Hepatoprotective Activity of Glycyrrhiza glabra Linn. on Experimental Liver Damage in Albino Rats

Rajesh M.G.¹* and Latha M.S.²

¹Navajyothi Sree Karunakara Guru Research Centre for Ayurveda and Siddha, Uzhavoor, Kottayam, Kerala- 686 634, India.

²School of Biosciences, Mahatma Gandhi University, Priyadarshini Hills P.O., Kottayam, Kerala- 686 560, India.

ABSTRACT:

Liver is a versatile organ in the body concerned with regulation of the internal chemical environment. Therefore, damage to the liver inflicted by hepatotoxic agents like xenobiotics, therapeutic agents and environmental pollutants is of grave consequences. There is still an unmet need for medicinal plants and phytopharmaceuticals with scientifically validated hepatoprotective activity. The present study evaluates the antihepatotoxic action of Glycyrrhiza glabra Linn. on carbon tetrachloride (CCl₄)–induced hepatic damage in Sprague-Dawley rats. The parameters assessed were liver function marker enzymes, total protein, bilirubin and lipid profile in serum. The lipid profile of different tissues was also estimated. The biochemical changes caused by CCl₄ were counteracted by the co-administration of G. glabra. The results suggest the antihepatotoxic activity of this medicinal plant.

KEYWORDS: Fabaceae, medicinal plant, antihepatotoxic, carbon tetrachloride.

INTRODUCTION:

Carbon tetrachloride (CCl₄) is a highly toxic chemical agent, the liver being its principal site of toxicity¹. CCl₄-induced hepatotoxicity in rats represents an adequate experimental model of cirrhosis in man and is used for the screening of hepatoprotective drugs^2-4. As the management of liver diseases is still a challenge to modern medicine, the role of herbal drugs in the management of various liver disorders is important^5,6. In Ayurveda, many indigenous plants have been used as hepatoprotective agents. However, the present age demands proof on a scientific basis to justify the various medicinal uses of plants⁷.

Glycyrrhiza glabra Linn. of the family Fabaceae is a tall perennial undershrub. Its underground stems and roots are used medicinally. It is used in the treatment of liver diseases⁸. Its hypocholesterolaemic and hypoglycemic activities have been reported ⁹. It is a component of many antihepatotoxic polyherbal preparations^10-13. So, the results of our attempt to explore the hepatoprotective effect of Glycyrrhiza glabra are presented in this communication.

MATERIAL AND METHODS:

Glycyrrhiza glabra was purchased from the raw drug market, Palai, Kottayam district, Kerala. The samples were identified with authentic literature and a voucher specimen was deposited in the institutional herbarium for future reference. The roots and underground stems were dried overnight at 45° C and powdered. Male albino rats of Sprague-Dawley strain were used for the experiment.

They were housed in polypropylene cages under standard conditions (23 ± 2° C, Humidity: 60-70%, 12hrs light and dark cycles), given a commercial rat food (M/s Hindustan Liver Ltd., Mumbai, India) and tap water for drinking on an ad libitum basis. Utmost care was taken to ensure that the animals were treated in the most humane and ethically acceptable manner. The rats were arranged into 3 groups as follows (each group with six rats): (1) Pair-fed control group; (2) CCl₄-treated group- this group was given a dose of 0.1ml of CCl₄ in groundnut oil (1:1, v/v) per 100g body weight through an intra-gastric tube twice a week for a period of 2 months; (3) CCl₄+ G. glabra – this group was given a dose of 1000mg/ kg body weight of G. glabra powder mixed with the feed for 60 days in addition to CCl₄. The dose of the medicinal plant was ascertained by a pilot study over a range of dosages varying from 500mg/ kg body weight to 1500mg/ kg body weight.

At the end of the experimental period (i.e., 60 days), rats were deprived of food overnight and sacrificed by decapitation. Blood was collected by excising the jugular vein. Serum samples were obtained by centrifuging the whole blood at 3000 rpm for 20 min., collected and left standing on ice until required. The tissues (liver and kidney) were excised and transferred into ice-cold containers for biochemical examinations.

Activities of serum enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), serum concentrations of protein, bilirubin and serum as well as tissue concentrations of total lipid, phospholipids, cholesterol and triglycerides were measured according to published procedures^14-22.

The protective effect of G. glabra was evaluated by comparing the above-mentioned biochemical parameters of group II with group I and group III with group II. Results are expressed as the mean ± SEM. Student’s t- test was used to assess statistical significance.

The results, expressed as per cent hepatoprotective activity (H) were calculated by the formula²³,

H = 1– (HC– N) / (C-N) X100,

Where, HC, C and N are the parameters measured in Herbal preparation + CCl₄-treated rats, CCl₄- treated rats and Normal control rats, respectively.

RESULTS:

In the present study, the antihepatotoxic effect of Glycyrrhiza glabra on CCl₄-induced liver damage was investigated in male Sprague-Dawley rats. Rats on CCl₄ treatment showed weight loss. Treatment with G. glabra prevented this change. Table I depicts the activities of (AST), (ALT), (ALP) and (GGT) in serum and serum concentrations of protein, bilirubin, total lipids, phospholipids, cholesterol and triglycerides. It shows significant increase in the activities of AST, ALT, ALP and GGT and the concentrations of bilirubin, total lipids, phospholipids, cholesterol and triglycerides in the CCl₄-treated group compared to normal control. But a decrease in protein content was observed in the rats of group II. Administration of G. glabra along with CCl₄helped to maintain the activities of the liver function marker enzymes and the concentrations of total protein, total lipids, phospholipids, cholesterol and triglycerides at near normal levels.

In table II, the concentrations of total lipids, phospholipids and cholesterol in tissues such as liver and kidney are presented. Substantial elevation in the concentration of total lipids and cholesterol was observed in the tissues of CCl₄-treated group. Significant increase in the concentration of phospholipids was noticed in the kidney of the above group. The level of phospholipids in liver decreased in the rats of group II. Administration of G. glabra, significantly prevented the CCl₄-induced alteration in the lipid profile.

DISCUSSION:

Since the liver is an organ with diverse functional activity, the hepatoprotective activity of a drug should be based on its ability to reduce the injurious effect or to preserve the architecture and physiological functions of the liver, disturbed by a hepatotoxin²³. Administration of CCl₄ is a conventional method for producing liver necrosis in the rat²⁴. Metabolism of CCl₄ involves electron and hemolytic cleavage catalysed by cytochrome P₄₅₀ of the mixed function oxidase system to yield a highly reactive trichloromethyl radical, CCl₃^• and trichloromethyl peroxy radical, CCl₃OO^{• 25}. These free radicals have two fates: they can act in a direct way by covalent bonding to membrane proteins and lipids especially to those of the endoplasmic reticulum leading to alkylation reactions and enzyme inactivation; or they can act in an indirect way through interactions with membrane unsaturated fatty acids and consequent promotion of lipid peroxidation, an important pathogenic mechanism of liver necrosis ²⁶.

Intracellular enzymes are normally tightly bound to particular organelles. Once the membrane integrity is lost due to the participation of membrane destabilizing agents, tissue destruction occurs and the enzymes leak out into the blood and their activity in serum increases ²⁷. The enzymes AST and ALT are of mitochondrial origin. The elevated activities of these hepatic enzymes in serum indicate cellular leakage and loss of the functional integrity of cell membrane of the liver²⁸. Co-administration of G. glabra at a dose of 1000mg/kg body weight to rats helped to maintain the activities of serum transaminases at normal levels.

The enzyme alkaline phosphatase is excreted by the liver through bile. Defective excretion of bile by the liver elicits increased levels of ALP in serum²⁹. Hepatotoxins can interfere with the metabolism of bile by: hemolytic, competing with serum albumin for binding with bilirubin or interference with the secretion of bilirubin by the liver cells³⁰.

Table 1: Effect of Glycyrrhiza glabra on rat serum parameters after CCl₄ intoxication

Parameters	Group
Parameters	I- Pair fed control	II- CCl₄-treated	III- CCl₄+ G. glabra
AST (IU/L)	36.09 ± 0.96	69.41 ± 2.44^***	35.94 ± 1.61^†††
ALT (IU/L)	27.76 ± 0.69	133.28 ± 4.69^***	37.69 ± 1.69^†††
ALP (IU/L)	84.31 ± 2.12	488.12 ± 17.14^***	161.03 ± 7.23^†††
GGT (U/L)	3.62 ± 0.09	7.64 ± 0.26^***	3.78 ± 0.17^†††
Total protein (mg/dl)	9.40 ± 0.24	7.40 ± 0.26^***	9.18 ± 0.41^†
Bilirubin (mg/dl)	1.60 ± 0.04	2.06 ± 0.05^***	1.64 ± 0.04^†††
Total lipid (mg/dl)	290.45 ± 7.25	556.67 ± 19.48^***	264.75 ± 11.97^†††
Phospholipids (mg/dl)	137. 50 ± 3.44	381.25 ± 13.34^***	136.47 ± 6.10^†††
Triglycerides (mg/dl)	5.70 ± 0.14	7.20 ± 0.25^***	5.79 ± 0.26^††
Cholesterol (mg/dl)	60.93 ± 1.52	89.41 ± 3.13^***	64.52 ± 3.02^†††

Group II has been compared with group I and group III has been compared with group II.

^*** P<0.01,^†††P<0.01, ^††P<0.02, ^†P<0.05. (Values are mean ± SEM, n=6)

Table 2: Effect of Glycyrrhiza glabra on lipid profile on rat liver and kidney after CCl₄ intoxication

Parameters	Group	Liver	Kidney
Total Lipids (mg/100g)	I- Pair fed control	4493.84 ± 112.84	4823.12 ± 122.02
	II- CCl₄-treated	5056.92± 176.92^*	6330.35 ± 218.78^***
	III- CCl₄+ G. glabra	4508.26 ± 196.38^†	4145.38 ± 187.54^†††
Phospholipids (mg/100g)	I- Pair fed control	1500.00 ± 37.67	3215.24 ± 80.38
	II- CCl₄-treated	1075.00± 37.74^***	3998.33 ± 99.46^***
	III- CCl₄+ G. glabra	1421.95 ± 56.16^†††	3285.63 ± 147.85^††
Triglycerides (mg/100g)	I- Pair fed control	450.80 ± 11.27	273.82 ± 6.85
	II- CCl₄-treated	542.80± 18.76^***	379.26 ± 13.27^***
	III- CCl₄+ G. glabra	439.45 ± 19.86^††	287.17 ± 12.92^†††
Cholesterol (mg/100g)	I- Pair fed control	351.18 ± 8.85	620.80 ± 15.52
	II- CCl₄-treated	473.91 ± 16.63^***	841.18 ± 29.36^***
	III- CCl₄+ G. glabra	342.57 ± 15.48^†††	504.88 ± 22.68^†††

Group II has been compared with group I and group III has been compared with group II.

^*** P<0.01, ^** P<0.02, ^*P<0.05, ^†††P<0.01, ^††P<0.02, ^†P<0.05. (Values are mean ± SEM, n=6)

Regulation of the metabolism of amino acids and proteins is one of the primary roles of the liver. The organ carries out four main functions in protein metabolism³¹: formation of plasma proteins including many clotting factors, albumin, thyroid binding globulin, VLDL apoB 100 and complement. Liver is the target site and is responsible for the metabolism of many hormones that have discordant effect on protein metabolism. So, chronic and acute liver diseases are associated with the dysfunction of many physiological processes and alter amino acid and protein metabolism. Gamma glutamyl transpeptidase (GGT), which is located in the plasma membranes of hepatocytes, is a component of the gamma glutamyl cycle and plays a significant role for the transport of extracellular amino acids through the outer membrane of the cells. In hepatotoxicity, hepatic amino acid metabolism is altered leading to the elevation of serum GGT activities³². Administration of CCl₄causes depression in protein synthesis. Defect in protein synthesis coincides with a substantial defect in the methylation of cytoplasmic ribosomal RNA. The defect in methylation was specific for 2’O position³³. The co-administration of G. glabra along with CCl₄ and to animals moderates the elevation in the activity of GGT the decrease in the level of proteins, in serum. This may be due to the restoration of hepatic amino acid metabolism and proper methylation of cytoplasmic ribosomal RNA. The increase in hepatocyte protein synthesis may also be due to the stimulation of the activity of ribosomal RNA polymerase by the medicinal herb³⁴.

Accumulation of fat in the liver during toxicity results from decreased mitochondrial fat oxidation ³⁵. Hepatic steatosis of the liver is a multifactorial phenomenon thought to be caused by the blockage of lipoprotein secretion, impaired synthesis or peroxidation of phospholipids or both, the toxic effects of free alkyl radicals on cell membranes and disturbances in methylation reaction³⁶. The action of free alkyl radicals on biomembranes causes haloalkylation-dependent blocking at the exit of the lipoprotein micelles from the Golgi apparatus. During toxicity, the lipid profile of serum and tissues like liver and kidney increases. CCl₄-poisoned rats appear to have deranged hepatic triglyceride secretory mechanism. Accumulation of triglycerides in liver during CCl₄-intoxication, according to Villela (1964), results not from a defect in the release of triglycerides into the plasma but is perhaps due to an increase in hepatic synthesis of triglycerides³⁷. Hepatotoxins interfere with hepatic phospholipids biosynthesis³⁸. This causes a decrease in the concentration of phospholipids in the liver of the CCl₄-treated group. Treatment with the medicinal herb resulted in significant improvement of the serum and lipid profile of CCl₄-intoxicated rats.

Membrane lipid peroxidation which stems from the interaction of free radicals elicited by CCl₄ is responsible for the leakage of cytosolic enzymes into the serum during toxicity. So, it can be concluded that G. glabra efficiently scavenges reactive oxygen species and free radicals, inhibits membrane lipid peroxidation and protects against chemical induced damage, for it normalized the activities of serum transaminase.

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Received on 04.05.2010

Accepted on 13.05.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(4): July-Aug. 2010, 313-316