Hepatoprotective and Antioxidant Effects of Mucuna Pruriens Against Acetaminophen-Induced Hepatotoxicity in Albino  Wister Rats

 

C. Hari Kumar¹*, Dr. A. Ramesh², Dr. G. Krishna Mohan³

¹Research Scholar, Jawaharlal Nehru Technological University Kakinada, Kakinada, A.P. India.

²Dept. of Pharmacology, Vishnu Institute of Pharmaceutical Education and Research, Narsapur, A.P. India.

³Centre for Pharmaceutical Sciences, JNTU-H, Hyderabad, A.P. India.

 

ABSTRACT:

The objective of the present study was to investigate the antioxidant and hepatoprotective activity of ethanolic seed extract of Mucuna pruriens against paracetamol (PCT) induced hepatotoxicity. The material was dried in shade, they were powdered and extracted with ethanol. Preliminary phytochemical tests were done. The hepatoprotective activity of the ethanol extract was assessed in paracetamol induced hepatotoxic albino wister rats. Paracetamol (3g/kg) has enhanced the levels of various biochemical markers of hepatic damage like SGOT, SGPT, ALP, bilirubin and antioxidant levels were tested in both Paracetamol treated and untreated groups. The various biochemical and Histopathological investigations done were aspartate aminotransferase (SGOT), Alanine Transaminase (SGPT), Alkaline phosphatise (ALP), Bilirubin, antioxidant activity by DPPH (1,1-diphenyl 2-picryl hydrazyl), NBT (Nitro Blue Tetrazolium), Hyderogen peroxide (H2O2), lipid perioxidation, hyderoxil radical and nitric oxide methods.  Treatment of ethanolic extract of M. pruriens seeds (100mg/kg, 200mg/kg and 400mg/kg body weight) has brought back the altered levels of biochemical markers to the near normal levels in the dose dependent manner. Our findings suggested that M. pruriens ethanol root extract possessed antioxidant and hepatoprotective activity.

 

 

INTRODUCTION:

Liver is considered to be the key organ in the metabolism, detoxification, and secretary functions of the body. Hepatic injury is a fundamental pathological process in most chronic hepatic diseases and long- standing hepatic injury leads to hepatic fibrosis, liver cirrhosis, and even hepatocellular carcinoma. Investigations have indicated that some herbal extracts and their chemical constituents can significantly inhibit these aforementioned pathologic processes and protect hepatocytes against the etiologies of chronic hepatic injury.¹ In absence of reliable liver-protective drugs in modern medicine, a large number of medicinal preparations are recommended for the treatment of liver disorders² and quite often claimed to offer significant relief. Attempts are being made globally to get scientific evidences for these traditionally reported herbal drugs.

 

Mucuna pruriens, known as velvet bean or cowitch, which is an annual, climbing shrub with long vines that can reach over 15 m in length. M. pruriens seeds have also been found to have antidepressant properties in cases of depressive neurosis when consumed and formulations of the seed powder have shown promise in the management and treatment of Parkinson disease. But To the best of our knowledge there is no scientific report is available in support of the Hepatoprotective activity of M. pruriens seeds. Therefore, to justify the traditional claims we have assessed the hepatoprotective effect of M. pruriens seeds using paracetamol - intoxicated rats.

 

MATERIALS AND METHODS:

Animals

Wister albino rats of either sex were used for the study of the crude extracts. Institution Animal Ethics Committee has approved the project (831/a/11/CPCSEA). The animals were kept at 27±2°C, relative humidity 44–56% and light and dark cycles of 10 and 14 h, respectively, for 1 week before and during the experiments. Animals were provided with standard diet (Lipton, India) and the food was withdrawn 18 h before the start of the experiment and water ad libitum. All the experiments were performed in the morning according to current guidelines for the care of the laboratory animals and the ethical guidelines for the investigation of experimental pain in conscious animals.³

 

Plant resources and preparation of crude drug extract

The seeds of M. pruriens were collected from Tirupati, Andhra Pradesh, India. And authentication was done by Dr. K. Madhava Chetty, Assistant professor, Department of Botany, Sri Venkateshwara University, Tirupati, Andhra Pradesh. The herbarium specimen has been submitted to Pharmacognosy Department of the College (Voucher specimen no- 002/Hari). Seeds were shade dried and defatted with petroleum ether. The defatted material was extracted with 95% ethanol using soxhlet apparatus and then vacuum dried.

 

Phytochemical studies

All the extracts were subjected for phytochemical study.⁴

 

Acute toxicity studies

The acute toxicity study for ethanolic of M. pruriens seeds were performed using albino rats. The animals were fasted overnight prior to the experiment and maintained under standard conditions. All the extracts were administrated orally in increasing dose and found safe up to dose of 2000 mg/kg for all extracts.

 

Experimental animal and design

The experiment was conducted according to the modified procedures described previously ⁵. PCT was dissolved in 0.5 % CMC for oral administration. Rats were randomly divided into six groups, each consisting of six rats. Group1 served as normal control and was orally given pure water for seven days, and then intraperitoneally injected with 10 ml/kg body weight isotonic 0.9% NaCl. Group 2 served as hepatotoxicity control and was orally given pure water for seven days and then orally intoxicated with 3 g/kg PCT. Group 3 served as standard, and received Standard drug Silymarin 25gm/kg, orally. Group 3, 4 and 5 were treated with the ethanol extract of M. pruriens seeds (with  concentrations of 100, 200 and 400 mg/kg respectively) for seven days. After 24 h of PCT intoxication, the rats were euthanized by ether and then sacrificed. The blood was collected by cardiac puncture in heparinized tubes. The liver was immediately taken out and washed with ice-cold saline. The blood and liver samples were assessed for their biochemical and antioxidant activities, as well as histological observation.

 

Biochemical determinations

The biochemical parameters like serum enzymes: aspartate aminotransferase (AST), serum glutamate pyruvate transaminase (ALT)⁶, serum alkaline phosphatase (ALP)⁷ and total bilirubin⁸ were assayed using assay kits (Span Diagnostic, Surat).

 

DPPH-scavenging activity

The free radical-scavenging activity of the extract was measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH⁹.  Solution of DPPH (0.1 mM) in ethanol was prepared and 1.0mL of this solution was added to 3.0mL of all the extracts solution in water at different concentrations (10–100 µg/mL). Thirty minutes later, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical-scavenging activity. Ascorbic acid was used as a standard drug.

 

Superoxide Radical Scavenging Activity

Each 3ml reaction mixture contained 50 mM sodium phosphate buffer (pH 7.6), 20 μg riboflavin, and 12 mM EDTA and 0.1 mg NBT and 1ml of sample solution. Reaction was started by illuminating the reaction mixture with different concentrations of plant extract and standard ascorbic acid solution viz. 10, 20, 40, 60, 80 and 100 μg/ml for 5min. Immediately after illumination, the absorbance was measured at 590 nm. Identical tubes with reaction mixture and 1ml of methanol were kept in the dark along and served as control. The percentage inhibition of superoxide anion generation was calculated from [(A0–A1)/A0] ×100, where A0 is the absorbance of the control, and A1 is the absorbance of the extract/standard. The antioxidant activity of the extract was expressed as IC50. All the tests were performed in triplicate and the graph was plotted with the average of three observations¹⁰.

 

Scavenging Of Hydrogen Peroxide (H₂O₂):

A solution of hydrogen peroxide (20mM) was prepared in phosphate buffer saline (pH-7.4), various concentrations of extract or standard in methanol (1ml) were added to 2ml of hydrogen peroxide solution in PBS .After 10 min absorbance was measured at 230nm¹¹.

 

Hydroxy Radical Scavenging Activity:

The scavenging activity for hydroxyl radical was measured according to the modified method. The assay was performed by adding 0.1 ml of 10mM Fecl₃, 0.1ml  of  different  dilutions of the extract (10-100mg/ml) dissolved in distilled water ,0.33 ml of phosphate buffer (50nM,Ph-7.4)  and  0.1ml of ascorbic acid in sequence. The  mixture  was  then  incubated at 37 for 1hr.A 1.0 ml portion of the incubated mixture was mixed with 1.0ml  of 10% TCA and 1.0 ml of 0.5%TBA to develop the pink chromogen measured at  532 nm. The hydroxyl radical scavenging activity of the extract is reported as % inhibition of deoxyribose degradation and is calculated ¹².

 

Nitric Oxide Radical Inhibition Assay:

The reaction mixture (6ml) containing sodium nitroprusside (10mM,4mL), and the extractor standard solution (1ml) was incubated at 25 C for 150 min. After incubation, 0.5ml of reaction mixture was removed, 1ml of sulphinalic acid reagent (0.33% in 20% glacial acetic acid) was mixed and allowed to stand for 5 min for completion of diazozitation reaction, 1ml of napthyl ethylene diamine dihydrochloride was added and the mixture was and mixture was allowed to stand for 30 min in diffused light. The absorbance is measured at 540nm¹³.

 

 

Table 1: Effects of extracts on superoxide, H₂O₂, Nitric oxide, and lipid perioxidation scavenging levels:

Group	Regimen (Dose)	SGOT (IU/L)	SGPT (IU/L)	ALP (IU/L)	Bilirubin (IU/L)
I	NORMAL	88.67  ± 1.085	64.83 ± 0.600	70.50 ± 0.763	0.246 ± 0.006
II	PARACETAMOL (25mg/kg)	242.5 ± 2.349	290.5 ± 0.763	209.5 ± 0.75	0.958  ± 0.007
III	STANDARD (25 mg/kg)	105.5 ± 0.763**	85 ± 0.577**	84.50 ± 0.763**	0.295 ± 0.007**
IV	MPS Extract (100 mg/kg)	208.2 ± 0.600	250.5 ± 0.763	186 ± 0.577	0.675 ± 0.09
V	MPS Extract (200 mg/kg)	190.5 ± 0.763**	206.2 ± 0.577**	155 ± 0.577**	0.551 ± 0.010**
VI	MPS Extract (400 mg/kg)	149.8 ± 0.600 **	125.5 ± 0.763 **	109.8 ± 0.945**	0.460 ± 0.004**

N= 6 animals in each group. **P < 0.001 when compared with Paracetamol. Values are expressed as mean SEM.

 

Table 2: Effects of extracts on scavenging activity

S.No	Concentration (µg/ ml)	% Reduction in H2O2 (%)	% Reduction in Lipid Peroxidation (%)	% Reduction in NO (%)	% Reduction in free radicals (%)	% Reduction in DPPH (%)	% Reduction in N.B.T (%)
1	10	80.92	8.8	40.1	15	33.15	12.81
2.	20	83.55	16.11	50.3	25.6	43.41	24.82
3.	40	59.53	19.5	55.1	39.7	51.67	37.18
4.	60	77.49	21.7	65.2	48.71	65.58	50.11
5.	80	83.45	25.7	70.1	53.8	75.94	58.00
6.	100	72.65	29.3	80.01	61.5	86.61	74.11
		IC 50 = 33.59 µg/ml	IC 50 = 24.37µg/ml	IC 50 = 68.75 µg/ml	IC 50 = 49.6 µg/ml	IC50 = 29.6 µg/ml.	IC50  = 53.8 µg/ml

 

 

Lipid Peroxidation Inhibitory Activity

Egg yolk was separated and washed with acetone until the yellow colour is removed the creamy white powder obtained was egg lecithin. Lipid peroxidation was induced by ferric chloride 10ml (400mM) and L-ascorbic acid 10ml (400mM) to a mixture containing egg lecithin (3mg/ml) in phosphate buffer solution and different concentrations of the extracts (100ml). After incubation of 1hr at 37  C the reaction was stopped by adding 2ml of 0.25N hydrochloric acid containing 0.375%w/v thio barbituric acid , boiled for 15 min, cooled, centrifuged and absorbance of supernatant was measured at 532nm¹⁴.

 

Histopathological studies

The liver tissue was dissected out and fixed in 10% formalin, dehydrated in gradual ethanol (50–100%), cleared in xylene, and embedded in paraffin. Sections were prepared and then

stained with hematoxylin and eosin (H–E) dye for photomicroscopic observation,  including cell necrosis, fatty change, hyaline regeneration, ballooning degeneration.

 

Statistical analysis

The data are expressed as mean±S.E.M. The difference among means has been analyzed by one-way ANOVA. A value of P < 0.05 was considered as statistically significant.

 

RESULTS:

Phytochemical study:

All extracts subjected for phytochemical study showed the presence of alkaloids, proteins, amino acids, phenolic compounds, glycosides and flavonoids.

 

Acute toxicity studies

Ethanolic and aqueous extracts did not show any sign and symptoms of toxicity and mortality up to 2000 mg/kg dose.

 

 

Effects of extracts on AST, ALT, ALP and total bilirubin

The results of hepatoprotective effect of extracts on PCT-intoxicated rats are shown in Table 1. The elevated levels of serum AST, ALT, ALP, and total bilirubin were significantly reduced in the animals groups treated with various extracts. Treatment with ethanolic extract showed highly significant activity (P < 0.001) with maximum inhibition. So, the ethanol extract treated group was superior to the other extracts but not as effective as the silymarin. Results are cited in Table 1.

 

Fig No: 1 - Normal	Fig No: 2 - Paracetamol (3g/kg)
Fig No: 3- Standard treated (25 mg/Kg Silymarin)	Fig No: 4- Ethanolic extract of M. pruriens seeds (100 mg)
	7
Fig No: 5- Ethanolic extract of M. pruriens seeds (200mg)	Fig No: 6- Ethanolic extract of M. pruriens seeds (400mg)

 

DPPH-scavenging activity

Table 2 illustrates a significant decrease in the concentration of DPPH radical due to scavenging ability of the extracts. The results indicate that ethanolic extract has the better scavenging activity that was enhanced with increasing concentration. The IC50 of ascorbic acid was found to be 29.6 ± 7.23µg/mL. Results are cited in Table 2.

 

Histopathological observations

Histology of the liver sections of the

Group 1.  Normal: Normal architecture of liver tissue with mild congestion and sensitivity. Fig no: 1

Group 2. Paracetamol (3g/kg) : Ballooning degeneration of hepatocytes with fatty liver tissue areas, indicating acute liver damage. Fig no:2

Group 3. Standard treated (25 mg/Kg Silymarin) : mild peripheral necrosis, less percentage of liver damage in comparison with other groups. Fig no: 3

Group 4. Ethanolic extract of M. pruriens seeds (100 mg): Ballooning degeneration in mid and peripheral zones, Cross linkage is observed. Fig no: 4

Group 5. Ethanolic extract of M. pruriens seeds (200mg): Two parenchyma are coming close. Fig no: 5

Group 6. Ethanolic extract of M. pruriens seeds (400mg): very less percentage of liver damage in comparison with all other groups. Fig no: 6

 

DISCUSSION:

The hepatotoxin is associated with changes at cellular levels that may lead to deterioration of organ functions. Therefore, any improvement in the treatment of hepatic function could be of potentially a great importance. The possible mechanism of herbal preparation as hepatoprotective agent against acetaminophen could be by substantially decreasing lipid peroxidation through the elevation of MDA level in liver homogenate.

 

Generally it is known that most of the acetaminophen is excreted by conjugating with glucuronate and sulphate, while metabolized by cytochrome p-450 system to produce a highly toxic N acetyl- p- benzoquinone- imine (NAPQI) which is readily detoxified by enzymatic conjugation with hepatic glutathione (GSH). But, when the detoxification process is disturbed, an active agent NAPQI is produced which in turn binds covalently to tissue macromolecules thereby causing severe hepatic damage.

 

Oral administration extract of seeds of M. pruriens prepared was standardized and has a significant hepatoprotective activity in preventive treatments against hepatotoxins induced hepatic damage and comparative normalization of serum enzymes against only Hepatotoxin administered one, strongly points out the possibility of seed extract of M. pruriens being able to condition the hepatocytes so as to protect the parenchymal cells¹⁵.

 

CONCLUSION:

Our aim was to develop Hepatoprotective preparation which could be safe with no interactions and beneficial in hepatoprotection, biochemical studies revealed a dose dependent significant fall in the levels of SGOT, SGPT, ALP, Bilirubin , an increase in the weight of liver in case of seed extract treated animals against  acetaminophen induced hepatotoxicity. The free radical scavenging activity was seen in the ethanolic root extracts, when compared with that of standard it was found that the ethanolic root extract has considerable antioxidant activity. Histopathological studies supplemented the findings by showing mild hepatic degeneration with absence of necrosis in comparison with the model control. Thus indicating the prominent significance of seeds of M. pruriens in hepatoprotection against acetaminophen induced hepatotoxicity.

 

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Received on 03.08.2013       Modified on 20.10.2013

Accepted on 01.11.2013      © RJPT All right reserved