Nephroprotective Action of Phoenix dactylifera. in Gentamicin-Induced Nephrotoxicity

Abstract

We investigated the effect of an extract of the flesh and pits of dates [Phoenix dactylifera. L (Arecaceae or Palmae)] on gentamicin (GM) nephrotoxicity in rats. The animals were given either the date flesh extract mixed with the food (50% w/w) or the pits extract mixed in the drinking water (2:1 w/v), and GM (80 mg kg⁻¹ day⁻¹ intramuscularly for 6 days) was injected during the last 6 days of treatment. Other groups of rats were given GM concomitantly with the date flesh extract or the date pits extract at the above doses. GM treatment significantly increased the plasma concentrations of creatinine and urea and induced a marked necrosis of the renal proximal tubules. The date flesh and pits were effective in significantly reducing the increases in plasma creatinine and urea concentrations induced by GM nephrotoxicity and ameliorating the proximal tubular damage. Antioxidant components in the date (e.g., melatonin, vitamin E, and ascorbic acid) were suggested to be the basis of the nephroprotection.

Keywords:

• Date palm
• gentamicin
• nephroprotection
• nephrotoxicity
• Phoenix dactylifera. L

Introduction

Gentamicin (GM) is a useful aminoglycoside antibiotic against serious and life-threatening infections caused by Gram-negative and/or Gram-positive bacteria. However, it has been reported that some signs of renal impairment are observed in up to 30% of patients treated with the drug for more than 7 days (Mathew, 1992). Several investigators have studied the factors that may influence GM-induced nephrotoxicity, including dietary components (Ali, 1995). It is established that protein is one of the risk factors associated with GM nephrotoxicity (Whiting, 1988) It has also been reported that iron supplementation in the diet aggravates GM-induced nephrotoxicity in rats (Kays et al., 1992). On the other hand, inclusion of fish oil (Ali & Bashir, 1994), ascorbic acid (Ben Ismail et al., 1994), and vitamin E (AbdelNaim et al., 1999) ameliorates biochemical and histologic signs of GM nephrotoxicity.

In view of its high nutritional content, date flesh has been used as an important dietary item in North Africa, Arabia, Persia, and some other parts of the Old World since ancient times. In some countries, the date pits are used crushed as an animal feed and as a hot drink instead of coffee.

The flesh and pits of dates have recently been investigated for their possible medicinal and nutritional benefits. The scientific basis of their benefit has started to be investigated. Recently, it has been shown that date flesh has a strong in vitro. antioxidant (Vayalil, 2002) and immunostimulant effect (Puri et al., 2000). There is a popular belief in our region that consumption of date flesh each morning helps in antagonizing the actions of toxic materials. The current work is one of a series of studies on the chemical constituents and pharmacological actions of date flesh and pits.

Materials and Methods

Animals

Thirty-six adult male Wistar albino rats weighing between 400 and 450 g were used in this work. They were kept at a controlled temperature of 25 ± 2°C, relative humidity of 60–80%, and a light regimen of 14 h light:10 h dark (lights on at 0600). General procedures for animal care and housing were in accordance with the U.S. Department of Agriculture through the Animal Welfare Act (7USC 2131) 1985 and Animal Welfare Standards incorporated in 9 CFR Part 3, 1991. Pelleted Purina chow and water were given ad libitum..

Plant material, preparation, and administration

The fruit of Phoenix dactylifera. L (Arecaceae or Palmae) was obtained from the date manufacturing plant in Buraydah, Al-Gaseem district, and classified in the Department of Botany, Faculty of Science, King Saud University, Saudi Arabia. Voucher specimens of the plant were deposited in the Department of Veterinary Medicine, Faculty of Agriculture and Veterinary Medicine, Bureidah University, Saudi Arabia. The date flesh was separated from the pits, and the flesh was soaked in distilled water (in the ratio of 1:2 w/v) for 48 h at 4°C. The extract was then thoroughly mixed with Purina chow, as a 50% w/w mixture, and given as a daily supply of food during the experimental period. The pits were washed free of any flesh, air-dried, and powdered. The powder was then soaked in cold distilled water (4°C) for 48 h in a ratio of 2:1 (w/v). The pit extract was given as the only source for drinking.

Toxicity testing

A separate experiment was performed to determine whether any toxic effects were produced by the aqueous extracts. The rats were fasted for 12 h and randomly divided into drug-treated test groups and vehicle-treated control group making up seven groups of six rats per cage. The aqueous flesh and pit date extracts (100, 200, 400, 800, 1600, and 3200 mg/kg body weight) were separately administered orally to the rats in each of the test groups. Each of the rats in the control groups was treated with vehicle alone (DMSO 0.5%; 1 mL/kg body weight). The rats in both the test and control groups were allowed access to food and water, and behavioral changes were observed over a period of 24 h to 21 days for signs of acute or chronic toxicity. The mortality number caused by the extracts within this period of time was observed. Log dose-response plots were constructed for each extract, from which the median lethal dose (LD₅₀) of the extracts was determined (Lorke, 1983).

Treatments

The rats were randomly divided into six equal groups as follows:

• Group 1: The animals were given Purina chow (100% w/w) for 28 consecutive days. During the last six days of the feeding period, normal saline was injected intramuscularly (i.m.) (2 mL kg⁻¹ day⁻¹ for 6 days). Rats were sacrificed 24 h after the last injection (day 29).

• Group 2: Rats were given Purina chow (100% w/w) for 6 days, and were concomitantly injected during this period with GM (80 mg kg⁻¹ day⁻¹ for 6 days). Rats were sacrificed 24 h after the last dose (day 7).

• Group 3: The animals were given Purina chow mixed with the date flesh (50% w/w) for 28 consecutive days. During the last 6 days of the feeding period, GM was injected i.m. (80 mg kg⁻¹ day⁻¹ for 6 days). Rats were sacrificed 24 h after the last dose (day 29).

• Group 4: Rats were given Purina chow mixed with the date flesh (50% w/w) for 6 days. During this period, rats were injected with GM (80 mg kg⁻¹ day⁻¹ for 6 days). Rats were sacrificed 24 h after the last dose (day 7).

• Group 5: The animals were given the aqueous date pits extract as the only source of drinking fluid for 28 consecutive days. During the last 6 days of the treatment period, GM was injected i.m. (80 mg kg⁻¹ day⁻¹ for 6 days). Rats were sacrificed 24 h after the last dose (day 29).

• Group 6: Rats were given the aqueous date pits extract as the only source of drinking fluid for 6 consecutive days, concomitantly with GM (80 mg kg⁻¹ day⁻¹ for 6 days). Rats were sacrificed 24 h after the last dose (day 7).

Injections of GM were made daily at 0800 to minimize the circadian variation in nephrotoxicity (Pariat et al., 1988). At the end of treatment, all animals were rapidly stunned and decapitated. Blood samples were taken from the trunk over heparin, centrifuged (900 × g. for 15 min at 5°C), and the plasma was separated and stored at −5°C until analysis.

Biochemical assay

Plasma creatinine and urea were determined by automated spectrophotometric methods (BM/Hitachi autoanalyzer-911; Boehringer Mannheim, Germany) according to the instructions of the manufacturer.

Drugs and chemicals

Gentamicin sulfate was a gift from the Saudi Pharmaceutical Company (Buraydah, Al Gaseem, Saudi Arabia). The kits were obtained from Boehringer Mannheim.

Statistical analysis

Recorded values are means ± SEM (n = 6). The significance of differences was estimated using the Student's t.-test. p. values less than 0.05 were considered significant.

Results

Oral administration of graded doses of the aqueous extracts of the date flesh and pits to male Wistar rats, in our acute and chronic toxicity study, did not produce any toxicity. Based on this observation, the extracts were considered to be safe in mammals.

The results are shown in Table 1. Treatment with the aqueous extract of the palm flesh resulted in an increase in the body weight of the animals during the treatment period. Pretreatment with the date flesh aqueous extract also induced a prophylactic action on GM-induced nephrotoxicity in the rats as evidenced by a significant decrease in the plasma concentrations of creatinine and urea. The same effect was seen when the palm flesh aqueous extract was given in conjunction with GM, resulting in a curative effect ameliorating the nephrotoxic action of GM.

Table 1 Effect of date flesh and pits aqueous extracts before and after treatments on gentamicin-induced nephrotoxicity changes in creatinine and urea in plasma of rats (n = 6).

Treatment	Body weight (g)	Change in weight (%)	Kidney weight (g)	Kidney weight (% body weight)	Creatinine concentration (mg/dL)	Urea concentration (mg/dL)
Group 1 Olive oil (5 mL/kg) + saline (2 mL/kg)	426.7 ± 0.87^b.	0.07	2.98 ± 0.02^a.	0.70 ± 0.01	0.53 ± 0.016^b.	21.03 ± 0.97 ^b.
Group 2 Olive oil (5 mL/kg) + gentamicin (80 mg/kg)	406.0 ± 4.34^c.	0.05	2.95 ± 0.02^a.	0.71 ± 0.02	1.23 ± 0.057 ^a.	58.60 ± 2.24 ^a.
Group 3 Palm flesh (28 days)	452.17 ± 3.84^a.		2.96 ± 0.02^a.		0.53 ± 0.016 ^b.	21.98 ± 1.65 ^b.
Group 4 Palm flesh (6 days)	456.33 ± 3.30^a.		2.93 ± 0.01^a.		0.59 ± 0.03^b.	22.32 ± 1.62^b.
Group 5 Palm pits (28 days)	426.17 ± 2.02^b.		2.98 ± 0.03^a.		0.56 ± 0.012^b.	23.16 ± 0.84 ^b.
Group 6 Palm pits (6 days)	424.00 ± 0.01^b.		2.97 ± 0.02^a.		0.63 ± 0.04^b.	23.83 ± 0.91^b.

Means having different superscripts are significantly different at p < 0.01.

Treatment with the palm pits did not affect the body weight of the animals. However, it showed a protective role against nephrotoxicity when given before and together with GM.

Discussion

The increase in the body weight of the animals fed with the date flesh aqueous extract could be attributed to the rich constituents seen in the form of sugar contents amounting to 70% of its weight (Fayadh & Al-Shwoiman, 1990).

GM nephrotoxicity is, at least partially, attributed to the generation of free radicals, enhanced lipid peroxidation, and decreased glutathione (GSH), in the kidney (Ramsammy et al., 1985; Walker & Shah, 1988; Ali et al., 1992). It is reasonable, therefore, to suggest that the observed nephroprotective action of the date flesh and pits may be due to an increase in activity of the free radical scavenging enzymes or counteraction of the free radicals. Date flesh and pits have recently been shown to possess strong antioxidant actions (Vayalil, 2002). Some of the constituents of dates have been shown to act as antioxidants. We have recently demonstrated the presence of melatonin, vitamin E, and ascorbic acid in date flesh and pits (AlQarawi et al., unpublished data). Melatonin was discovered to be a direct free radical scavenger (Reiter et al., 2000; Karbownik et al., 2001). The work carried out by AbdelNaim et al. (1999) indicated that the antioxidant activity of vitamin E initiated a potential protective effect against gentamicin-induced nephrotoxicity through its inhibition of lipid peroxidation. Ben Ismail et al. (1994) found that ascorbic acid, in a moderate dose (100 mg/kg), acted as an antioxidant reducing lipid peroxidation and, thus, induced a protective role against the GM-induced nephrotoxicity. It is of interest to point out that a recent investigation carried out by AlQarawi et al. (2005) indicated that the chemical composition of the dates was implicated in a protective role against gastric ulcer through an anti-oxidant action.

In conclusion, this work has shown that date flesh and pits extract possess substances that may have protective action against GM nephrotoxicity in rats.

Acknowledgments

This work was supported by a research grant from the College Research Board. Thanks to Professor S. Al Yahya for his interest and support of this project, and Al Gaseem Date Factory for providing free samples of dates for this study.