Protective effect of Aqueous extracts of Sacharum officinarum leaves against Rifampicin-induced Hepatotoxicity in Rats

 

Nitin M.*, Syeda Sana, Amreen Begum, Ansari Firdous

Department of Pharmacology, HKES Matoshri Taradevi Rampure Institute of Pharmaceutical Sciences,

Sedam Road, Gulbarga-585105, Karnataka, India.

*Corresponding Author E-mail:- allnitin@yahoo.com; syedasanaismail@yahoo.com

 

Background: Liver diseases remain one of the serious health problems that effects considerable number of people in the world. In the absence of reliable liver protective drugs in allopathic medical practices. Herbs play important role in the management of various liver disorders. Aims: To investigate the hepatoprotective activity of aqueous extract of leaves of Saccharum officinarum belonging to family Gramineae against rifampicin induced liver damage in albino rats.

Materials and Methods: Hepatotoxicity was induced in albino rats by oral administration of rifampicin (1 g/kg b.w for 10 days).The aqueous extracts of leaves of S. officinarum was administered to the experimental animals at two selected doses (200 and 400 mg/kg) for 10 days. The hepatoprotective activity of aqueous extracts was evaluated by the liver function marker enzymes in serum as, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), Alkaline Phosphatase ALP, Total Bilirubin, Liver weight and by histopathological studies. Statistical analysis: The results were calculated using Dunnet ‘t’ test. The results were found to be highly significant p<0.001.

Results: Elevated levels of liver function marker enzymes in serum following rifampicin induction were significantly lowered in pretreatment with aqueous leaves extract of Saccharum officinarum The higher dose of the extract (400 mg/kg, p.o) prevented the increase in liver weight when compared to hepatotoxin treated group, while the lower dose was less effective . The (400 mg/kg po) of S. officinarum aqueous leaves extracts was found to be more effective than (200 mg/kg po).

Conclusion: Thus the present study suggests that the oral administration of aqueous extract of S. officinarum leaves (200 and 400 mg/kg) significantly possesses good hepatoprotective activity.

 

 

INTRODUCTION:

Liver diseases, such as jaundice, cirrhosis and fatty liver are very common worldwide. The liver occupies the pivotal position in body plays an essential role in drug and xenobiotic metabolism and in maintaining the biological equilibriumof the organism. The role played by this organ in the removal of toxic substances from the portal circulation makes it susceptible to first and persistent attack by offending foreign (xenobiotics) compounds culminating in liver dysfunction ^[1].

 

Metabolism of drugs or toxins occur in 2 phases in liver^[2].In the phase 1 reaction, the drug is made polar by oxidation or hydroxylation.

 

All drugs may not undergo this step, and some may directly undergo the phase 2 reaction. The cytochrome P₄₅₀ enzymes catalyze phase 1reactions^[3,4]. Most of these intermediate products are transient and highly reactive. These reactions may result in the formation of metabolites that are far more toxic than the parent substrate and may result in liver injury. Phase 2 reactions may involve conjugation with a moiety (i.e., acetate, amino acid, sulfate, glutathione, glucuronic acid)^[5]. Subsequently, drugs with high molecular weight may be excreted in bile, while the kidneys excrete the smaller water soluble molecules.

 

Drug induced hepatotoxicity is the most commonly reported adverse reaction of isoniazid and rifampicin, resulting in acute liver failure and elevated enzyme levels^[6].Rifampicin is a potent inducer of cytochrome P₄₅₀ enzymes resulting in drug induced liver injury, a major hurdle in treatment of tuberculosis. Recent studies indicate the existence of a strong correlation between hepatic injury in experimental animals treated with antitubercular drugs.

Liver diseases are still a worldwide health problem. Unfortunately, conventional or synthetic drugs used in the treatment of liver diseases are inadequate and sometimes can have serious side effects^[7]. In the absence of a reliable liver protective drug in modern medicine there are a number of medicinal preparations in Ayurveda recommended for the treatment of liver disorders^[8]. In view of severe undesirable side effects of synthetic agents, there is growing focus to follow systematic research methodology and to evaluate scientific basis for the traditional herbal medicines that are claimed to possess hepatoprotective activity.

 

Saccharum officinarum (sugarcane) is a tropical perineal grass belonging to family Gramineae. It is extensively cultivated  throughout India and world. The plant contains amongst many others flavonoids, alkaloids, amino acids, carbohydrates, starch and vitamins. The use of the plant include as a cardiotonic, expectorant, haemostatic, tonic, diuretic and useful in urinary disorders. The plant also has better antioxidant effects. The present study was undertaken to evaluate the hepatoprotective activity of leaves of this plant in experimental animals ^[9].

 

MATERIALS AND METHODS:

Animals:

Adult albino rats of either sex (200–250 g) were used for this study. The rats were housed in polypropylene cages and maintained under standard conditions (12 h. light and dark cycles, at 25 ± 5°C and 35 – 60% humidity) standard pelletised feed and potable water were provided ad libitum. The animals were acclimatized to laboratory conditions for 48 hours prior to the experimental protocol to minimize any nonspecific stress. Prior permission from IAEC was obtained for conduction of experiments.

 

Drugs and chemicals:

Pure drug samples of Silymarin and Rifampicin  were obtained as a gift sample from Micro Labs.(Bangalore), Lupin Limited (Aurangabad). The kits for the biochemical estimation were purchased from Erba Diagnostics. Mumbai, Maharashtra, India. Ethanol solvents and other chemicals were procured from reputed manufacturers.

 

Plant Material:

Leaves of Saccharum officinarum (Sugarcane) were purchased from the local market of Gulbarga, Karnataka and were authenticated at Pharmacognosy department of HKES’s MTR Institute of Pharmaceutical Sciences, Gulbarga. A specimen of the plant is stored in the herbarium with specimen No. HKEcogcol – 06.

 

Preparation of Extract:

The coarse powder of the shade dried leaves of Saccharum officinarum was subjected to maceration. For each 100 g of crushed leaves, 300 ml of water of added. The crushed leaves were  soaked in water for about7  days. After 7 days the water was strained through a muslin cloth and the extract was concentrated on water bath to obtain thick pasty mass. Standard methods were used for preliminary phytochemical screening of the extract to know the phytoconstituents ^[10].It was found that the extract contains amongst many others flavonoids, alkaloids, amino acids, carbohydrates, starch and vitamins.

 

Acute toxicity studies:

Acute oral toxicity study was performed as per OECD-423 guidelines (Ecobichon. 1997). The methonolic extract of S. officinarum was administered orally in doses of 5, 50, 300, 2000, 4000 mg/kg bodyweight to groups of rats (n=3) and the percentage mortality was recorded over a period of 24 h. During the first 1 h of drug administration, rats were observed for gross behavioural changes as described by Irwin et al.(1968)^[11].The extracts were found to be safe and fall under GSH 5.

 

Hepatoprotective Activity:

The animals were divided into five groups of six animals each. Silymarin was dissolved in 2% gum acacia suspension and herbal extract was dissolved in water. The treatment protocol was planned to study the effect of herbal extract in preventive aspect of rifampicin induced hepatotoxicity. The dose of rifampicin to induce hepatic damage was selected as 1 g/kg body weight for ten days^[12].The treatment protocol was summarized as shown below:

 

Group 1-   Normal control: 2% w/v suspension of gum acacia (1mg/kg) p.o.

Group 2-   Toxicant: rifampicin (1 g/kg) p.o for every 72 hours for 10 days.

Group 3-   Standard: silymarin (25 mg/kg) p.o + after 30 minrifampicin(1 g/kg) p.o for every 72 hours for 10 days.

Group 4-   (AESO) Saccharum officinarum  aqueous extract (200 mg/kg) p.o + after 30 min rifampicin (1 g/kg) p.o for every 72 hours 10 days.

Group 5- (AESO) Saccharum officinarum  aqueous extract (400 mg/kg) p.o + after 30 min rifampicin (1 g/kg) p.o for every 72 hours for 10 days.

 

On 0 and 11^th day the blood samples were collected from all animals by retro orbital puncture. Serum was separated by centrifugation at 2500 rpm for 15 min. and analyzed for various biochemical parameters. Immediately after collection of blood the animals were euthanized with an over dosage of ether. The livers were removed, washed in saline and the wet weight and volume was determined then transferred into 10% formalin for its histopathological studies^[13].

 

Histopathological studies:

Histopathological study of livers was performed in his­topathology Laboratory by consultant histopathologist. Haematoxylin-eosin was used for staining in histopa­thology studies.

 

 

 

Table 1:  Influence of aqueous extract of leaves of Saccharum  officinarum (AESO) on selected serum biochemical and physical parameters in Rifampicin-induced hepatotoxic rats.

G	Treatment	Dosep.o	ALT(IU/L)	AST(IU/L)	ALP (IU/L)	BIT (mg/dL)	Mean liver weight (g/100g)
1	Normal control	1mg/kg gum acacia	43.3±0.99	63.6±1.74	143.7±0.53	0.55±0.17	3.98± 0.05
2	RIF Toxicant	1 g/kg	163.4±3.43***	275.7±6.40***	481.1±2.83***	3.33±0.17***	6.42±0.21***
3	SIL +RIF	25 mg/kg + 1 g/kg	56.0±2.86***	73.9±1.25***	185.1±3.35***	0.86±0.15***	4.08±0.04***
4	AESO+RIF	200 mg/kg + 1 g/kg	94.2 ±1.24***	100.7±1.33***	207.4±2.38***	1.23±0.14***	3.26±0.06***
5	AESO+RIF	400 mg/kg + 1 g/kg	55.5±3.54***	79.3±3.04***	166.4±1.46***	0.70±0.04***	3.77±0.06***

p.o: per oral; n = 6.

***P<0.001Rifampicin Vs Normal control,

***P<0.001 (SIL + RIF) , (AESO+ RIF 200 mg/kg) Vs Rifampicin group and(AESO+ RIF 400 mg/kg) Vs Rifampicin group.

G-Group, RIF- Rifampicin , SIL-Silymarin , AESO-Aqueous extract of leaves of Saccharum officinarum.

 

Figure 1: (Normal control): Microphotograph Showing normal histology of rat liver.		Figure 2: Microphotograph of RIF   induced rat liver shows profound inflammation and congestion along with degeneration around the central vein
Figure 3: Microphotograph of RIF  + standard Silymarin treated rat liver showing normal and affected areas.	Figure 4:Microphotograph rat liver treated with AESO (200 mg/kg + rifampicin ) showing mild degeneration and inflammation		Figure 5: Microphotograph of   rat liver t treated with AESO (400 mg/kg + Rifampicin) showing mild degeneration and no inflammation.

 

Statistical Analysis:

The data obtained in the experiment was expressed in terms of mean ± SEM. Statistical significance of data was assessed by one way analysis of variance (ANOVA) followed by a comparison between different groups using Dunnet ‘t’ test. Values with (***P<0.001) were considered to be statistically significant. The rifampicin group was compared with the normal control group and all other treatment groups were compared with the rifampicin group.

 

RESULTS:

The dose of AESO (400 mg/kg, p. o ) and silymarin (25 mg/kg, p.o ) produced a significant reduction in serum marker enzymes (P<0.001), as dose of AESO (200 mg/kg, p.o) also produced a significant reduction in SGOT, SGPT, ALP and total bilirubin when compared to toxicant group, but it was less effective(Table 1).Administration of rifampicin produced non-significant increase in liver weight. Silymarin and the dose of AESO (200 mg/kg, p.o) did not affect the liver weight, when compared to toxicant group. Whereas, dose of AESO (400 mg/kg, p.o) showed a significant reduction in the liver weight (P<0.001) when compared with the toxicant group (Table1). Histological examination of the liver tissue from toxicant group animals revealed that hepatocytes of the normal control group showed a normal histology of the liver. In the rifampicin treated group the liver showed loss of lobular architecture, extensive central vein dilation and inflammation. Silymarin and AESO pretreated groups showed mild central vein dilation and architecture of the liver was maintained.      (Fig 1-5)

 

DISCUSSION:

Rifampicin causes transient elevations in hepatic enzymes AST and ALT indicating the liver necrosis. Clinically, these enzyme levels are elevated in acute hepatitis, chronic hepatitis, chronic alcoholic hepatitis, diffuse intrahepatic cholestasis, extrahepatic obstruction and focal intrahepatic disease. Serum level of transaminases returns to normal once the healing of hepatic parenchyma and regeneration of hepatocytes occurs^[14].

 

Assessment of liver function can be made by estimating the activities of serum ALT, AST, ALP and Bilirubin which are enzymes originally present in higher concentration in cytoplasm. When there is hepatotoxicity, these enzymes leak into the blood stream in conformity with the extent of liver damage^[15].

 

In the present study extensive liver damage was induced by rifampicin as a toxicant, the activities of serum enzymes were found to increase in the toxicant group animals, and were significantly reduced in groups of aqueous leaves extract of Saccharum officinarum as compared to that of toxicant group. The extent of hepatic damage was assessed by histological evaluation along with the level of various biochemical parameters in circulation. The animals in the toxicant group showed severe hepatotoxicity evidenced by necrosis, degeneration and fibrosis as compared to the normal hepatic architecture of the normal group animals which is clearly seen in Figure.  Higher dose of the extract (400 mg/kg, p.o) showed the healing of damaged parenchyma which was comparable to that of the standard group treated with silymarin. The effect was more pronounced with 400mg/kg extract.

 

Bilirubin is one of the most useful clinical clues to the severity of necrosis and its accumulation is a measure of binding, conjugation and excretory capacity of hepatocyte. Decrease in serum bilirubin after treatment with the extracts in liver damage induced by rifampicin, indicated the effectiveness of the extract in normal functional status of the liver.

 

CONCLUSION:

Both the extract groups doses of AESO (400 mg/kg, p.o ) and AESO (200 mg/kg, p.o) show the hepatoprotective effect. In all the tests of AESO (400 mg/kg, p.o) showed a better protective activity than  of AESO (200 mg/kg, p.o) .From the above preliminary study, we conclude that aqueous extracts of Saccharum officinarum , is proved to be one of the herbal remedies for liver ailment.

 

ACKNOWLEDGEMENT:

Authors are thankful to the authorities of H.K.E.S’s MTRIPS, Gulbarga for providing facilities to carry out this study.

 

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Accepted on 18.06.2013                © RJPT All right reserved