Evaluation of therapeutic effect of aqueous extract of stem bark of Mangifera indica Linn. on gentamicin-induced nephrotoxicity

Abstract

The objective of the study was to evaluate the effect of aqueous extract of stem bark of Mangifera indica (M. indica) on gentamicin (GM)-induced nephrotoxicity. A total of 32 male Sprague–Dawley rats were divided in four equal groups. Group 1 was kept as normal healthy control and Group 2 was maintained as GM control in which the animals were intraperitoneally administered GM at 80 mg/kg for 8 days. Groups 3 and 4 were administered with M. indica at 100 mg/kg b.w. and 500 mg/kg b.w., respectively from day 9 to day 30. Nephrotoxicity was evaluated by an increase in the serum creatinine, urea and lipid peroxidation and a decrease in the superoxide dismutase (SOD), catalase (CT) and reduced glutathione (RG) levels. The aqueous extract of stem bark of M. indica at 100 mg/kg and 500 mg/kg reduced (P<0.05) serum creatinine, urea and lipid peroxidation and increased (P<0.05) SOD, CT and RG levels in a dose dependent manner. It reduced the GM-induced changes of serum Na and K upto 15 days. However, the histopathological examination revealed that the aqueous extract of stem bark of M. indica could not provide a complete therapeutic effect in GM-induced nephrotoxicity as evidenced by subcapsular tubular necrosis, dilatation of distal convoluted tubules and hyaline cast formation in the treated groups. To conclude, the results of the present study indicated that the aqueous extract of stem bark of M. indica prevented the GM-induced free radical formation.

Keywords:

• biochemical parameters
• free radical scavenging
• histopathology
• Mangifera indica
• nephrotoxicity

Introduction

Nephrotoxicity is of great concern to kidney since they are routinely exposed to high concentrations of drugs or their metabolites or both (Guo and Nzerue 2002). It has been one of the major side effects of aminoglycosides especially gentamicin (GM), cisplatin, amphotericin B, radiocontrast agents, pentamidine, cocaine and intravenous immunoglobulins. Drugs having direct or indirect antioxidant properties have been identified to prevent GM-induced nephrotoxicity (Balakumar et al. 2010). Mangifera indica Linn. belonging to the family Anacardiaceae has many medicinal properties. The bark is used in the treatment of uterine haemorrhagic ailments (Coe 2008). The major constituent of the stem bark is mangiferin and it has been reported to have radioprotective, immunomodulatory, anti-allergic, anti-inflammatory, antitumour, anti-diabetic, lipolytic, antibone resorption, antibacterial, antifungal, antiparasitic and monoamine oxidase-inhibition activity (Wauthoz et al. 2007). The present study was carried out to evaluate the therapeutic effect of aqueous extract of stem bark of M. indica on GM-induced nephrotoxicity in course of its antioxidant property in male Sprague–Dawley rats.

Materials and methods

The plant materials for the study were collected from the campus of College of Veterinary and Animal Sciences, authenticated by Dr. K.T. Prasanna Kumari, Professor, Department of Plant Breeding and Genetics, College of Horticulture, Kerala Agricultural University and a voucher specimen (No: 2069) was deposited at Department of Pharmacology, College of Veterinary and Animal Sciences. The stem bark of M. indica was air-dried at room temperature and coarsely powdered. A total of 100 g of the powder was mixed with 1 L of distilled water and kept undisturbed for 24 hours. Then they were subjected to boiling for 30 min with constant stirring. The extracts were filtered through a muslin cloth and then kept in a boiling water bath for complete evaporation of water. The yield of the extract was 30.3% on dry matter basis.

Acute toxicity study of the plant extract was conducted using acute toxic class method as per Organisation of Economic Co-operation and Development (OECD) guidelines 423.

The aqueous extract of stem bark of M. indica were tested for the presence of various active chemical constituents namely steroids, alkaloids, tannins, phenolic compounds, flavonoids, glycosides, diterpenes, triterpenes and saponins as per the procedure quoted by Harborne (1991).

GM sulphate (TTK Pharma Limited, Raja Annamalaipuram, Chennai, India) was administered at 80 mg/kg i.p. for 8 days to induce nephrotoxicity. This dose was proved to be nephrotoxic (Vijaykumar et al. 2000).

Thirty two adult male Sprague–Dawley rats weighing 200–250 g were purchased from a Small Animal Breeding Station, College of Veterinary and Animal Sciences (COVAS), Mannuthy, Thrissur, Kerala, India. The animals were maintained as per Committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines for the experimentation of animals. The experimental design was approved by the Institutional Animal Ethics Committee of COVAS, Mannuthy.

The animals were randomly divided into four groups comprising eight animals each. The experiment was conducted for a period of 30 days. Eight rats (Group 1) were retained as healthy control. The rest of the rats were treated with GM sulphate at 80 mg/kg i.p. for 8 days. These rats were either given no treatment (Group 2) or given aqueous extract of stem bark of M. indica at 100 mg/kg (Group 3) or 500 mg/kg (Group 4) p.o. from day 9 to day 30.

A total of 0.2 ml of blood was collected from all the animals from the retro orbital plexus under mild ether anaesthesia on 0, 9th, 15th and 30th day and serum was separated and used for the estimation of biochemical parameters.

Serum creatinine and urea were estimated using the standard enzyme kits from Merck Specialities Pvt. Ltd, India. Sodium (Na) and potassium (K) concentrations in serum were determined by flame photometry using ‘Systronics flame photometer 128’.

On 30th day, the rats were sacrificed and both the kidneys were dissected out, weighed and the right kidney was used for the estimation of antioxidant enzymes-superoxide dismutase (SOD) (Mimami and Yoshikawa 1979), lipid peroxidation (Fraga et al. 1988), catalase (CT) (Cohen et al. 1970). The left kidney was used for the estimation of reduced glutathione (RG) (Ellman 1959) and for conducting histopathological studies..

Kidney samples were processed as described by Sheehan and Hrapchak (1980). The sections were stained with haematoxylin and eosin. The sections were examined in detail under light microscope.

The results of creatinine, urea, sodium and potassium obtained were analysed using analysis of covariance followed by Duncan's multiple range test for comparison between the groups as described by Snedecor and Cochran (1985). The cytoprotective enzymes were analysed using analysis of variance (ANOVA).

Results and discussion

In the oral acute toxicity study, the aqueous extract of M. indica did not produce any signs of toxicity and mortality. Phenolic compounds, tannins, glycosides and diterpenes were detected in the phytochemical screening. The stem bark of M. indica contained polyphenols, triterpenes, flavonoids and phytosterols. Treatment with aqueous extract of M. indica increased (P<0.05) SOD, CT and RG levels and reduced (P<0.05) lipid peroxidation (Table 1). Many investigators have described the role of reactive oxygen species (ROS) including hydroxyl radicals in GM-induced nephrotoxicity (Abdel-Raheem et al. 2009; Khan et al. 2009). The decoction of stem bark of M. indica caused an increase in SOD level in 12-o-tetradecaecanoylphorbol-13-acetate (TPA)-induced oxidative damage in mice (Sanchez et al. 2000). QF808 formulation, prepared as decoction of stem bark of M. indica, was proved to be antioxidant due to its ability to scavenge free radicals involved in microsome lipid peroxidation (Martinez et al. 2001). The results of the antioxidant enzymes suggested that the aqueous extract of stem bark of the M. indica possessed free radical scavenging activity in a dose dependent manner and it may be attributed to its polyphenolic content, mangiferin.

Table 1.  Effect of aqueous extract of stem bark of M. indica on SOD, lipid peroxidation, CT and RG levels.

Group	SOD levels of kidney tissue, units/mg of protein	Lipid peroxidation in kidney tissue, mM/100 mg of tissue	CT levels of kidney tissue, units/assay mixture (250 mg)	RG levels of kidney tissue, mg/100 g of tissue
1	2.494±0.12^a	24.367±0.43^b	0.988±0.02^b	133.525±3.23^d
2	1.612±0.05^a	36.400±0.95^a	0.722±0.01^d	81.337±2.97^c
3	5.914±0.13^b	15.912±0.45^d	2.964±0.02^c	183.825±2.70^b
4	10.821±0.33^c	9.688±0.87^c	5.543±0.03^a	188.487±3.75^a
Note: Means bearing the same superscript letter within the column do not differ significantly at P<0.05. All the values are expressed as Mean±SE.

Serum creatinine and urea are considered as the index of nephrotoxicity (Afzal et al. 2004). A significant reduction (P<0.05) in the serum creatinine and urea values were observed in the treatment groups on day 15 and the mean serum creatinine values of the two treatment groups were also comparable on day 15 and day 30 (Table 2). Similar pattern of observation was noted on serum urea in which the treatment groups showed comparable values on day 15 and day 30 (Table 2). Nephroprotection could be achieved by reduction in serum creatinine and urea levels in GM-induced nephrotoxicity (Singh et al. 2009). The results of the present study also indicated the therapeutic efficacy of the aqueous stem bark extract in GM-induced nephrotoxicity by the reduction of serum creatinine and urea values. Serum Na and K levels did not show a definite trend (Table 3).

Table 2.  Effect of aqueous extract of M. indica on serum creatinine (mg/dl) and urea (mg/dl) levels.

	Creatinine (mg/dl)				Urea (mg/dl)
Group	0 day	9th day	15th day	30th day	0 day	9th day	15th day	30th day
1	0.54±0.05	0.67±0.03^c	0.52±0.04^c	0.58±0.05^b	46.88±1.90	50.25±2.02^c	48.50±2.81^b	50.38±1.73^a
2	0.45±0.03	2.81±0.36^a	2.13±0.23^a	1.56±0.18^a	28.63±1.92	125.63±4.74^b	67.63±2.62^a	56.63±2.54^b
3	0.44±0.04	2.60±0.12^ab	0.89±0.02^b	0.62±0.05^b	31.88±1.88	166.25±2.99^a	41.75±2.27^bc	45.50±2.51^ac
4	0.44±0.06	2.06±0.17^b	0.88±0.03^b	0.61±0.05^b	33.25±1.89	83.75±1.94^d	44.87±2.34^b	43.63±1.59^ac
Note: Means bearing the same superscript letter within the column do not differ significantly at P<0.05. All the values are expressed as Mean±SE.

Table 3.  Effect of aqueous extract of M. indica on serum Na (mEq/L) and serum K (mEq/L) levels.

	Na (mEq/L)				K (mEq/L)
Group	0 day	9th day	15th day	30th day	0 day	9th day	15th day	30th day
1	153.19±2.81	170.86±6.89^a	162.01±1.66^b	148.94±1.54^b	192.60±1.28	149.16±2.43^a	106.87±0.64^a	80.13±0.34^b
2	172.58±2.01	170.52±3.13^a	192.14±2.59^a	166.78±5.52^ab	83.95±0.29	150.32±2.56^a	93.04±0.69^c	84.14±0.32^a
3	162.61±2.51	176.56±3.52^a	151.00±3.10^b	166.19±5.87^ab	99.68±1.64	145.87±2.84^a	108.50±0.84^ab	88.43±0.24^a
4	104.78±2.44	130.03±2.12^a	124.78±3.30^b	169.71±2.29^ab	99.44±2.67	169.54±1.82^a	102.44±0.90^bc	85.15±0.67^a
Note: Means bearing the same superscript letter within the column do not differ significantly at P<0.05. All the values are expressed as Mean±SE.

The microscopic examination of the kidney of Group 1 revealed the usual histological parameters. The tubular structures were largely intact without the presence of any mononuclear infiltrates in the interstitium (Figure 1). GM-induced nephrotoxicity was histologically confirmed by proximal tubular necrosis (Bibu et al. 2010; Jeyanthi and Subramanian 2010). In the present study, in Group 2, predominantly subcapsular extensive proximal tubular necrosis and loss of the lining epithelium were observed. Besides, there were interstitial oedema, perivascular oedema and multiple focal collections of mononuclear cells in the interstitium. The glomerular changes were also quite marked (Figure 2). In Group 3, variable degrees of tubular degeneration, vascular congestion, perivascular oedema and mononuclear cell infiltration and aggregation in the tubulo-interstitial spaces were observed (Figure 3). In Group 4, a few small foci of subcapsular tubular necrosis were observed and some of the tubules of distal convoluted tubules showed varying degrees of dilatation and hyaline cast formation in their lumen (Figure 4). Studies by Dhanarajan et al. have shown that pretreatment with Ebselen in GM-induced nephrotoxicity showed mild tubular necrosis and normal medullary architecture and concluded that the pretreatment ameliorated GM-induced nephrotoxicity (Dhanarajan et al. 2006). In the present study also, aqueous extract of stem bark of M. indica at 500 mg/kg showed moderate therapeutic efficacy in GM-induced nephrotoxicity.

Figure 1.  Histopathological examination of normal group, GM group and aqueous stem bark extract of M. indica treated groups. Group 1 (H&E×100): (A) intact glomerulus; (B) intact proximal convoluted tubules.

Figure 2.  Histopathological examination of normal group, GM group and aqueous stem bark extract of M. indica treated groups. Group 2 (H&E×100): (A) marked glomerular changes; (B) extensive tubular necrosis; C: mononuclear cell infiltration in the interstitium.

Figure 3.  Histopathological examination of normal group, GM group and aqueous stem bark extract of M. indica treated groups. Group 3 (H&E×400): (A) vacuolar changes in proximal convoluted tubules; (B) scattered mononuclear cell infiltration in the cortex.

Figure 4.  Histopathological examination of normal group, GM group and aqueous stem bark extract of M. indica treated groups. Group 4 (H&E×400): (A) mild tubular degeneration and necrosis in the subscapular area; (B) multiple hyaline cast formation in the distal convoluted tubule; (C) pronounced dilatation of distal convoluted tubules.

The results of the present study indicated that the aqueous extract of stem bark of M. indica prevented the GM-induced free radicals but it could not provide a complete therapeutic effect.

Acknowledgements

The authors are thankful to Dr. K.T. Prasanna Kumari for authenticating the plant material. The financial support provided by Kerala Agricultural University is duly acknowledged. The authors are also grateful to the Dean, Faculty of Veterinary and Animal Sciences for providing the facilities for conducting the studies.