Nephroprotective activity of Vigna mungo (Linn.) Hepper on gentamicin-induced renal damage in albino rats.

Nitin M.¹, Ifthekar S.²*

Department of Pharmacology, H.K.E Society’s College of pharmacy, Sedam road, Gulbarga – 585105, Karnataka, India

ABSTRACT:

The present study was undertaken to investigate the preliminary phytochemical studies and nephroprotective activity of aqueous and methanolic extracts of seeds of Vigna mungo in Gentamicin-induced nephrotoxicity in rats. The seed powder of Vigna mungo was successively extracted with methanol and water. Preliminary phytochemical tests were done, the extracts showed the presence of amino acids, alkaloids, ascorbic acid, carbohydrates, flavonoids, glycosides, proteins, phytic acid, total phenolic compounds, reducing sugars, saponins and tannins. The protective activity of the extracts was justified by physical (decrease in body weight) and biochemical (increased blood urea nitrogen, serum creatinine, serum uric acid and decreased serum total protein) changes induced by gentamicin in kidney parameters. When compared to normal control group. The groups treated with both the extracts had significantly prevented the above changes produced by gentamicin in rats. In general both the extracts possessed protective activity though methanolic extract was found to exhibit greater protection.

KEYWORDS: Gentamicin, Nephroprotective Activity, Vigna mungo, Phytochemistry.

INTRODUCTION:

Kidney is one of the important targets 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.^1,2 Gentamicin is an extensively used aminoglycoside antibiotic for the treatment of gram negative bacterial infections. Nephrotoxicity is the major side effect of aminoglycosides, accounting for 10-15% of all cases of acute renal failure.³Previous studies have demonstrated, an increase in renal cortical lipid peroxidation in gentamicin treated rats and in vitro generation of hydrogen peroxide by renal cortical mitochondria.^4,5It has been reported that the use of hydroxyl scavengers may mitigate gentamicin-induced kidney function in rats.⁶

Vigna mungo (Linn.) Hepper, Family Fabaceae commonly known as black gram (Urid, Mash). The seeds are extensively cultivated all over the India. It has been used for various medicinal purposes in Ayurvedic and Unani systems of medicine. The seeds are sweet, laxative, aphrodisiac, tonic, appetizer, diuretic, galactagogue and styptic; useful in piles, asthma, scabies, leucoderma, gonorrhea, pains, epistaxis, paralysis, rheumatism and affections of the nervous system, liver and cough. It is also prescribed for dropsy and cephalalgia.⁷Oxygen free radicals have been implicated in several biological processes potentially important in glomerular diseases.⁸ The Vigna mungo have been reported for its antioxidant activity.⁹The drug is found to be potent diuretic which causes excretion of sodium and potassium. These observations made us to investigate the plant material for its nephroprotective activity in rats. However there are no scientific and methodical investigations so far been reported in literature regarding their actions on kidney.

Table 1: Preliminary phytochemical screening of aqueous and methanolic extracts of seeds of Vigna mungo.

Sl no.	Tests	AEVM	MEVM	Inference
1	Tests for alkaloids			Alkaloids present in both the extracts
	Dragendorff`s test	++	++
	Mayer`s test	+	+
	Hager`s test	++	+
	Wagner`s test	+	+
2	Tests for carbohydrates and Glycosides			Reducing sugars and glycosides present in aqueous extract. Non-reducing sugars and glycosides present in Methanolic extract
	Molish`s test	++	++
	Fehling`s test	++	-
	Benedict`s test	++	-
	Hydrolyse (Fehling & Benedict)	+	++
	Barfoed`s test	++	+
3	Tests for proteins			Proteins present in both the extracts
	Biuret`s test	++	++
	Million`s	++	++
4	Tests for Amino acids			Amino acids present in both the extracts,aqueous (low) and Tryptophan in methanolic extract
	Ninhydrin test	+	++
	Test for tryptophan	-	++
	Test for cysteine	-	-
5	Tests for Phenolic compounds and tannins			Phenolic compounds and tannins present in both the extracts
	Fecl3 test	++	++
	Lead acetate test	++	++
	Gelatin test	++	++
6	Tests for Flavonoids			Flavonoids present in both extracts aqueous (low)
	Shinoda test	++	++
	Aqueous NaOH	+	++
	Conc. H₂SO₄	+	++
7	Tests for Vitamin C (Ascorbic acid)			Ascorbic acid present in aqueous extract and absent in methanolic ext.
	Sodium nitroprusside test	++	-
	Dichlorophenolindophenol test	++	-

Table 2: Influence of methanolic (MEVM) and aqueous (AEVM) extracts of seeds of Vigna mungo on physical and biochemical parameters against gentamicin-induced nephrotoxicity in rats.

Group	Treatment	Dose	Physical parameter	Biochemical parameters
Group	Treatment	Dose	Change in wt. (g)	BUN (mg/dl)	Serum Creatinine (mg/dl)	Serum Uric acid (mg/dl)	Total Protein (g/dl)
1	Normal control	Equivalent volumes (DW)	8.17±0.70	22.85±1.08	0.54±0.02	2.64±0.20	5.23±0.06
2	GENT control	80mg/kg (GENT)	-13.83±0.60^***	54.20±3.93^***	1.90±0.03^***	7.07±0.18^***	3.50±0.14^***
3	GENT + CYST	80mg/kg + 500mg/kg	4.50±0.43^***	26.90±2.26^***	0.67±0.02^***	2.80±0.11^***	5.57±0.16^***
4	GENT+ AEVM	80mg/kg + 500mg/kg	4.33±0.42^***	28.13±1.50^***	0.66±0.04^***	2.83±0.23^***	5.34±0.21^***
5	GENT+ MEVM	80mg/kg + 500mg/kg	5.67 ±0.33^***	26.45±2.81^***	0.55±0.34^***	2.57±0.20^***	5.71±0.29^***

Values are represented as Mean ± SEM; (n = 6).

^***p<0.001 Gentamicin control Vs Normal control,

^***p<0.001 (GENT + CYST), (GENT + AEVM) and (GENT + MEVM) Vs Gentamicin control.

DW-Distilled water, GENT-Gentamicin, CYST-Cystone tablet, AEVM-Aqueous extract of seeds of Vigna mungo, MEVM- Methanolic extract of seeds of Vigna mungo.

MATERIALS AND METHODS:

Drugs and chemicals: Cystone tablets (Himalaya Cystone), Gentamicin, were purchased from medical shop. The kits for biochemical estimation were purchased from ERBA manufactured by Transasia Bio-medicals LTD. Baddi, Dist: Solan, India (HP). The solvents and other chemicals were obtained from the store of H.K.E. Society`s College of pharmacy Gulbarga that were supplied by standard manufacturers.

Plant material 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 College of Pharmacy, Gulbarga.

For aqueous extract the powdered material was subjected to maceration process with distilled water for 7 consecutive days with occasional stirring. For methanolic extract the seeds were powdered and defatted with petroleum ether and then subjected to successive extraction with methanol (95%) using Soxhlet apparatus for 36 h. After completion of extraction process both the extracts were evaporated at 60^oC on a water bath to have thick pasty mass referred to as seed extracts, the % yield was 23 g. (aqueous extract) and 13 g. (methanolic extract).

Phytochemical screening:

Preliminary phytochemical screening of MEVM was carried out as described by Khandelwal.¹⁰

Animals and housing parameters:

Normal adult albino wistar rats of either sex weighing 200-250 g. were used for this study. The rats were procured from animal house of H.K.E.S`s College of Pharmacy Gulbarga. Standard environmental conditions such as temperature (26±2^o), relative humidity (45-55%) and 12 h light and dark cycle were maintained in the quarantine. Standard pelletised feed and water was allowed ad libitum under hygienic conditions. 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.

Acute toxicity studies:

According to earlier reports the dose of Vigna mungo seed extract 500mg/kg body weight p.o. was used as Hepatoprotective.¹¹ 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.

Figure 1: Influence of aqueous (AEVM) and methanolic (MEVM) extracts of seeds of Vigna mungo on selected physical and serum biochemical parameters in gentamicin-induced nephrotoxicity in rats.

Nephroprotective Activity:

Nephroprotective activity study was carried out as described by Vijay.¹²Albino rats of either sex (200- 250 g) were selected and divided in to five groups of six animals each.

Group 1: Normal control; received equivalent volumes of vehicle (distilled water)

Group 2: Gentamicin control; received Gentamicin (80 mg/kg/day i.p.) for 8 days.

Group 3: Standard; received Cystone (500 mg/kg.p.o) after 30 m. gentamicin (80 mg/kg/day i.p.)

Group 4: received AEVM (500 mg/kg.p.o.) after 30 m. gentamicin (80 mg/kg/day i.p.)

Group 5: received MEVM (500 mg/kg.p.o.) after 30 m. gentamicin (80 mg/kg/day i.p.)

The study was carried out for 8 days, Body weight was noted before and after 8 days. On 9^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.¹³ 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, serum creatinine, serum uric acid and serum total proteins.

Figure 2: Effect of aqueous (AEVM) and methanolic (MEVM) extracts of seeds of Vigna mungo on histopathological examination of rat kidney in gentamicin-induced nephrototoxicity.

a. Group 1 (Normal control): Showing normal

b. Group 2 (Gentamicin control): PC- histology of rat kidney. Peritubular congestion and Inflammation.

Histopathological studies:

Histopathological studies of kidney were performed in Farooqui 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 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 gentamicin control group was compared with the normal control group and all other treatment groups were compared with the gentamicin control group.

c. Group 3 (Standard): CMC-Cortical mild congestion.

d. Group 4 (AEVM): MCON&I-Mild congestion and Inflammation.

e. Group 5 (MEVM): VMC-Very mild congestion.

RESULTS:

Preliminary phytochemical studies revealed the presence of amino acids, alkaloids, ascorbic acid, carbohydrates, flavonoids, glycosides, proteins, phytic acid, phenolic compounds, reducing sugars, saponins and tannins in aqueous extract and the methanolic extract additionally showed the presence of non reducing sugars, tryptophan and higher concentration of amino acids and flavonoids (Table 1).

Parameters assessed for kidney functions:

The body weight of the rats treated with gentamicin were found to be significantly reduced as compared to normal control group and the groups treated with cystone, AEVM and MEVM showed significant elevation in body weight 4.50g, 4.33g and 5.67g respectively (Table 2, Figure 1).

Biochemical parameters such as Blood urea nitrogen, serum creatinine, and serum uric acid were found to be significantly increased and serum total protein was significantly decreased in group treated with gentamicin as compared to the normal control group. Whereas groups treated with cystone, AEVM and MEVM had significantly prevented the above biochemical changes induced by gentamicin compared to gentamicin control group (Table 2, Figure 1).

Histopathological studies:

Normal control group showed the normal histology of rat kidney whereas gentamicin control group showed cortical glomerular, peritubular, blood vessel congestion and interstitial inflammation. Groups treated with cystone, AEVM and MEVM were found to reduce such histological changes produced by gentamicin in rat’s kidney as shown in Figure 2.

DISCUSSION:

Aminoglycoside antibiotics including gentamicin are widely used in the treatment of gram-negative infections. However the major complication of the use of these drugs is nephrotoxicity, accounting for 10-15% of all cases of acute renal failure. The nephrotoxicity of gentamicin is well established in man & experimental animals. Renal tubular cell injury produced by gentamicin evolves subacutely over several days & is characterized by following

· Cellular necrosis

· Large lysosomes & myeloid bodies

· Mitochondrial structural alteration like swollen & ruptured mitochondria,

· Accumulation of gentamicin in renal proximal convoluted tubules.

· Suppression of free radical defence mechanism

· Phospholipidosis

Gentamicin in a dose of 80 mg/kg i.p. has already been shown to produce nephrotoxicity.¹⁴Various 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.¹⁵ 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.¹⁶In gentamicin treated rats, there was a significant increase in lipid peroxidation products (MDA) suggesting the involvement of oxidative stress. A role of lipid peroxidation in gentamicin-induced acute renal failure has been described by evaluating the effect of diphenyl-phenylenediamine and vitamin E.¹⁷ Gentamicin enhances the production of hydrogen peroxide in isolated mitochondria, Pretreatment of rats with hydroxyl radical scavengers protects against gentamicin induced acute renal failure.⁶Phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl, thus inhibiting the oxidative mechanism that lead to degenerative diseases.^18,19

In the present study the group treated with only Gentamicin 80mg/kg body weight i.p. produced nephrotoxicity as evidenced by decrease in body weight, increase in blood urea nitrogen, serum creatinine, serum uric acid and decrease in serum total protein levels as compared to normal control group. On the other hand the groups pretreated with AEVM and MEVM significantly increased body weight, decreased blood urea nitrogen, serum creatinine, serum uric acid and increased serum total protein levels as compared to gentamicin control group. The histological changes such as cortical glomerular, peritubular blood vessel congestion and interstitial inflammation produced by gentamicin were significantly prevented the groups pretreated with AEVM and MEVM. All the histological changes observed were in correlation with the physical and biological parameters of the kidney.

The aqueous and methanolic extracts showed the presence of potent antioxidants such as ascorbic acid, polyphenols like flavonol glycosides, flavonoids, total phenolic compounds, tannins, saponins etc. it has been reported that saponins from Vigna mungo seeds possess diuretic activity.²⁰Antioxidant activity.⁹And it has been proved that pedalium murex has a diuretic and antioxidant activity possesses a nephroprotective activity against cisplatin induced nephrotoxicit.¹⁶

CONCLUSION:

From the above summary it can be concluded that due to the presence of strong antioxidant and potent diuretic effect in the Vigna mungo, the extracts possess a nephroprotective activity. Though methanolic extract showed greater activity than aqueous extract.

ACKNOWLEDGEMENT:

The authors are thankful to authorities of H.K.E.S and MTR institute of pharmaceutical Science for providing the necessary facilities to carry out the work.

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Received on 03.07.2012

Modified on 18.07.2012

Accepted on 27.07.2012

Research J. Pharmacology and Pharmacodynamics. 4(5): September –October, 2012, 299-303