Protective effects of Allium paradoxum against gentamicin-induced nephrotoxicity in mice

Abstract

Allium paradoxum (M.Bieb.) G. Don is a locally known vegetable and is utilized for preparation a variety of local foods in northern of Iran. This study was conducted to quantitatively evaluate the recovery effects of A. paradoxum on gentamicin-induced nephrotoxicity in mice. The curative effect of extracts from the aerial parts and bulbs of A. paradoxum against gentamicin-induced renotoxicity in mice was determined. Both extracts at the dose 200 mg kg⁻¹ day⁻¹ offered nephroprotective effect by change in the blood urea nitrogen and creatinine. No statistical difference could be obtained in serum creatinine and blood urea nitrogen of bulb-treated mice compared to normal mice. The result show that bulb extract at 200 mg kg⁻¹ day⁻¹ has better activity than aerial parts.

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Introduction

For many years, aminoglycoside antibiotics such as gentamicin have been used for treatment of gram-negative infections.¹ Renal dysfunction is one of the most important side effects of aminoglycoside antibiotics, that may occur in 10–20% of patients.² The mechanism of gentamicin-induced renotoxicity is now unclear. It is caused by direct tubular necrosis that is usually localized in proximal tubules.³ Numerous reports show that oxidative stress caused by oxygenate free radicals has an important role in gentamicin-mediated renotoxicity. Antioxidant administration shows a protective role against gentamicin-induced rhinopathy.³ Plants are a rich source of antioxidant compounds.⁴ Among them, many attentions have been paid to endemic and edible species. The genus Allium (Alliaceae family) contains more than 700 species, but just a few Allium species have been shown so far as vegetables and food additives.⁵Allium paradoxum (M.Bieb.) G. Don is known as a wild edible vegetable in northern Iran. It is locally called alezi and is used for preparation of several local foods in northern Iran, especially in Mazandaran province.⁶ This plant has a limited distribution in Caucasus and Hyrcanian area, northern Iran, with a preferred forest and shady habitats.⁷ Previously studies reports cysteine sulfoxides, alliinase activity and antioxidant activity of A. paradoxum.^4,5 Because of its good antioxidant potential, its nephroprotective activity needs to be investigated using gentamicin-induced nephrotoxicity. The goal of this study was to determine the renoprotective effect of the aerial parts and bulb extracts of A. paradoxum.

Materials and methods

Plant materials and preparation of extract

Allium paradoxum aerial parts and bulbs were collected from lowland to submountain forest areas of Semeskandeh protected area, Sari (Mazandaran), Iran, and identified using Flora Iranica.⁷ A voucher (No. 1512) has been deposited in herbarium of University of Mazandaran, Babolsar, Iran. The plant materials were dried at 25 °C for 2 weeks and then in oven (35 °C for 2 days). Dried plant materials were coarsely ground (2–3 mm) before extraction procedures. Allium paradoxum aerial parts and bulbs were extracted using methanol/water (80/20 w/w) by percolation method for 24 h at 25 °C. Extracts were filtered and concentrated under reduced pressure at 40 °C using a rotary evaporator until crude extracts were obtained which were then freeze-dried for complete solvent removal. The yields were 15.5 and 27% for bulbs and aerial parts, respectively.

Animals

The present study was performed on male NMRI mice of approximately the same age group and body weight (2–3 weeks; 20–25 g), housed in ventilated animal rooms at a temperature of 24 ± 2 °C with a 12/12 h light/dark cycle and 60 ± 5% humidity. Animals were fed with standard animal diets, manufactured by Pasture institute of Iran, Tehran, Iran. Water was provided ad libitum. All of the experiments were performed according to the norms of the Ethical Committee of University of Mazandaran.

Renoprotective activity

Animals were divided in to the six groups which containing 10 mice. Group I, served as normal group which received isotonic normal saline intraperitoneally. Group II received 100 mg kg⁻¹ day⁻¹gentamicin (intraperitoneally) for 8 consecutive days and served as control group. Group III and IV, received 100 mg kg⁻¹ day⁻¹gentamicin (intraperitoneally) for 8 consecutive days and 200 mg kg⁻¹ day⁻¹ each extracts (intraperitoneally) for 10 consecutive days.⁸ At the end of animal treatments, animals were anesthetized with ketamine (60 mg kg⁻¹) and xylazine (5 mg kg⁻¹) given intraperitoneally. Blood samples were collected from cardiac puncture. The serums were rapidly separated and processed for evaluation of blood urea nitrogen (BUN) and creatinine using commercially available kits (Ziest Chem. Diagnostic kit, Tehran, Iran).

Statistical analysis

The values are presented as means ± SD. Differences between group means were estimated using a one-way ANOVA followed by Duncan's multiple range test. Results were considered statistically significant when p < 0.05.

Results and discussion

Renoprotective effect of A. paradoxum bulbs and aerial parts extracts against gentamicin-induced renal injury have been showed in Table 1. Gentamicin (100 mg kg⁻¹i.p.) when injected for 8 consecutive days caused marked nephrotoxicity showing significant (P < 0.001) increase in serum creatinine (45.03%), and blood urea nitrogen (100.3%) as compared to normal control animals. The extract of A. paradoxum aerial parts-treated mice differed from normal control mice by an elevated concentration of serum creatinine (20.00%, P < 0.05), blood urea nitrogen (63.10%, P < 0.01). These parameters were found to be statistically significantly different as compared to normal mice. The extract of A. paradoxum bulbs-treated mice differed from normal control by an elevated concentration of serum creatinine (15.59%, P > 0.05), blood urea nitrogen (8.56%, P > 0.05). No statistical significant difference could be obtained in serum creatinine and blood urea nitrogen of bulbs-treated mice compared to normal mice.

Table 1 Effect of Allium paradoxum on serum creatinine, serum urea, and blood urea nitrogen levels in gentamicin-induced renotoxic mice^a

Groups	Serum creatinine/mg dl^−1	Blood urea nitrogen/mg dl^−1
a Values are mean ± SD (n = 10). Data for normal animals are considered as base-line data; there was no significant base-line difference between the groups. Percentage increase (in parentheses) is calculated with reference to normal control. b P > 0.05 versus control group. c P < 0.05 versus control group. d P < 0.001 versus control group.
Normal	0.272 ± 0.014	27.45 ± 2.89
Control (100 mg kg^−1, i.p.)	0.394 ± 0.028^d(45.03)	54.99 ± 1.31^d(100.3)
Bulbs extract-treated (200 mg kg^−1, i.p.)	0.314 ± 0.007^b(15.59)	29.80 ± 0.87^b(8.56)
Aerial parts extract-treated (200 mg kg^−1, i.p.)	0.338 ±.0.021^c(24.40)	44.77 ± 1.2^d(63.10)

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The estimation of glomerular filtration rate (GFR) is best index for assessment of renal function. On the other hand, it is almost difficult to estimate GFR, so most of clinicians estimate the GFR from creatinine concentration. In the glomerulus, serum creatinine is freely filtered, not reabsorbed and so is gold standard for GFR.⁹

The renotoxic effects induced by gentamicin were significantly ameliorated by the bulb extracts, adding further evidence that A. paradoxum has the potential to be used to protect against gentamicin-induced renotoxicity. Oxygenate free radicals such as hydroxyl radicals have an important role in the initiation and progression of gentamicin-induced renotoxicity.^10,11

Aminoglycoside-iron complexes and their related oxidative stress caused by Fenton's reaction have been known as the major mechanisms in the progression of gentamicin -induced acute renal failure.¹² Previously, Walker and Shah¹³ demonstrated that gentamicin increases the production of hydrogen peroxide by renal cortical mitochondria and metal chelators and hydroxyl-radical scavengers have ameliorating role against gentamicin-induced renotoxicity.¹⁴ On the other hand, important role of peroxynitrite and nitric oxide in the renal dysfunction has been reported¹⁵ especially in gentamicin-induced acute renotoxicity. Previous studies demonstrated that nitric oxide and peroxynitrite scavengers are better than Inducible nitric oxide synthase inhibitors as a curative mediation.¹⁶ Previously, we reported good antioxidant and chelating activity from extracts of A. paradoxum bulbs and aerial parts.⁴ So, iron-chelating and free-radical scavenging activity of the extracts may be the possible mechanism of their renoprotective action that may be result of existence of phytochemical compounds such as phenols and flavonoids.

Conclusions

The tested extracts show good activity in recovery of gentamicin-induced renotoxicity. These results can be useful as a starting point for further applications of this plant or its constituents in pharmaceutical preparations after performing clinical researches.

Acknowledgements

The authors are grateful for financial support from the National Elite's Foundation of Iran (Tehran, Iran) for this study. This paper is dedicated to Seyed Maryam Nabavi and the memory of Seyed Ali Asghar Nabavi.

Notes and references

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