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 Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 1  |  Issue : 2  |  Page : 85-91

Evaluation of hepatoprotective and nephroprotective activity of aqueous extract of Vigna mungo (Linn.) Hepper on rifampicin-induced toxicity in albino rats


Department of Pharmacology, HKES`s MTR Institute of Pharmaceutical Sciences, Sedam Road Gulbarga-585105, RGUHS Karnataka, Bangalore, India

Date of Web Publication27-Sep-2012

Correspondence Address:
M Nitin
Department of Pharmacology, HKE Society`s MTR Institute of Pharmaceutical Sciences, Sedam Road Gulbarga-585105, RGUHS Karnataka, Bangalore
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.101695

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  Abstract 

Aim: The objective of this study was to investigate the hepatoprotective and nephroprotective activity of aqueous extract of seeds of Vigna mungo (AEVM) (fabaceae) against rifampicin-induced liver and kidney damage in rats. Settings and Design: Albino rats of either sex (200-250 g) were selected and divided in to eight groups of six animals in each. Four groups for hepatoprotective activity and four groups for nephroprotective activity. Group 1 was normal control, group 2 was positive control, group 3 was treated with standard drug, group 4 was treated with AEVM. Similarly it was done for nephroprotective activity. The results are evidenced on the basis of physical, biochemical, histological, and functional parameters. Materials and Methods: Drugs used are rifampicin, silymarin, diagnostic kits (SGPT, SGOT, ALP, and BIT) for hepatoprotective activity. BUN, serum creatinine, and serum uric acid for nephroprotective activity. Seed powder of Vigna mungo was extracted with water. Preliminary phytochemical tests were done to identify the phytoconstituents. The hepatoprotective and nephroprotective activity of the AEVM were assessed in rifampicin-induced hepatotoxic and nephrotoxic rats. Statistical Analysis Used: One-way analysis of variance (ANOVA) followed by "Tukey-Kramer" multiple comparison tests. Results: The AEVM showed the presence of amino acids, alkaloids, carbohydrates, flavonoids, glycosides, proteins, phytic acid, total phenolic compounds, saponins, and tannins. Rifampicin produced significant changes in physical (increased liver weight, decreased body weight), biochemical (increase in serum glutathione pyruvate transaminase (SGPT), oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP) and total bilirubin (BIT) level, increase in blood urea nitrogen (BUN), serum creatinine, and serum uric acid level), histological (damage to hepatocytes, nephrons), and functional (barbiturates-induce sleeping time) induced by rifampicin in liver and kidney parameters, respectively. Pretreatment with AEVM significantly prevented the physical, biochemical, and histological changes induced by rifampicin in the liver and kidney, respectively. Conclusion: The AEVM possessed statistically significant hepatoprotective and nephroprotective activity.

Keywords: Hepatoprotective, nephroprotective, rifampicin, Vigna mungo


How to cite this article:
Nitin M, Ifthekar S Q, Mumtaz M. Evaluation of hepatoprotective and nephroprotective activity of aqueous extract of Vigna mungo (Linn.) Hepper on rifampicin-induced toxicity in albino rats. Int J Health Allied Sci 2012;1:85-91

How to cite this URL:
Nitin M, Ifthekar S Q, Mumtaz M. Evaluation of hepatoprotective and nephroprotective activity of aqueous extract of Vigna mungo (Linn.) Hepper on rifampicin-induced toxicity in albino rats. Int J Health Allied Sci [serial online] 2012 [cited 2024 Mar 29];1:85-91. Available from: https://www.ijhas.in/text.asp?2012/1/2/85/101695


  Introduction Top


Liver, the most versatile but complex internal organ, plays a vital role in metabolic activities in human body. Its importance also lies in its impetus in management of internal environment and biochemical conversion of endogenous and exogenous chemicals to harmless and excretable compounds. Therefore, being a vital organ, its protection has a special status in therapeutics. [1] Liver is the main organ involved in the metabolism of biological toxins and medicinal agents. Such agents are always associated with the disturbance of hepatocytes resulting in generation of reactive oxygen species (ROS). [2] Jaundice and hepatitis are two major hepatic disorders that account for a high death rate. [3]

Kidney is also the important target organ for the toxic effects of drugs xenobiotics and oxidative stress. Oxygen-free radicals have been implicated in several biological processes potentially important in glomerular diseases. [4],[5]

Rifampicin over dosage may result in "red man syndrome" in which brownish orange discoloration of the skin, urine, sweat, feces, tears, and saliva occurs and is proportional to the amount ingested. [6] Rifampicin produces hepatic dysfunction and elevation of liver enzymes and some fatalities have occurred. [7] Rifampicin also reported to produce nephrotoxicity. [8] Acute renal failure. [9] Herbal medicines have recently attracted much attention as alternative medicines useful for treating or preventing life style-related disorders and relatively very little knowledge is available about their mode of action. There has been a growing interest in the analysis of plant products which has stimulated intense research on their potential health benefits. [10] The Vigna mungo seeds are much used in medicine, both internally and externally, in paralysis, rheumatism, and affections of the nervous system, in fever, considered hot and tonic, useful in piles, affections of liver and cough, the seeds considered diuretic, and used in dropsy. [11] It's another species green gram seeds are used in kidney diseases. [11] The seeds are rich in total phenolic compounds, tannins, saponins, flavonoids, carbohydrates, proteins, amino acids, lipids, ascorbic acid, and enzymes. [12] Vigna mungo seeds have been reported for its hepatoprotective activity; [13] antioxidant activity. [14] However, no other models were used for screening of its hepatoprotective activity. And also there is no scientific and methodical investigations so far been reported in literature regarding their actions on kidney.

Several plants containing antioxidant properties exhibited nephroprotective activity against gentamicin and cisplatin [15] and also the drug has been found to be potent diuretic which causes excretion of sodium, potassium, and other metabolites, etc. Therefore, the objective of this study was to evaluate the hepatoprotective and nephroprotective activity against rifampicin-induced toxicities on aqueous extract of seeds of Vigna mungo (Linn.) Hepper.


  Materials and Methods Top


Drugs and chemicals

Rifampicin was obtained as a gift sample from Shreya life science Pvt Ltd. Roorkee. Silymarin was obtained from Micro Labs. Bangalore. The kits for all biochemical estimation were purchased from Pathozyme diagnostics Kagal, Dist. Kolhapur, India. The solvents and other chemicals were obtained from the store of HKES's MTR Institute of pharmaceutical sciences, Gulbargathat were supplied by standard manufacturers.

Plant materials and extraction

The seeds of Vigna mungo (Linn.) Hepper were purchased from the local market of Gulbarga, Karnataka; were authenticated at Pharmacognosy department of HKES's MTR Institute of pharmaceutical sciences, Gulbarga. The seeds are powdered and subjected to maceration process with distilled water for 7 consecutive days with occasional stirring, during this period 1-2 drops of chloroform was added to avoid fungal growth. On 8 th day, this is filtered and the solvent was evaporated at 60°C on a water bath to have thick pasty mass referred to as seed extract, the % yield was 23 g.

Phytochemical screening

Preliminary phytochemical screening of AEVM was carried out as described by Khandelwal. [16]

Animals and housing parameters

Healthy adult albino wistar rats of either sex weighing 200-250 g were used for this study. The normal adult rats of either sex were procured from animal house of H.K.E.S's MTR Institute of pharmaceutical sciences, Gulbarga. Standard environmental conditions such as temperature (26±2°), relative humidity (45-55%) and 12 h light and dark cycle were maintained in the quarantine. Standard pelletized feed and water was allowed ad libitum under hygienic conditions. The animals were habituated to laboratory conditions for 48 h 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/IACE/32/2010-11.

Acute toxicity studies

According to earlier reports, the dose of Vigna mungo seed extract 500 mg/kg body weight p.o. was used as hepatoprotective. [13] As Vigna mungo seeds being used as food can be consumed in daily life therefore the above dose was considered as non-toxic and hence used same dosage in this study.


  Settings and Design Top


Hepatoprotective activity

Hepatoprotective activity study was carried out as described by Agrawal et al.[17] Albino rats of either sex (200-250 g) were selected and divided into four groups of six animals in each.

Group 1: Normal control; received only gum acacia (5 mg/kg p.o.),
Group 2: Positive control; received rifampicin (1 g/kg p.o) every 72 h,
Group 3: Standard; received silymarin (25 mg/kg p.o) after 30 min. rifampicin (1 g/kg p.o.) every 72 h.
Group 4: received AEVM (500 mg/kg p.o.) after 30 min. rifampicin (1 g/kg p.o) every 72 h.

The study was carried out for 10 days. On 11 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 [SGPT, [18] SGOT, [18] ALP, [19] and BIT (Tot. Bilirubin) [20] ]. Immediately after collection of blood the animals were euthanized with an overdosage of ether, livers were removed, washed in saline and the wet weight and volume was determined then transferred into 10% formalin for its histopathological studies. [21]

Nephroprotective activity

Nephroprotective activity study was carried out as described by Shelke et al.[22] Albino rats of either sex (200-250 g) were selected and divided into four groups of six animals each.

Group 1: Normal control; received equivalent volumes of distilled water.
Group 2: Positive control; received rifampicin (1 g/kg p.o.) every 72 h.
Group 3: Standard; received cystone (500 mg/kg p.o) after 30 min. rifampicin (1 g/kg p.o.) every 72 h.
Group 4: received AEVM (500 mg/kg p.o.) after 30 min. rifampicin (1 g/kg p.o) every 72 h.

The study was carried out for 2 weeks. [8] Body weight was noted before and after 2 weeks. On 15 th day all the animals were anaesthetized by overdosage of ether and sacrificed. The blood samples were collected by cardiac puncture method and kidneys were dissected out immediately and transferred into 10% formalin for its histopathological studies. [23] The blood samples were centrifuged at 2500 rpm for 15 min. and then subjected for the estimation of biochemical parameters such as blood urea nitrogen, [24] serum creatinine, [25] and serum uric acid. [20]

Histopathological studies

A histopathological study of both livers and kidneys were performed in histopathology laboratory by consultant histopathologist.

Statistical analysis

The data obtained in the experiment were expressed in terms of mean±SEM. Statistical significance of data was assessed by using one-way analysis of variance (ANOVA). Post-hoc comparisons were done by using "Tukey-Kramer" multiple comparison tests. P values <0.05 were considered significant. The positive control group was compared with the normal control group and all other treatment groups were compared with the positive control group.


  Results Top


Preliminary phytochemical studies revealed the presence of Ascorbic acid, amino acids, carbohydrates, flavonoids, proteins, total phenolic compounds, reducing sugars, tannins, saponins, etc.

Parameters assessed for liver functions

Treatment of rats with rifampicin (group 2) produced an increase in liver weight and volume. Whereas rats pretreated with silymarin and AEVM showed a significant decrease in liver weight and volume compared to the positive 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 positive control [Table 1].
Table 1: Influence of aqueous extract of seeds of Vigna mungo (AEVM) on selected physical and functional parameters in rifampicin-induced hepatotoxic rats

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Rifampicin administration resulted in significant elevation of SGPT, SGOT, ALP, and BIT (Tot. Bilirubin) levels compared to the normal control group. Pretreatment with silymarin and AEVM significantly prevented the biological changes induced by rifampicin [Table 2].
Table 2: Influence of aqueous extract of seeds of Vigna mungo (AEVM) on selected serum biochemical parameters in rifampicin-induced hepatotoxic rats

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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, focal hepatocytes drop out, focal necrosis, and extensive portal traid inflammation. However, silymarin and AEVM-pretreated groups had significantly prevented the above histological changes induced by rifampicin [Figure 1].
Figure 1: Effect of aqueous extract of seeds of Vigna mungo (AEVM) on histopathological examination of rat liver in rifampicin-induced hepatotoxicity, (H and E stained cells). (a) Group 1 (Control): Showing normal histology of rat liver. (b) Group 2 (Positive control): N-Focal Necrosis, PTI-Extensive portal triad inflammation, CVC-Central vein congestion. (c) Group 3 (Standard): CVC-Central vein congestion, RH-Regenerating hepatocytes. (d) Group 4 (AEVM): MCD-Mild central vein dilation, VMI-Very mild inflammation

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Parameters assessed for kidney functions

The body weight of the rats treated with only rifampicin were found to be significantly reduced as compared to normal control group and the groups treated with cystone and AEVM showed significant elevation in body weight compared to the positive control group [Table 3].
Table 3: Influence of aqueous extract of seeds of Vigna mungo (AEVM) on selected physical and serum biochemical parameters in rifampicin-induced nephrotoxic rats

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Biochemical parameters such as blood urea nitrogen, serum creatinine, and serum uric acid were found to be significantly increased in group treated with only rifampicin as compared to the normal control group, on the other hand groups treated with cystone and AEVM showed significantly low values when compared with the positive control group [Table 3].

The normal control group showed normal histology of rat kidney whereas, rifampicin (group 2) showed cortical glomerular, peritubular, blood vessel congestion, and interstitial inflammation. Groups treated with cystone and AEVM were found to reduce such changes in kidney histology induced by rifampicin [Figure 2].
Figure 2: Effect of aqueous extract of seeds of Vigna mungo (AEVM) on histopathological examination of rat kidney in rifampicin-induced nephrototoxicity, (H and E stained cells) (a) Group 1 (Control): Showing normal histology of rat kidney. (b) Group 2 (Positive control): C&I-Congestion and inflammation. (c) Group 3 (Standard): MCC-Mild cortical congestion (d) Group 4 (AEVM): MI&C-Mild inflammation and congestion

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  Discussion Top


Hepatoprotective activity

The liver gets injured by many chemicals and drugs. In the present study rifampicin was selected as a hepatotoxicant to induce liver damage. Rifampicin is a safe, effective, and widely used antituberculic drug; however, an overdosage can induce severe hepatotoxicity in experimental animals and humans. Excessive administration of rifampicin can induce enzymes CYP3A4 and CYP3A7 mRNAs in adult's human hepatocytes in culture. [26] It was evidenced that biotransformation of rifampicin into its active metabolite, 25-desacetyl rifampicin, reduces the drug metabolizing enzymes and specifically binds to RNA polymerase which inhibits the nucleic acid and protein synthesis responsible for hepatotoxicity. [27] The hepatic injury leads to elevation of serum levels of SGPT (ALT), SGOT (AST), ALP, and BIT (Tot. Bilirubin), 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 in to the serum, resulting in increased concentrations. [28] Vigna mungo (L.) Hepper was reported to be used in a variety of disease conditions of liver in Indian traditional system of medicine. [29]

In the present study, rifampicin administration for 10 days resulted in morphological changes such as enlargement of liver, scratches, dark brown coloration, and increased volume. Barbiturates are a class of xenobiotic that are extensively metabolized in the liver, deranged liver function leads to delay in the clearance of barbiturates, resulting in a longer duration of the hypnotic effect. [30] In the present study, administration of thiopentone sodium to rats treated with rifampicin resulted in an increased duration of thiopentone-induced sleeping time. Whereas the AEVM pretreated animals showed similar morphology of livers compared to that of normal control animals that were healthy in appearance and significantly decreased volume and also decreased thiopentone-induced sleeping time, an indirect evidence of their hepatoprotective effect.

Rifampicin administration also produces significantly increased serum enzymes, namely SGPT, SGOT, ALP, and BIT (Tot. Bilirubin). Whereas the AEVM-pretreated animals had significantly prevented the elevation of these enzyme levels indicating their hepatoprotective effect against rifampicin-induced liver cell damage.

Histological changes such as loss of lobular architecture, extensive central vein dilation, focal hepatocyte drop out, focal necrosis, and extensive portal traid inflammation were observed in the rifampicin-treated (positive) control group. 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. The extract of Vigna mungo successive water extract showed a significant hepatoprotective effect.

It has been found that AEVM could prevent rifampicin-induced biochemical changes of liver toxicity. The hepatoprotective activity of AEVM was monitored by estimating serum SGPT, SGOT, ALP, BIT (Tot. Bilirubin), which gives a good idea about the functional state of the liver.The increase in the levels of serum bilirubin reflected the level of jaundice, severity of hepatic necrosis and ALP was the clear indications of cellular leakage and loss of functional integrity of cell membrane.

Rifampicin was found to produce hepatotoxicity. [7] In the present study, the evaluation has been carried out for extract of Vigna mungo. This ability of AEVM to protect the liver from rifampicin-induced damage might be attributed to this ability to restore the activity of antioxidative enzymes. Our experiment showed the successive AEVM posses the good hepatoprotective activity.

Nephroprotective activity

Various environmental toxicants and clinically useful drugs, like acetaminophen and gentamicin, can cause severe organ toxicities through the metabolic activation to highly reactive free radicals including the superoxides and oxygen reactive species. [31] A relationship between oxidative stress and nephrotoxicity has been well demonstrated in many experimental animal models. Administration of vitamin E significantly reduced the nephrotoxic symptoms produced by adriamycin. [32] In recent times, there has been an upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in reducing tissue injuries caused by free radicals. [33] It has been proved that Pedalium murex has a diuretic and antioxidant activity and possesses a nephroprotective activity against cisplatin-induced nephrotoxicity. [22] Phenolic compounds function as high-level antioxidants because they possess the ability to absorb and neutralize free radicals as well as quench reactive oxygen species. Plant flavonoids which show an antioxidant activity in vitro also function as antioxidants in vivo. Again, a strong relationship between the total phenolic content and antioxidant activity in fruits, vegetables, grain products, and plant subjects of ethnopharmacological treatments has also been reported. [34] Flavonoids, tannins, and saponins have been reported to exert profound in vitro and in vivo stabilizing effect on the lysosomes of experimental animals. [35] Tannins and saponins stabilize the erythrocyte membrane by binding cations and other biomolecules. [36] It has been reported that Vigna mungo seeds possess an antioxidant activity [14] and diuretic activity because of the presence of saponins in Vigna species. [37]

In the present study, rifampicin administration for 14 days significantly decreased body weight however increased serum enzymes such as blood urea nitrogen, serum creatinine, and serum uric acid, whereas AEVM pretreated animals had significantly increased body weight and also prevented the elevation of these enzyme levels compared to the positive control group and hence indicating their nephroprotective effect against rifampicin-induced kidney damage.

Histological changes such as cortical glomerular, peritubular blood vessels congestion, and interstitial inflammation were observed in the rifampicin-treated (positive) control group. The AEVM pretreated animals had significantly prevented these histological changes, further indicating their nephroprotective activity. All the histological changes observed were in correlation with the physical and biological parameters of the kidney.

From the above reports, the Vigna mungo seeds possess antioxidant and diuretic activity. And it has been proved that P. murex has a diuretic and antioxidant activity possesses a nephroprotective activity against cisplatin induced nephrotoxicity. [22] Our results are parallel to this report. From the above experimental study, we conclude that the AEVM produces adequate nephroprotective activity on albino wistar rats. It showed potent nephroprotective activity.


  Conclusion Top


Hepatoprotective and nephroprotective activity of the AEVM was studied. In the present study, the AEVM produced adequate hepatoprotective and nephroprotective activity on albino wistar rats as evidenced by physical, biochemical, histological, and functional parameters of liver and kidney, respectively.

The hepatoprotective and nephroprotective activity may be due to the presence of strong antioxidants such as total phenolic compounds, phytic acid, tannins, and flavonoids. And the potent diuretic such as saponins in the extract, which causes excretion of sodium, potassium, drug metabolites toxins, etc. thereby protecting the liver and kidneys from toxic effects of rifampicin.

From the above studies it can be concluded that aqueous extract of seeds of Vigna mungo (Linn.) Hepper (AEVM) possess a hepatoprotective and nephroprotective activity against rifampicin-induced hepatotoxicity and nephrotoxicity, respectively.

 
  References Top

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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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