Protective Effect of Enicostemma littorale. against CCl₄-Induced Hepatic Damage in Rats

Abstract

The alcohol extract of the whole plant of Enicostemma littorale. Blume was evaluated for its antihepatotoxic activity against CCl₄-induced hepatic damage in rats. The activity was evaluated by using biochemical parameters, such as serum glutamate pyruvate transaminase, serum glutamate oxaloacetate transaminase, alkaline phosphatase, total bilirubin, and γ.-glutamate transpeptidase. The histopathological changes of liver sample were compared with respective controls. The extract showed a remarkable hepatoprotective effect.

Keywords:

• Biochemical parameters
• carbon tetrachloride
• Enicostemma littorale
• hepatoprotective
• histopathological studies

Introduction

Liver disease remains a serious health problem and is caused by drugs, chemicals, and alcohol. Even though hepatoprotective drugs are available in allopathic medicine, herbs play a vital role in the management of various liver disorders. Various medicinal plants and their formulations are used in the Indian traditional system of medicine for their hepatoprotective effect. Most of the herbal drugs speed up the natural healing process of liver. Enicostemma littorale. Blume (Gentianaceae), found throughout India, is traditionally used in the treatment of rheumatism, ulcer, hypoglycemia, and insect poisoning (Anon., 2001). It is found to be active against hyperglycemia, inflammation, and tumor (Sadique et al., 1987; Kavimani & Mani Senthilkumar, 2000; Maroo et al., 2002). Preliminary phytochemical studies have revealed the presence of alkaloids, glycosides, and flavonoids. The current study was undertaken to evaluate the hepatoprotective effect of alcohol extract of whole plant of Enicostemma littorale. against CCl₄-induced hepatic damage in rats.

Materials and Methods

Plant material and extraction

E. littorale., collected in and around Trichirapalli District in the month of December 2002, was authenticated by Dr. G. Murthy, Botanical Survey of India, Coimbatore, Tamilnadu, India. A voucher specimen (PEL-21) has been kept in our laboratory for future reference. The whole plants were shade-dried and pulverized. The powder was treated with petroleum ether for defatting as well as to remove chlorophyll. The powder was packed into a Soxhlet apparatus and subjected to hot continuous percolation using alcohol (95% v/v) as solvent. The extract was concentrated under vacuum and dried in a vacuum desiccator (yield 6.5% w/w) and then suspended in 5% gum acacia and used for the hepatoprotective studies.

Animals

Male Wistar rats weighing between 150–175 g were used. The animals were maintained in the college animal house under standard laboratory conditions with commercial pellet diet and water ad libitum..

Hepatoprotective studies

Rats were divided into four groups, each group consisting of six animals. The hepatoprotective activity of E. littorale. was evaluated using a CCl₄-induced model. Group 1 was kept on normal diet and served as control, the second group received CCl₄ (1.25 ml/kg) by the oral route, the third and fourth groups received silymarin (100 mg/kg; p.o.) and alcohol extract of E. littorale. (100 mg/kg p.o.), respectively, once daily, for 7 days. On the seventh day, CCl₄ was given by the oral route 30 min after the administration of silymarin and test drug. After 36 h of CCl₄ administration, blood was collected and separated serum was analyzed for various biochemical parameters like serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvate transaminase (SGPT) (Reitman & Frankel, 1957), alkaline phosphatase (ALP) (Kind & King, 1954), total bilirubin (Mallay & Evelyn, 1937), protein (Lowry et al., 1951), and γ.-glutamate transpeptidase (GGTP) (Szaszi, 1969).

The livers were examined grossly, weighed, and stored in formalin 10% and were processed for paraffin embedding using the standard micro technique (Galigher & Kozloff, 1971). Sections of the liver (5 µm) stained with alum hemotoxylin and eosin were observed microscopically for histopathological studies.

Statistical analysis

All values are expressed as mean±SEM. Data were analyzed by Student's t.-test.

Results and Discussion

The results of biochemical parameters revealed the elevation of enzyme levels in the CCl₄-treated group, indicating that CCl₄ induces damage to the liver (Table 1). Liver tissue rich in both transaminases increases in patients with acute hepatic diseases. SGPT, which is slightly elevated by cardiac necrosis, is a more specific indicator of liver disease (Rodwell et al., 1983). A significant reduction was observed (p < 0.001) in SGPT, SGOT, ALP, total bilirubin, GGTP, and increased protein levels in the groups treated with silymarin and the alcohol extract of E. littorale.. The enzyme levels were nearly restored to the normal level.

Table 1.. Effect of alcohol extract of E. littorale. on CCl₄-treated rats.

Treatment	Dose (mg/kg)	SGPT U/l	SGOT U/l	ALP U/l	Total bilirubin mg%	Protein mg%	GGTP U/l
Control	—	131.5 ± 1.98	45 ± 0.80	165.6 ± 3.79	0.70 ± 0.03	6.5 ± 0.10	123.0 ± 3.10
CCl4	1.25 ml/kg	482.3 ± 6.5	324.5 ± 3.8	876 ± 17.46	2.7 ± 0.06	4.5 ± 0.14	251.0 ± 5.31
Silymarin + CCl4	100	138 ± 2.27**	74 ± 3.10*	210 ± 4.3*	0.9 ± 0.07*	5.8 ± 0.20***	109.6 ± 2.20*
Alcohol extract + CCl4	100	170 ± 4.3*	115 ± 2.79*	296 ± 4.5*	1.0 ± 0.02*	5.5 ± 0.12*	146.8 ± 2.72*

SGPT, serum glutamic pyruvate transaminase; SGOT, serum glutamic oxaloacetic transaminase; ALP, alkaline phosphatase; GGTP, γ.-glutamate transpeptidase.

n = 6 animals in each group; *p < 0.001; **p < 0.05; ***p < 0.01 when compared with control.

It was observed that the liver was enlarged in CCl₄-intoxicated rats but is was normal in drug-treated groups. A significant reduction (p < 0.001) in liver weight supports this finding (Table 2).

Table 2.. Effect of the alcohol extract of E. littorale. on liver weight variation.

Design of treatment	Dose (mg/kg)	Liver wt/100 g body weight (g)
Control	—	3.5 ± 0.10
CCl4	1.25 ml/kg	6.9 ± 0.28
Silymarin + CCl4	100	3.9 ± 0.16**
Alcohol extract + CCl4	100	4.8 ± 0.05*

n = 6 animals in each group; *p < 0.001; **p < 0.05 when compared with control.

Histopathological examination of the liver section of the rats treated with toxicant showed intense centrilobular necrosis and vascuolization. The rats treated with silymarin and the extract, along with toxicant, showed signs of protection against these toxicants to a considerable extent as evident from formation of normal hepatic cards and absence of necrosis and vacuoles.

Carbon tetrachloride is one of the most commonly used hepatotoxins in the experimental study of liver diseases. The hepatotoxic effect of CCl₄ are largely due to its active metabolite, trichloromethyl radical (Johnson & Kroening, 1998). 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 (Kaplowitz et al., 1986). This is evidenced by an elevation in the serum marker enzymes, namely, SGPT, SGOT, ALP, total bilirubin, GGTP, and decrease in protein.

The efficacy of any hepatoprotective drug is dependent on its capacity of either reducing the harmful effect or restoring the normal hepatic physiology that has been disturbed by a hepatotoxin. The silymarin and extract of the plant decreased the CCl₄-induced elevated levels of the enzymes in groups 3 and 4, indicating the production of structural integrity of hepatocytic cell membrane or regeneration of damaged liver cells by the extract.

Decrease in serum bilirubin after treatment with the extract in CCL₄-intoxicated rats indicated the effectiveness of the extract in normal functional status of the liver. Histopathological analyses are in good agreement with biochemical changes. The chemical constituents of E. littorale. responsible for its hepatoprotective activities are not known. However, the preliminary phytochemical studies reveal the presence of flavonoids in alcoholic extract of E. littorale.; various flavonoids have been reported for its hepatoprotective activity (Scevola et al., 1984; Wegener & Fintelmann, 1999). Therefore, the possible mechanism of hepatoprotective effect of E. littorale. may be due to its flavonoids content. On the basis of our result, it can be concluded that E. littorale. exhibited significant hepatopotective activity. Further studies are needed to isolate the active principle of the E. littorale. and establish the chemical nature that are responsible for the hepatoprotective properties.