate was prepared in 0.15 M Tris-Hcl buffer and processed for the estimation of lipid peroxidation (9). A part of homogenate after precipitating proteins with Trichloroacetic acid (TCA) was used for estimation of glutathione (10). The rest of the homogenate was centrifuged at 1500 rpm for 15 min at 40 C. The supernatant thus obtained was used for estimation of SOD and CAT activities (11, 12).

Serum hepatospecific markers:

Activities of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) were estimated by the method of Reitman and Frankel (4). 0.05 ml of serum with 0.25 ml of substrate (aspartate and α-ketoglutarate for SGOT; alanine and α - keto glutarate for SGPT, in phosphate buffer pH 7.4) was incubated for an hour in case of SGOT and 30 min. for SGPT. 0.25 ml of DNPH solution was added to arrest the reaction and kept for 20 min in room temperature. After incubation 1 ml of 0.4 N NaOH was added and absorbance was read at 505 nm in uv-vis spectrophotometer. Activities were expressed as IU/dl.

Based on the method of King and Armstrong (5) alkaline phosphatase activity was assayed using disodium phenyl phosphate as substrate. The colour developed was read at 510 nm in uv-vis spectrophotometer after 10 min. Activities of ALP was expressed as IU/dl. Serum total bilirubin level was estimated based on the method of Malloy and Evelyn (6) Diazotised sulphonilic acid (0.25 ml) reacts with bilirubin in diluted serum (0.1 ml serum + 0.9 ml distilled water) and forms purple colored azobilirubin, which was measured at 540 nm in uv-vis spectrophotometer. Activities of total bilirubin were expressed as mg/dl. Total cholesterol was determined by method of Richmond [7].Serum total protein level was estimated based on the method of Gornall et al ( 8) . Biuret reagent (1.0 ml) reacts with serum (10 μL) and the colour developed was read at 578 nm in uv-vis spectrophotometer. Activities of total protein were expressed as mg/dl.

Determination of Thiobarbituric Acid Reactive Substances (TBARS):

Lipid peroxidations in liver tissues were estimated colorimetrically by measuring thiobarbituric acid reactive substances (TBARS) by the method of Ohkawa et al (9). To 0.2ml of sample, 0.2ml of 8.1% Sodium dodecyl sulfate, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% TBA were added. The volume of the mixture was made up to 4 ml with distilled water and then heated at 950 °C in a water bath for 60 min. After incubation the tubes were cooled to room temperature and the final volume was made upto 5 ml in each tube. Then 5 ml of n-butanol: Pyridine mixture was added and the contents were vortexed thoroughly for 2 min. After centrifugation at 3000 rpm for 10 min the upper organic layer was taken and its OD was read at 532 nm against an appropriate blank without the sample.

Determination of reduced glutathione (GSH):

Reduced glutathione (GSH) was determined by the method of Ellman (10). To 0.1 ml of different tissue homogenate 2.4 ml of 0.02 M EDTA solution was added and kept on ice bath for 10 min. Then 2 ml of distilled water and 0.5 ml of 50 % TCA were added. This mixture was kept on ice for 10-15 min and then centrifuged at 3000 rpm for 15 min. 1 ml of supernatant was taken and 2ml of Tris-Hcl buffer was added. Then 0.05 ml of DTNB solution (Ellman’s reagent) was added and vortexed thoroughly. OD was read (within 2-3 min after the addition of DTNB) at 412 nm against a reagent blank. Absorbance values were compared with a standard curve generated from known GSH.

Assay of super oxide dismutase (SOD):

Superoxide dismutase (SOD) activity was determined by the method of (9). Prepared 10 % w/v tissue homogenate in 0.15 M Tris HCl .Centrifuged at 15000 rpm for 15 min at 4 °C. Supernatant (0.1 ml) was taken consider it as sample and 1.2 ml sodium pyrophosphate buffer (pH 8.3, 0.052 M) + 0.1 ml phenazine methosulphate (186 μM) + 0.3 ml of 300 μM Nitroblutetrazolium + 0.2 ml NADH (750 μM) were added. Incubated at 30°C for 90 s .0.1 ml glacial acetic acid was added. Stirred with 4.0 ml n-butanol.Allowed to stand for 10 min Centrifuged and separated butanol layer. OD at 560 nm was taken (taken butanol as blank) and concentration of SOD was expressed as units/g of liver tissue. Absorbance values were compared with a standard curve generated from known SOD.

Assay of Catalase (CAT):

Catalase was assayed according to the method of Aebi (12). The estimation was done spectrophotometrically following the decrease in absorbance at 240 nm. The liver tissue was homogenized in M/150 phosphate buffer (pH 7.0) at 1-40 C and centrifuged at 5000 rpm. The reaction mixture contained 0.01 M phosphate buffer (pH 7.0), 2 mM H₂O₂ and the enzyme extract. The specific activity of catalase was expressed in terms of units/gram of liver tissue. Absorbance values were compared with a standard curve generated from known CAT.

Histology: The tissues of liver were removed from animals, washed with normal saline to remove blood, fixed in 10% formalin and embedded in paraffin wax. Sections of 5 μm thickness were made using rotary microtome and stained with haematoxylin-eosin and histological observations were made under light microscope (13, 14).

Statistical analyses:

The experimental results were expressed as the Mean ± S.D for six animals in each group. Statistical analyses were performed using the unpaired t test. A p value of 0.05 or less was considered to indicate a significant difference between groups.

RESULTS:

The effect of Chenopodium album Linn. on serum marker enzymes is presented in Table 1 and 3. The levels of serum SGPT, SGOT, ALP, total bilirubin, total cholesterol were markedly elevated and that of protein decreased in CCl₄treated animals, indicating liver damage. Administration of Chenopodium album Linn .extract at the doses of 200, 400 and 600 mg/kg remarkably prevented CCl₄-induced hepatotoxicity in a dose dependent manner.

Table 1: Effect of hydroalcholic extract of Chenopodium album Linn. on serum, SGPT, SGOT, ALT and Total bilirubin on carbon tetrachloride induced hepatotoxicity in rats:

Groups	Treatment	SGPT(IU/dl)	SGOT(IU/dl)	ALP(IU/dl)	Total Bilirubin (mg/dl)
I	Normal Control	42.4±4.742	55.1±5.855	102.4±7.004	0.49±0.057
II	CCl₄treated	98.6±6.347***	113.7±7.301***	209.3±8.403***	1.30±0.063***
III	Silymerin(200 mg/kg)+CCl₄treated	54.9±6.143⁺⁺⁺	62.8±6.622⁺⁺⁺	115.3±8.108⁺⁺⁺	0.55±0.056⁺⁺⁺
IV	Chenopodium album Linn.(200mg/kg)+CCl₄ treated	82.1±5.108⁺⁺	90.8±6.443⁺⁺	170.6±10.076⁺⁺⁺	1.12±0.061⁺⁺
VI	Chenopodium album Linn.(400mg/kg)+CCl₄ treated	66.5±4.401⁺⁺⁺	74.1±6.266⁺⁺⁺	139.9±7.722⁺⁺⁺	0.69±0.061⁺⁺⁺
VI	Chenopodium album Linn.(600mg/kg)+CCl₄ treated	63.0±5.659⁺⁺⁺	68.7±4.869⁺⁺⁺	132.6±4.833⁺⁺⁺	0.67±0.069⁺⁺⁺

All values are represents mean ± SD; n = 6 animals.

P values: ***‹ 0.0001 when compared with control untreated rats; +++ ‹ 0.0001; ++ ‹ 0.001 when compared with carbon tetrachloride treated rats.

Table 2: Effect of hydroalcholic extract of Chenopodium album Linn. on SOD, GSH, LPO and Catalase in carbon tetrachloride induced hepatotoxicity in rats:

Groups	Treatment	SOD (unit/mg tissue)	GSH (mmol/mg tissue)	LPO (nmol MDA/mg tissue)	Ctalase (unit/mg tissue)
I	Normal Control	11.65±0.602	4.59±0.356	1.52±0.099	14.02±0.685
II	CCl₄treated	3.05±0.200^***	0.30±0.026^***	5.15±0.462^***	3.95±0.252^***
III	Silymerin(200 mg/kg)+CCl₄treated	10.11±0.652⁺⁺⁺	4.12±0.383⁺⁺⁺	1.66±0.216⁺⁺⁺	13.09±0.247⁺⁺⁺
IV	Chenopodium album Linn.(200mg/kg)+CCl₄ treated	6.23±0.358⁺⁺⁺	1.17±0.351⁺⁺⁺	4.13±0.392⁺	7.20±0.762⁺⁺⁺
V	Chenopodium album Linn.(400mg/kg)+CCl₄ treated	8.16±0.159⁺⁺⁺	2.61±0.348⁺⁺⁺	2.18±0.259⁺⁺⁺	11.21±0.479⁺⁺⁺
VI	Chenopodium album Linn.(600mg/kg)+CCl₄ treated	8.90±0.375⁺⁺⁺	3.62±0.383⁺⁺⁺	2.02±0.244⁺⁺⁺	12.03±0.617⁺⁺⁺

All values are represents mean ± SD; n = 6 animals.

P values: ***‹ 0.0001 when compared with control untreated rats; +++ ‹ 0.0001; ++ ‹ 0.001 when compared with carbon tetrachloride treated rats.

Table 3: Effect of hydroalcholic extract of Chenopodium album Linn. on serum Total Protein and Total Cholesterol on carbon tetrachloride induced hepatotoxicity in rats:

Groups	Treatment	Total Protein(ug/mg)	Total Cholestrol (mg/dl)
I	Normal Control	88.3±3.531	89.9±5.619
II	CCl₄treated	43.6±5.793***	168.6±6.471***
III	Silymerin (200 mg/kg)+CCl₄treated	80.1±4.662⁺⁺⁺	102.8±7.106⁺⁺⁺
IV	Chenopodium album Linn.(200mg/kg)+CCl₄ treated	56.7±4.309⁺	139.6±6.302⁺⁺⁺
V	Chenopodium album Linn.(400mg/kg)+CCl₄ treated	67.6±4.106⁺⁺⁺	123.4±5.637⁺⁺⁺
VI	Chenopodium album Linn.(600mg/kg)+CCl₄ treated	72.5±6.115⁺⁺⁺	135.6±6.782⁺⁺⁺

All values are represents mean ± SD; n = 6 animals.

P values: ***‹ 0.0001 when compared with control untreated rats; +++ ‹ 0.0001; ++ ‹ 0.001; +<0.01 when compared with carbon tetrachloride treated rats.

Analysis of LPO levels by thiobarbituric acid reaction showed a significant (P<0.0001) increase in the CCl₄ treated rats. Treatment with Chenopodium album Linn. (200 mg/kg, 400 mg/kg and 600 mg/kg) significantly (P<0.0001) prevented the increase in LPO level which was brought to near normal. CCl₄ treatment caused a significant (P<0.0001) decrease in the level of SOD, Catalase, GSH in liver tissue when compared with control group (Table 2). The treatment of Chenopodium album Linn. at the doses of 200,400 and 600 mg/kg resulted in a significant increase of SOD, Catalase, and GSH when compared to CCl₄ treated rats. The liver of silymarin treated animals also showed a significant increase in antioxidant enzymes levels compared to CCl₄ treated rats.

Morphological observations showed an increased size and enlargement of the liver in CCl₄treated groups. These changes were reversed by treatment with silymarin and also Chenopodium album Linn. at the doses tested.

Histopathological studies, showed CCl₄to produce extensive vascular degenerative changes and centrilobular necrosis in hepatocytes. Treatment with different doses of Chenopodium album Linn. extract produced mild degenerative changes and absence of centrilobular necrosis when compared with control. All these results indicate a hepatoprotective potential of the extract.

DISCUSSIONS:

Carbon tetrachloride is one of the most commonly used hepatotoxins in the experimental study of liver diseases. The hepatotoxic effects of CCl₄ are largely due to its active metabolite, trichloromethyl radical (15). These activated radicals bind covalently to the macromolecules and induce peroxidative degradation of membrane lipids of endoplasmic reticulum rich in polyunsaturated fatty acids. This leads to the formation of lipid peroxides. This lipid peroxidative degradation of biomembranes is one of the principle causes of hepatotoxicity of CCl₄ (16). This is evidenced by an elevation in the serum marker enzymes namely SGPT, SGOT, ALP, total bilirubin, total cholestrol and decrease in protein.

In the assessment of liver damage by CCl₄ the determination of enzyme levels such as SGPT and SGOT is largely used. Necrosis or membrane damage releases the enzyme into circulation and hence it can be measured in the serum. High levels of SGOT indicates liver damage, such as that caused by viral hepatitis as well as cardiac infarction and muscle injury, SGOT catalyses the conversion of alanine and glutamate and is released in a similar manner. Therefore SGOT is more specific to the liver, and is thus a better parameter for detecting liver injury. Elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver (17). Serum SGPT, bilirubin and total protein levels on other hand are related to the function of hepatic cell. Increase in serum level of SGPT is due to increased synthesis, in presence of increasing biliary pressure (18). The increase in LPO level in liver induced by CCl₄suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanism to prevent formation of excessive free radicals. Treatment with Chenopodium album Linn .significantly reverses these changes.

Decrease in enzyme activity of superoxide dismutase (SOD) is a sensitive index in hepatocellular damage and is the most sensitive enzymatic index in live injury. Curtis and Mortiz (19), SOD has been reported as one of the most important enzymes in the enzymatic antioxidant defense system. It scavenges the superoxide anion to form hydrogen peroxide and thus diminishing the toxic effect caused by this radical. In Chenopodium album Linn. causes a significant increase in hepatic SOD activity and thus reduces reactive free radical induced oxidative damage to liver.

Catalase (CAT) is an enzymatic antioxidant widely distributed in all animal tissues, and the highest activity is found in the red cells and liver. CAT decomposes hydrogen peroxide and protects the tissues from highly reactive hydroxyl radicals (20). Therefore reduction in the activity of CAT may result in a number of deleterious effects due to the assimilation of superoxide radical and hydrogen peroxide.

Glutathione is one of the most abundant tripeptide, non-enzymatic biological antioxidant present in the liver. It removes free radical species such as hydrogen peroxide, superoxide radicals and maintains membrane protein thiols. Also it is substrate for glutathione (21). Decreased level of GSH is associated with an enhanced lipid peroxidation in CCl₄treated rats. Administration of Chenopodium album Linn. significantly (P<0.0001) increased the level of GSH in a dose dependent manner.

Extensive vascular degenerative changes and centrilobular necrosis in hepatocytes was produced by CCl₄. Treatment with different doses of ethanolic extract of leaves of Chenopodium album Linn. produced only mild degenerative changes and absence of centrilobular necrosis, indicating its hepatoprotective efficiency.

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Received on 06.03.2015 Modified on 15.03.2015

Res. J. Pharmacology & P’dynamics. 7(1): Jan.-Mar. 2015; Page 29-34

DOI: 10.5958/2321-5836.2015.00007.5