Amelioration of fluoride induced oxidative stress by L. fruit Florürün oluşturduğu oksidatif stresin iyileştirilmesi
L. ile
Rupal A. Vasant, Amaravadi V. R. L. Narasimhacharya Laboratory for Animal Sciences, Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar -388 120, Gujarat, India SUMMARY AIM: The mitigative effects of L. fruit powder as a dietary supplement in fluoride toxicity were investigated with reference to tissue lipid peroxidation and antioxidant metabolism. METHODS: Fluoride toxicity was induced in laboratory rats through drinking water (100 ppm sodium fluoride) and the rats were fed diet supplemented with mango fruit powder with three different doses (2.5, 5 and 10 g %) for 30 days. RESULT: Exposure to fluoride resulted in significant elevation of hepatic and renal tissue lipid peroxidation with a reduction in antioxidant profiles. Administration of mango fruit powder reduced both hepatic and renal tissue lipid peroxidation, with a significant increase in antioxidant profiles (TAA, SOD, CAT, GSH and GPX). CONCLUSION: fruit possesses considerable antiperoxidative and antioxidant potential to mitigate the fluoride toxicity. Key words: Antioxidants, fluoride, mango, oxidative stress, lipid peroxidation. ÖZET GİRİŞ: Besin takviyesi olarak L. meyve tozunun Florür toksisitesinde hafifletici etkileri, doku lipit peroksidasyonu ve antioksidan metabolizma baz alınarak araştırılmıştır. YÖNTEM: Florür toksisitesi, içme suyu sayesinde (100 ppm sodyum florür) laboratuar sıçanlarında oluşturuldu ve sıçanlar 30 gün süresince üç farklı dozda (%2.5, %5 ve %10 gram) mango meyve tozu ile takviye olarak beslendi. BULGULAR: Florüre maruz kalım, antioksidan profillerde bir azalma ile birlikte hepatik ve renal lipit peroksidasyonunda belirgin yükselmeye neden oldu. Mango meyve tozunun verilmesi, antioksidan profillerde (TAA, SOD, CAT, GSH ve GPX) belirgin bir artışla birlikte, hepatik ve renal doku lipit peroksidasyonunu azalttı. SONUÇ: meyvesi, florür toksisitesini hafifletmede dikkat çekici antiperoksidatif ve antioksidan potansiyele sahiptir. Anahtar kelimler: Antioksidanlar, florür, mango, oksidatif stres, lipit peroksidasyonu.
Corresponding Author: Dr. A. V. R. L. Narasimhacharya Laboratory for Animal Sciences, Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388 120 Gujarat, India E-mail:
[email protected]
Received October 02, 2011; accepted November 15, 2011 DOI 10.5455/spatula.20111115030048 Published online in ScopeMed (www.scopemed.org). Spatula DD. 2011; 1(4): 181-188.
INTRODUCTION Fluorosis is an endemic health problem in at least 25 nations spanning several continents- Australia, Asia, Africa, North and South Americas. The chief natural source of fluoride in soil is the parent rock itself and virtually, all foodstuffs contain at least trace amounts of fluoride as it is ubiquitous in the environment. Fluoride also exists in natural waters and enters the human and animal bodies through intake and to a lesser extent through food. Besides, the sources of fluoride also include industries, medicaments and cosmetics [1]. Fluorosis is a welldefined clinical disorder generally caused by ingesting excessive amounts of fluoride through potable water, is a serious rural health problem. Fluoride interacts with a wide range of cellular processes such as gene expression, cell cycle, proliferation and migration, respiration, metabolism, ion transport, secretion, endocytosis, apoptosis, necrosis, and oxidative stress [2,3]. Chronic exposure to fluoride is reported to result in the development of cloudy swellings, tubular epithelial degeneration, tissue necrosis, tubular vacuolization, glomerular hypertrophy and interstitial edema in kidneys leading to nephritis [4]. Excessive consumption of fluoride is also known to induce pathological changes in the spleen affecting the hematopoetic progenitor cells [5]. It is well documented that fluoride toxicity causes oxidative stress in reproductive organs leading to impaired reproductive functions [6,7]. Field surveys on endemic fluorotic areas too indicated that fluoride ingestion induces chromosomal aberrations and sister chromatid exchanges in different tissues [8, 9]. Further, it has also been demonstrated that fluoride causes not only oxidative stress but also decreases the antioxidant enzymes, mRNA and protein expression in rat hippocampal neurons [10,11]. L. (F: Anacardiaceae) fruit is well known throughout the world and, in India this fruit is used in preparation of pickles and salads. Dehydrated mango powder replaces tamarind and lime as a spice in Indian foods. The ripe mangoes and their juice are consumed as desserts or as such or the juice is used in preparation of milk shakes or the juice is dried and made into a cake [12]. Mango fruit is considered a cardiotonic, hypotensive, hepato- and gastro-protective agent and is reported to possess antidiabetic, hypolipidemic, antioxidant, anti-viral, antibacterial, anti fungal, anthelminthic, anti parasitic and anti-inflammatory properties [13]. The mango peel extracts have been shown to contain polyphenols and carotenoids and prevent the RBC
membrane spectrin degradation thereby protecting the RBCs from oxidative stress [14]; the flavonoids of the fruit are reported to decrease tissue lipid peroxidation and improve antioxidant profiles [15,16]. The composition of diet plays an important role in management of health in general and the diets incorporating natural ingredients (antioxidants, polyphenols) are gaining importance in treatment of various ailments such as cardiovascular diseases, diabetes, atherosclerosis and oxidative stress etc., [17] to supplement the concurrent therapies. It is widely accepted that diets rich in phytochemicals and nutrients are superior to synthetic drugs due to their minimum or no side effects. Recent reports also indicated the effective use of plants/ plant based products in reducing the oxidative stress caused by long term exposure to fluoride [18-21]. Our earlier work indicated that high protein diets and dietary supplementation normalized the disturbed tissue lipid peroxidation and antioxidant metabolism in fluoride exposed rats [22, 23]. Although fruit is well known for its culinary usage and in medicinal preparations in India, there are no reports on its possible use as a dietary supplement in fluoride toxicity. Therefore, the objective of the present study was to determine the efficacy of fruit powder on tissue lipid peroxidation and antioxidant profiles in fluoride exposed male rats.
MATERIALS AND METHODS fruit powder preparation and analysis The unripe fruits of were purchased from local market; pulp was extracted, air dried, ground to powder and stored in an air tight container. This powder was used at the rate of 2.5, 5 and 10 g per 100 g diet amounting to 2.5, 5 and 10g% supplementation. Phytoconstituents of fruits were analyzed and quantified by standard methods. The fruit powder was extracted in petroleum ether to remove fat and subjected to acid and alkaline treatment and, the fiber content was estimated by gravimetric analysis [24]. The polyphenol content of the fruit was analysed using Folin-Ciocalteu reagent against catechol as a standard. The flavonoid content was estimated using vanillin-sulphuric acid reagent with phloroglucinol as a standard [24]. Phytosterol and saponin contents of fruits were estimated using ferric chloride-sulphuric acid and vanillin-sulphuric acid methods with β-sitosterol and
saponin as standards [25, 26]. The total ascorbic acid content was estimated using 2, 4-dinitrophenyl hydrazine reagent [27]. The FRAP (ferric-reducing ability of plasma) assay of the fruit was performed to measure the concentration of total antioxidants. FRAP was determined in aqueous extracts by the method of Benzie and Strain [28]. The method is based on the measurements of absorption changes that appear when TPTZ-Fe3+ (2, 4, 6-tri-pyridyl-striazine) complex is reduced to TPTZ-Fe2+ form in presence of antioxidants. The results were expressed as the concentration of antioxidants having a ferric reducing ability equivalent to that of 1 mmol/L FeSO4. Animals Colony bred male Albino rats ( ; three mo, 200-260 g bw) housed individually in well-ventilated animal unit (26 ± 2 °C, humidity 62%, and 12-h light/dark cycle) were provided standard diet (Pranav Agro Industries, Vadodara, India) and water . The experiment was conducted following the guidelines of Institutional Animal Ethics Committee (MoEF/CPCSEA/Reg. 337). Experimental protocol After 10-day adaptation period, 30 animals were randomly segregated into 5 groups of 6 animals each as followed: Normal control (NC) - normal animals without any treatment; Fluoride control (FC) - 100 ppm NaF administered through drinking water; FMi 2.5- fluoride administered animals fed fruit powder (2.5 g %); FMi 5 - fluoride administered animals fed fruit powder (5 g %); FMi 10- fluoride administered animals fed fruit powder (10 g %). The composition of the diets used in the present experiment is given in Table 1.
At the end of 30 days period, animals were fasted overnight and sacrificed under mild ether anesthesia. Blood was collected by cardiac puncture; plasma was separated by centrifugation and stored at low temperature. Liver and kidneys were excised and kept frozen until analyzed. Biochemical studies !" " # $ "$ $ % & Malondialdehyde concentration was used as a marker of lipid peroxidation and determined by the thiobarbituric acid reactive substances (TBARS) method [29]. In brief, liver and kidney tissues were (0.25 g in 2.5 ml of PBS- 50mM, pH 7.0) homogenized and centrifuged at 3000 rpm for 10 min; 2 ml of homogenate was treated with 4 ml of TCA (15%)- TBA (0.37%)- HCl (0.25N) reagent and placed in a water bath at 100 ºC for 15 min. The mixture was then centrifuged at 1500 rpm for 6 min and the absorbance of the clear supernatant was measured at 535 nm and expressed as nM TBARS. ! $ !" ! Total ascorbic acid content of the homogenate was measured by the method of Schaffert and Kingsley [27]. Hepatic and renal tissues were homogenized (0.25 g in 2.5 ml of norit reagent), centrifuged at 3000 rpm for 8 min. 1 ml of supernatant was mixed with 250 µl of DNPH reagent (2 % dinitro phenyl hydrazine in 9 N H2SO4) and 8 µl thiourea (10% in 50 % ethyl alcohol) and incubated in water bath at 100º C for 15 min. After cooling the mixture, 1.25 ml of 85 % chilled H2SO4 was added, incubated for 15 min in ice bath. The color developed was read at 515 nm and the concentration of ascorbic acid was expressed as µg/g of tissue.
Table 1: Composition of the experimental diet (g %) NC
FC
F Mi 2.5
F Mi 5
F Mi 10
Moisture
8.75
8.75
8.53
8.31
7.88
Crude protein
22.12
22.12
21.57
21.01
19.91
Crude carbohydrates
55.67
55.67
54.28
52.89
50.10
Crude fat
4.06
4.06
3.96
3.86
3.65
Crude fiber
3.76
3.76
3.67
3.57
3.38
Mineral mixture
5.64
5.64
5.50
5.35
5.08
-
-
2.50
5.0
10.0
fruit powder
!' $( $ Superoxide radicals were generated by the PMS/ NADH system according to the method of Kakkar . [30]. Hepatic and renal tissues were homogenized (0.25 g in 5 ml of 0.9 % KCl) and centrifuged at 3000 rpm for 10 min. Assay mixture was prepared with 1.2 ml of sodium pyrophosphate buffer (52mM; pH 8.3), 100 µl of phenazine methosulphate (188 µM) and 300 µl nitro blue tetrazolium (300 µM). The reaction was initiated by addition of 200 µl of NADH (780 µM). After 90s incubation at 37 ºC, the reaction was stopped by addition of 1 ml of glacial acetic acid. The mixture was stirred vigorously with 4 ml of n-butanol. Color density of the chromogen in the butanol was measured at 560 nm and concentration of SOD was expressed as U/mg of protein. One unit of SOD activity was defined as the enzyme concentration required for 50% inhibition of chromogen production per minute under assay conditions. ) $ ) Catalase activity was measured following the method of Aebi [31]. The homogenate was made in PBS as mentioned before and the 250 µl clear supernatant was added to the cuvette containing 1.8 ml of phosphate buffer (50 mM; pH 7.0). Reaction was started by addition of freshly prepared H2O2 (30mM in phosphate buffer). The rate of decomposition of H2O2 (nM H2O2 decomposed s-1 g1 ) was measured at 240 nm. * ! * + Assay of non-enzymatic antioxidant glutathione was determined according to the method of Jollow [32]. Briefly, 0.1 g of liver and kidney tissues were homogenized in 1 ml of phosphate buffer (0.1 M, pH 7.4), centrifuged at 3000 rpm for 6 min. The reaction mixture contained 200 µl supernatant, 800 µl of distilled water and 500 µl of sulphosalicylic acid (10%). After vortexing for 5 min in cyclomixer; the preparation was kept in the refrigerator for 5 min; centrifuged at 3000 rpm for 10 min. 500 µl of clear supernatant was treated with 4.5 ml of tris buffer (0.5 M; pH 8.23) and 500 µl of DTNB (10mM, 5, 5’- dithiobis-2-nitrobenzoic acid) and the color intensity was read at 412 nm. The concentration of GSH was expressed as µg/g of tissue. * ! GPx activity method of Flohe homogenate was
!' $ *,' was determined following the and Gunzler [33]. The tissue made in phosphate buffer as
mentioned above. Reaction mixture was prepared by adding 300 µl of phosphate buffer (0.1 M; pH 7.4), 100 µl sodium azide (10mM), 200 µl glutathione (2mM), 300 µl supernatant and 100 µl of H2O2 (1mM). The preparation was then incubated for 3 min at room temperature; 500 µl of 5 % TCA was added to the mixture, after mixing well it was kept for 5 min and then centrifuged at 3000 rpm for 10 min. A 100 µl of supernatant was added to the cuvette containing 700 µl of phosphate buffer (0.1 M; pH 7.4) and 700 µl of DTNB (10mM) and then immediately the color intensity was measured at 412 nm. The GPx activity was calculated using 6.22 ×103 M-1 cm-1 molar extinction coefficient and results were expressed as U/mg of protein. !' !$ .& , # FRAP assay was measured according to the procedure described by Benzie and Strain [28]. The FRAP reagent contained 2.5 ml of 10mM TPTZ2,4, 6-Tri (2´-pyridyl) 1,3,5-triazine in 40mM of HCl, 2.5 ml of 20mM FeCl3·6H2O and 25 ml of acetate buffer (pH 3.6). In short, 900 µl of FRAP reagent was mixed with 20 µl of sample, standard (ascorbic/gallic acid: 10mg %) or reagent blank. The mixture was then incubated at 37 ºC for 10 min; the absorbance was read at 593 nm. The results were expressed as µM of antioxidant activity equivalent to ascorbic acid or gallic acid per liter of plasma. Statistical Evaluation Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) with Tukey’s significant difference post hoc test was used to compare differences among groups. Data were statistically analyzed using Graph Pad Prism 3.0 statistical software. P values
