The ethanol, solvents and other chemicals were procured from reputed manufacturers.

The seeds of Vigna mungo (Linn.) Hepper were purchased from the local market of Gulbarga, Karnataka; were authenticated at Pharmacognosy department of HKES’s College of Pharmacy, Gulbarga.

For aqueous extract the seeds were powdered and the powdered material was subjected to maceration process with distilled water for 7 consecutive days with occasional stirring, 0.5-1 ml of chloroform was added daily to prevent microbial growth.

Phytochemical screening:

Preliminary phytochemical screening of aqueous extract of seeds of Vigna mungo was carried out as described by Khandelwal.⁷

Experimental 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±5⁰C and 35-60% humidity) standard pelletised feed and tap water were provided ad libitum. The animals were habituated to laboratory conditions for 48 hour prior to the experimental protocol to minimize any nonspecific stress. The Institutional Animal Ethics Committee of H.K.E.S`s College of pharmacy, Gulbarga, India, approved the experimental protocol in accordance with the guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) with registration no. (142/1999 CPCSEA 5 ^TH July 1999). IAEC No. HKECOP/IAEC/32/2010-11.

Acute toxicity studies:

According to earlier reports the dose of Vigna mungo seed extract 500mg/kg body weight p.o. was used as hepatoprotective.⁴ As Vigna mungo seeds are used as food and can be consumed in daily life therefore the above dose was considered as non-toxic and hence used in same dosage in this study.

Hepatoprotective activity:

The albino rats of either sex (200- 250 g) were selected and divided into four groups of six animals. The treatment protocol is summarized and given below.⁸

Group 1- Vehicle control: 2% Gum acacia suspension (5mg/kg) p.o. once daily for 25 days

Group 2- Toxicant control: 40% Ethanol (3.76 g/kg) twice daily, p.o. for 25 days

Group 3- Standard: Silymarin (100 mg/kg) p.o. 30 min later ethanol (3.76 g/kg) p.o. for 25 days

Group 4- AEVM (500 mg/kg) p.o. 30 min later ethanol (3.76 g/kg) p.o. for 25 days

The study was carried out for 25 days. On 26^th day thiopentone sodium (40 mg/kg, i.p.) was injected and the sleeping time was recorded. After complete recovery, the blood samples were collected from all animals by retro orbital puncture method. Serum was separated by centrifugation at 2500 rpm for 15 min. and analyzed for various biochemical parameters such as serum glutathione pyruvate transaminase (SGPT), oxaloacetate transaminase (SGOT), alkaline phosphatase (SALP), and total bilirubin (SBIT). Immediately after collection of blood the animals were euthanized with an over dosage of ether and sacrificed by cervical dislocation. The livers were removed, washed in saline and the wet weight and volume was determined then transferred into 10% formalin for its histopathological studies.⁹

Histopathological studies:

Histopathological study of livers was performed in histopathology Laboratory by consultant histopathologist.

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 “Tukey-Kramer” multiple comparison test. A value of P<0.05 was considered to be statistically significant. The toxicant control group was compared with the normal (vehicle) control group and all other treatment groups were compared with the toxicant control group.

RESULTS:

Preliminary phytochemical studies revealed the presence of ascorbic acid, total phenolic compounds, tannins, saponins, flavonoids, carbohydrates proteins, amino acids and phytic acid.Rats treated with ethanol at a dose of 3.76 g/kg body weight (group 2) significantly (P<0.001) increased the weight of wet liver and volume as compared to vehicle control group. Whereas the rats pretreated with silymarin and aqueous extract of seeds of Vigna mungo (AEVM) significant (P<0.001) decreased wet liver weight and volume compared to toxicant control group [Table 1]. In thiopentone induced sleeping time studies, AEVM also increased onset time (in sec) and decreased duration (in minutes) of sleeping time as compared to ethanol control [Table 1].

Ethanol (3.76 g/kg) administration resulted in significant (P<0.001) elevation of SGPT, SGOT, ALP, and BIT levels compared to vehicle control group. Groups pretreated with silymarin and AEVM significantly (P<0.001) prevented the above biological changes induced by ethanol. [Table 2].

Hepatocytes of the vehicle control group showed a normal histology of the liver. In the ethanol treated group the liver showed loss of lobular architecture, extensive central vein dilation, sinusoidal congestion and inflammation. Silymarin-and AEVM-pretreated groups showed mild central vein dilation, architecture of the liver was maintained, and hepatocytes showed regeneration. [Figure 1]

a. Group 1 (Vehicle control): Showing normal histology of rat liver.

b. Group 2 (Toxicant control): CVC-Central vein congestion, INF-Inflammation.

c. Group 3 (Standard): CandINF- mild Congestion and inflammation.

d. Group 4 (AEVM): MCVD-Mild central vein dilation.

Figure 1: Effect of aqueous extract of seeds of Vigna mungo (AEVM) on histopathological examination of rat liver in ethanol-induced hepatotoxicity

DISCUSSION:

Alcoholic beverages are used universally and alcohol is the world`s most widely used psychoactive drug, but chronic, excessive alcohol consumption leads to permanent organ damage or death.¹⁰ Acute and chronic diseases that affects liver architecture or function markedly affect hepatic metabolism of some drugs, such conditions include alcoholic hepatitis, active or inactive alcoholic cirrhosis, hemochromatosis, chronic active hepatitis, biliary cirrhosis and acute viral or drug-induced hepatitis. Depending on their severity, these conditions may significantly impair hepatic drug-metabolizing enzymes, particularly microsomal oxidases and thereby markedly affect drug elimination.¹¹Recent study indicates that oxidative stress is involved in the pathogenesis of liver diseases including drug induced hepatic damage, alcohol hepatitis and viral hepatitis or ischaemic liver injury.¹² Increased formation of lipoperoxides, conjugated dienes and malondialdehyde and reduced levels of antioxidants like vitamin E and glutathione in the tissues have been demonstrated in experimental animals administered with ethanol as well as in alcoholic human subjects. The increased level of SGPT, SGOT, ALP, and total bilirubin is conventional indicator of liver injury.¹³ Oxidative stress is one major factor in etiology of ethanol injury, mainly by Kupffer cell derived reactive oxygen species (ROS) and Ethanol activates Kupffer cells primarily through the action of a substance called endotoxin, which is released by certain gram-negative bacteria present in the intestine which activates Kupffer cell to generates ROS and pro inflammatory cytokines (TNF alpha, IL 1), both of them can lead to liver damage.¹⁴ The hepatic injury leads to elevation of serum levels of SGPT (ALT), SGOT (AST), ALP, and BIT in rats and are used as markers for assessing toxicant effect and also hepatoprotective agents. During hepatic damage these enzymes present in the liver cells leak into the serum, resulting in increased concentrations.¹⁵

In the present study ethanol administration for 25 days resulted in morphological changes such as enlargement of liver, scratches, dark brown coloration and increased volume. Barbiturates are a class of xenobiotics that are extensively metabolized in the liver. Deranged liver

function leads to delay in the clearance of barbiturates, resulting in a longer duration of hypnotic effect.¹⁶ In the present study, administration of thiopentone sodium to rats treated with ethanol resulted in an increased duration of thiopentone-induced sleeping time. Whereas the Vigna mungo seed extract (AEVM) pretreated animals showed similar morphology of livers compared to that of normal (vehicle) control animals that were healthy in appearance and significantly decreased the weight and volume, and also decreased thiopentone-induced sleeping time, an indirect evidence of their hepatoprotective effect.

Ethanol administration also significantly increased serum marker enzymes such as SGPT, SGOT, ALP and BIT in toxicant control group compared to vehicle control group. Pretreatment with AEVM significantly decreased the enzymes SGPT, SGOT, ALP and BIT levels as compared to toxicant control group. All the above parameters indicate their hepatoprotective effect against ethanol-induced liver cell damage.

Histological changes such as loss of lobular architecture, extensive central vein dilation, sinusoidal congestion and inflammation were observed in ethanol treated (toxicant control) group. The Vigna mungo seed extract (AEVM) pretreated animals had significantly prevented these histological changes, further indicating their hepatoprotective activity. All the histological changes observed were in correlation with the physical, biochemical and functional parameters of the liver.

Ethanol is metabolized largely by sequential hepatic oxidation, first to acetaldehyde by alcohol dehydrogenase (ADH) and then to acetic acid by aldehyde dehydrogenase (ALDH).¹⁷Acetaldehyde is thought to have a number of adverse effects like decreased transport and secretion of proteins owing to tubulin polymerization, enhanced vitamin metabolism and trace metals. Drugs like paracetamol cause severe acute liver injury which is sometimes fatal.^18,19,20 Antioxidants exhibit hepatoprotective activity by blocking the conversion of ethanol to acetaldehyde.²¹

Liver diseases are often associated with edema and ascites in conjunction with elevated portal hydrostatic pressure and reduced plasma oncotic pressure, the mechanisms for retention of sodium by the kidney are complex, they probably involve a combination of factors, including diminished renal perfusion resulting from systemic vascular alteration, diminished plasma volume as a result of ascites formation and diminished oncotic pressure from hypoalbuminemia. In addition, there may be primary sodium retention by the kidney, diuretic therapy is useful to overcome the ascites and edema.²²From the previous studies it was found that Vigna mungo has exhibited diuretic activity.⁶Due to the presence of strong antioxidants like ascorbic acid, total phenolic compounds, tannins, flavonoids etc. and potent diuretic such as saponins in the extract may be responsible for the hepatoprotective activity.

CONCLUSION:

From the above studies it can be concluded that aqueous extract of seeds of Vigna mungo (Linn.) Hepper (AEVM) possesses a hepatoprotective activity against ethanol-induced hepatotoxicity.

ACKNOWLEDGMENT:

The authors are thankful to authorities of H.K.E.S and MTR Institute of Pharmaceutical Sciences, for providing the necessary facilities to carry out the work.

Table 2: Influence of aqueous extract of seeds of Vigna mungo on selected serum biochemical parameters in ethanol-induced hepatotoxic rats.

Group	Treatment	Dose	SGPT(IU/L)	SGOT (IU/L)	ALP(IU/L)	BIT(mg/dL)
1.	Vehicle control	5mg/kg (acacia suspension)	43.05±1.16	62.48±0.98	144.70±1.54	0.61±0.02
2.	Toxicant control	3.76 g/kg (ETH)	144.32±6.23^***	236.62±8.66^***	441.28±9.98^***	2.48±0.29^***
3.	SIL + ETH	100mg/kg + 3.76 g/kg	48.57±2.06^***	70.18±1.65^***	147.05±2.75^***	0.54±0.04^***
4.	AEVM + ETH	500mg/kg + 3.76 g/kg	52.90±0.71^***	78.25±0.69^***	152.08±0.63^***	0.90±0.06^***

Values are expressed as mean ± SEM; n = 6. ^***P<0.001 Toxicant control Vs Vehicle control, ^***P<0.001 (SIL + ETH) and (AEVM + ETH) Vs Toxicant control. ETH-Ethanol, SIL-Silymarin, AEVM-Aqueous extract of seeds of Vigna mung

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Received on 10.04.2012 Modified on 05.05.2012

Research J. Pharm. and Tech. 5(6): June 2012; Page 780-784

Group	Treatment	Dose	Mean liver weight (g/100g)	Mean liver volume (ml/100g)	Thiopentone induced sleeping time
Group	Treatment	Dose	Mean liver weight (g/100g)	Mean liver volume (ml/100g)	Onset (s)	Duration (min)
1.	Vehicle control	5mg/kg (acacia suspension)	3.88±0.07	3.91±0.07	170.50±0.98	85.54±1.35
2.	Toxicant control	3.76 g/kg (ETH)	6.42±0.21^***	6.46±0.20^***	92.40±2.06^***	150.50±1.05^***
3.	SIL + ETH	100mg/kg + 3.76 g/kg	4.05±0.07^***	4.08±0.07^***	160.30±0.98^***	98.18±0.91^***
4.	AEVM + ETH	500mg/kg + 3.76 g/kg	4.14±0.11^***	4.18±0.11^***	155.57±0.81^***	102.09±0.92^***