Hepatoprotective Activity of Borreria hispida on Paracetamol induced Liver Damage

 

D. Benito Johnson*, C.Senthil Kumar R. Rajesh, R. Venkatnarayanan and Mohammed Ansar VK

Department of Pharmacology,  R.V.S. College of Pharmaceutical Sciences, Sulur, Coimbatore, Tamil Nadu.

 

ABSTRACT:

Hepatocyte are the functional cells of the liver and perform a wide range of metabolic, secretory and endocrine functions. Hepatotoxicity implies chemical-driven liver damage. The liver plays a major role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ. Other chemical agents, those used in laboratories and industries, natural chemicals and herbal remedies can also cause hepatotoxicity. Borreria hispida seed flavonoid-rich fraction possesses free radical scavenging and antioxidant activity both in vitro and in vivo Borreria hispida Linn has been in use in the Indian system of medicine. Various part of the plant are useful in the treatment of antifertility, appetite, Bleeding in child birth, body ache, Gum trouble, scabies and skin disease, Stomach compliance, Ulcers, hepatitis, Wounds, head ache and tooth ache. The hepatotoxicity is induced by the paracetamol overdose, and the methanolic extract of Borreria hispida shows a good reduction of hepatotoxicity.

 

 

INTRODUCTION: ^[1]

Hepatotoxicity implies chemical-driven liver damage. The liver plays a major role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ¹. Other chemical agents, those used in laboratories and industries, natural chemicals and herbal remedies can also cause hepatotoxicity. Chemicals that cause liver injury are called Hepatotoxins¹³.

·        NSAIDs (Acetaminophen, Aspirin, Ibuprofen, etc.)

·        Glucocorticoids

·        Anti-Tubercular drugs (Isoniazid, Rifampin, Pyrazinamide, etc.)

·        Industrial toxins (arsenic, carbon tetrachloride and vinyl chloride)

·        Natural Products (many amanita mushrooms, aflatoxins)

 

SYMPTOMS OF HEPATOTOXICITY²:

The list of signs and symptoms mentioned in various causes for Hepatotoxicity includes the symptoms as listed below:

·        Nausea

·        Vomiting

·        Abdominal pain

·        Loss of appetite

·        Diarrhea

·        Tiredness

·        Weakness

·        Jaundice

·        Yellow eyes

·        Yellow skin

·        Enlarged liver

·        Abnormal liver function test results

·        Swelling in feet

·        Weight increase due to water retention

·        Prolonged bleeding time

 

MATERIALS AND METHODS:

Drugs Used: Paracetamol, Borreria hispida, Chemicals: Methanol, Instruments: Soxhlet apparatus, Animals: Healthy adult Albino rats of both sexes (150-220g).

 

TABLE – 1: STUDY SCHEDULE

I.N. CONTROL	Distilled Water	Distilled Water	Distilled Water	Distilled Water	Distilled Water	Distilled Water	Distilled Water	Blood collection, Euthanasia & collection of liver
II.P. CONTROL	Distilled Water	Distilled Water	Distilled Water	Distilled Water	Distilled Water	Distilled Water + Paracetamol	Distilled Water	Blood collection, Euthanasia collection of liver
III.DOSE - A	Dose- A	Dose- A	Dose- A	Dose- A	Dose- A	Dose A + Paracatemol	Dose-A	Blood collection, Euthanasia & collection of liver
IV. DOSE-B	Dose-B	Dose-B	Dose-B	Dose-B	Dose-B	Dose B+ Paracetamol	Dose-B	Blood collection, Euthanasia & collection of liver
V.STANDARD	Std	Std	Std	Std	Std	Std + Paracetamol	Std	Blood collection, Euthanasia & Liver is collected

 

METHODOLOGY

Animals were divided into five groups of 6 animals (3 Males + 3 Females) in each group. The first group received Distilled water 1 ml/kg for one week (control). The group II received distilled water 1 ml/kg for first five days in a week (positive control). The groups III and IV received 200 mg/kg and 400 mg/kg of Borreria hispida ethanolic extract respectively once a day for seven days. The groups V received Liv-52 once a day for 7 days.  On the fifth day, after the administration of the respective treatments, all the animals of groups II, III, IV and V were administered with paracetamol 2 gm/kg orally. On the seventh day after 2 hr of respective treatments the blood samples were collected for the estimation of biochemical marker enzymes. Then animals under ether anesthesia were sacrificed. The data’s were given in Table – 1.

 

PARAMETERS MONITRED

·        Change in body weight

·        Alanine Transaminase ( ALT )

·        Aspartate Transaminase ( AST)

·        Alkaline Phosphatase ( ALP)

·        Histopathological Studies

 

CHANGES IN BODY WEIGHT

Animal are weighed daily during the study. The treated (Group III, IV, V) group weights were increased when compared with that of control Group.

 

ALANINE TRANSAMINASE (ALT)

The ALT method is an adaptation of the recommended procedure of the IFCC as described by Bergmeyer³. The procedure is based on the principles outlined by Wroblewski and LaDue but is modified to contain pyridoxal-5-phosphate (P5P) as an activator and to replace phosphate buffer with tris (hydroxymethyl) amino methane. Significant elevations of ALT are found in diseases of the liver, such as hepatitis, necrosis, jaundice and cirrhosis. ALT levels can be elevated even before clinical jaundice appears⁴. The results were given in Table – 2.

 

ASPARTATE TRANSAMINASE (AST) ⁵

The AST method is an adaptation of the methodology recommended by the International Federation of Clinical Chemistry (IFCC). The method uses the coenzyme Pyridoxal-5-Phosphate (P5P) to activate the apoenzyme and lactic acid Dehydrogenase (LDH) to eliminate pyruvate interference. Significant elevations of AST are found in diseases of the liver such as hepatitis, necrosis, jaundice and cirrhosis. AST levels can be elevated even before clinical jaundice appears. The results were given in Table -3.

 

Alkaline Phosphatase (ALP)

Measurements of ALP are used in the investigation of hepatobiliary and bond disease⁶. The ALP method is based on a procedure published by Bowers and McComb and more recently reviewed by Rej. This method responds to all ALP isoenzymes in human serum. The results were given in Table – 4.

 

Histopathology Study ^{10, 11, 12}

Rats from all the treatment groups and control groups were euthanized on the day 7. After gross observation, liver was collected and fixed in 10% Neutral Buffer Formalin¹³.

Trimming: Tissues were trimmed from all the lobes of liver.

Processing: Processing is done with the help of Automated Tissue Processor (ATP) (Leica ASP 300) for 16 hours.

Embedding: Processed tissues were embedded in paraffin with the help of paraffin embedding station (Leica EG 1150 H).

Sectioning: Initially blocks were trimmed at 25 microns and then sectioned at 4 microns with the help of semi automatic Microtome (Leica RM 2245)

Staining: Slides were stained by H&E stain at Multistainer (Leica ST 5020)

All the H&E stained slides were observed for pathological findings.

 

RESULTS:

Table – 2: Results of ALT

S.No	Groups	ALT (IU/L)
1	Negative Control	64.3± 5.87
2	Positive Control	167.1± 12.6
3	Dose A	125 ±3 7.6
4	Dose B	103 ±4.56
5	Standard	98.0 ±7.68

 

Table – 3 : Results of AST

S.NO	Groups	AST (IU/L)
1	Negative Control	98.21± 1.73
2	Positive Control	228.8± 4.18
3	Dose A	159.5± 2.615
4	Dose B	140.0± 1.467
5	Standard	132.0 ±1.71

 

Table – 4: Results of ALP

S.No.	Groups	ALP (IU/L)
1	Negative Control	102.4± 10.6
2	Positive Control	254.2 ±11.49
3	Dose A	179.3 ±7.6
4	Dose B	142.7± 1.83
5	Standard	139.3± 5.66

 

Fig.01   Comparison of ALT levels against different groups

 

 

 Fig.02   Comparison of AST levels against different groups

 

 

Fig.03   Comparison of ALP levels against different groups

Histopathological Study

 

Fig.04  Group I (Normal control) Section of liver with normal cell structure

 

 

Fig.05  Group II (Paracetamol) Section of liver showing centrilobular necrosis

 

 

Fig.06  Group III (Borreria hispida 200mg/kg)

 

Summary of Histopathology

·        In control animals, no pathological abnormality was seen in any animal

·        In Paracetamol induced animals, mild to moderate centrilobular necrosis was seen

·        Group 3 animals were showing centrilobular necrosis  of minimal Severity (Fig.06)

·        Group 4 animals were showing near to normal pathology of control animal (Fig.07)

·        Group 3&4 animals are compared to the Group - 2 but occurrence and severity were less in these groups.

 

 

Fig.07   Group IV (Borreria hispida 400mg/kg)

 

 

Fig.08   Group V (Standard-Liv - 52)

 

DISCUSSION:

Paracetamol is a common analgesic and antipyretic drug. Several studies have demonstrated the induction of hepatocellular damage or necrosis by paracetamol higher doses in experimental animals and humans⁸. Due to the presence of Flavonoids in the methanolic extract of Borreria hispida it can be said that it has showed the Hepatoprotective activity and may be responsible for the decrease of the elevated parameters⁹.

 

CONCLUSION:

Animals of group 2 which were treated with high dose of paracetamol have exhibited prominent hepatotoxicity effect. The groups (3&4) treated with 200mg & 400mg/kg of methanolic extract of Borreria hispida  along with paracetamol, has shown the reversal effect of hepatotoxicity which is in line with the standard Group 5. Group 3, 4, 5 were found comparable to that of the control group. Therefore it can be said that the Borreria hispida extract may be able to reduce the hepatotoxicity effect of paracetamol.

 

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Received on 31.10.2012       Modified on 23.11.2012

Accepted on 18.12.2012      © RJPT All right reserved