Reagents
All reagents used in this study were purchased from Sigma Chemicals Co. (St. Louis, MO, USA): Alloxan, Folin-Ciocalteu phenol reagent, 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB), L-Glutathione (reduced form) and thiobarbituric acid (TBA). Insulin kit was provided by Gentaur, France.
Laboratory animals and experimental diabetes induction
Swiss mice (supplied by Tunisia Central Pharmacy), weighting about 30 g, were divided into batches of 6 animals. Animals were kept in a breeding farm at 22 °C with a stable hygrometry, under constant (10 h darkness/24 h) photoperiod. They have been fed on 14%-proteins commercial food pellets (Sico, Sfax, Tunisia. Animals were maintained in accordance with the guidelines for animal care approved by the Science Faculty of Sfax (Tunisia).
Mice were injected intraperitoneally with a freshly prepared solution of alloxan monohydrate in sterile normal saline (0.9% sodium chloride) at a dose of 120 mg/kg of body weight [5]. Because alloxan is able to induce fatal hypoglycaemia as a result of massive pancreatic insulin release, mice were treated with 20% glucose solution (5 to 10 mL) orally 6 hours post alloxan administration. The mice were then kept for the next 24 h with 5% glucose solution bottles in their cages to prevent hypoglycaemia [6]. After 2 weeks, mice with moderate diabetes having glycosuria and hyperglycaemia (i.e., with blood glucose levels of 200 to 300 mg/dL) were chosen for the experiment.
Experimental design
Mice were divided into 4 groups: Control group (C), Diabetic mice group (D), Diabetic mice treated with insulin injections and given distilled water as sole beverage group (D+I) and Diabetic mice treated by EGCG group (D+E). (C) and (D) groups received distilled water at the same period. At day 15 eight animals from each group were rapidly sacrificed by decapitation in order to minimize the handling stress. Liver and kidney were rapidly removed, cleared from fat, and frozen at –20 °C until use. Blood plasma was recovered and used for the estimation of glucose levels.
Evaluation of in vitro antioxidant effects
Determination of DPPH radical scavenging activity
The free radical scavenging activity of the EGCG was determined by the 1,1 Diphenyl–2-picrylhydrazyl (DPPH) assay described by Koleva et al. (2002) [7]. Inhibition of free radical DPPH, in percentage was calculated as: Scavenging activity (%) = ([A0 - A1]/A0] × 100.
A0 is absorbance of blank at 517 nm and A1 is the absorbance of the sample in the presence of the extract. IC50 in this test was defined as the concentration of EGCG that was able to inhibit 50% of the total DPPH radicals.
Determination of total antioxidant capacity
The antioxidant activity of the extracts was evaluated by the phosphomolybdenum method following [8]. Extract (0.3 mL) was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 min and cooled to room temperature. Then, the absorbance of the solution was measured at 695 nm using a UV-VIS spectrophotometer (UVmini–1240) against blank after cooling to room temperature. Methanol (0.3 mL) as a control. The total antioxidant activity is expressed as the number of gram equivalent of ascorbic acid. The calibration curve was prepared by dissolving ascorbic (1000; 500; 250; 125; 62.5 and 31.25 µg/mL) in methanol.
Determination of Fe+ chelating activity
The reducing power was determined according to the method of Megías et al. (2009) [9]. Sample (1 mg/mL) was mixed with 1 mL of 200 mM sodium phosphate buffer (pH = 6.6) and 1 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min and then 1 mL of 10% trichloroacetic acid (w/v) was added. The mixture was centrifuged at 2000 rpm for 10 min. The upper layer solution (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of fresh ferric chloride (0.1%). Absorbance was measured at 700 nm; a higher absorbance was taken to indicate a higher reducing power.
Determination of NO• Chelation activity
Nitric oxide was produced by the spontaneous decomposition of sodium nitroprusside (20 mM) in phosphate buffer (pH = 7.4). Once NO is generated, it interacts with oxygen to produce nitrite ions, which were measured by the Griess reaction. The entrapment of nitric oxide (NO •) can be determined by the method described by Shirwaikar et al. (2006) [10]. A solution of sodium nitroprusside (20 mM) was prepared in phosphate buffer (0.5 M, pH = 7.4). The reaction mixture is composed of 2 ml (20 mM) and 250 μl of each extract, and incubated at 25° C for 150 min. After incubation, a volume of 1 ml of each solution was taken and diluted with 1 ml of Griess reagent (1% sulfanilamide, 2% H3PO and 0.1% N–1-anphthyl ethylenediamine). The mixture is again incubated for 30 min at room temperature (25° C), then the absorbance is measured at 546 nm against the blank. Ascorbic acid was used as standard. The percentage of NO trapping of the extract was calculated according to the following formula:
NO• (%) = [(A0 - A1)/ A0] × 100
- A0: absorption of control
- A1: l’absorbance of « EGCG »
Estimation of seric glucose
Seric levels of glucose were determined using commercial diagnostic kit (Biomaghreb, Tunis, Tunisia). The absorbance was read at 620 nm. Glucose was expressed as milligrams per ml of blood.
Anti-diabetic activity of the EGCG
Anti-diabetic activity assay kit (REF 80023, Biolabo-France (Maizy, France)) was used to determine the amylase activity.
Anti-lipidimic activity of the EGCG
Anti-lipidemic activity assay kit (REF 95801, Biolabo-France (Maizy, France)) was used to determine the lipase activity.
Serum biochemical factors
The serum levels of lipids [total triglyceride (TG), total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C)], liver enzymes [aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alanine aminotransferase (ALT), as well as urea and creatinine were measured by commercial kits purchased from Biomérieux Laboratory (Marcy-L’étoile, France) and Beckman Coulter Laboratory (Brea, CA).
Estimation of lipid peroxidation
The level of lipid peroxidation in animal tissues was estimated by measuring thiobarbituric acid reactive species (TBARS) according to Yagi (1976) [11]. About 1g of rat organs, namely liver and kidney, was cut into small pieces and immersed into 2 ml ice-cold lysis Tris-buffered saline (TBS, pH = 7.4). The mixture was then sonicated (10 seconds, twice) and centrifuged (5000xg, 30 min, 4°C). Supernatants were collected and stored at –80°C until use. For the assay, 125 µl of supernatants were homogenized by sonication with 50 µl of TBS, 125 µl of TCA-BHT in order to precipitate proteins and centrifuged (1000xg; 10 min; 4°C). 200 µl of the resulting supernatant were mixed, with 40µl of HCl (0.6M) and 160 µl of TBA (dissolved in Tris), and the mixture heated at 80°C for 10 minutes. The absorbance of the supernatant was then read at 530 nm. The amount of 2-thiobarbituric acid-reactive material (TBA-rm) was calculated using an extinction coefficient of 156 mM–1 cm–1.
Antioxidant enzyme activities
Catalase activity was measured following the procedure of Aebi, 1984 [12]. The reaction mixture (1 mL) contained 100 mM phosphate buffer (pH = 7), 100 m M H2O2and 20µl (about 1–1.5 mg of protein) of liver homogenate. H2O2 decomposition was followed by measuring the decrease in absorbance at 240 nm for 1 minute. The enzyme activity was calculated using an extinction coefficient of 0.043 mM−1 cm−1 and expressed in international units (IU), i.e., in µmoles H2O2 destroyed /mn/mg of protein, at 25°C.
SOD activity was determined by measuring inhibition of photo reduction of NBT [13]. Briefly, one milliliter of supernatant was mixed with 50mM phosphate buffer (pH 7.8), 39mM methionine, 2.6 mM NBT and 2.7 mM EDTA. Riboflavin, at a final concentration of 0.26mM, was added at last and light switched on allowing the initiation of the reaction. Changes in absorbance at 560 nm were, then, recorded for 20 min. The activity was expressed as units/mg protein, at 25°C. In this assay, one unit of SOD is defined as the enzyme amount required to inhibit the photo reduction of NBT by 50%.
Glutathione peroxidase activity was measured according to Paglia (1967) [14]. One millilitre of 50 mM phosphate buffered saline (PBS) (pH 7.4) containing 5 mM Ethylene-diaminetetra-acetic acid (EDTA), 2 mM reduced -nicotinamide adenine dinucleotide phosphate (NADPH), 20 mM glutathione (GSH), 10 mM sodium azide (NaN3), and 23 mU of oxidized glutathione (GSSG) reductase was incubated at 37°C for 5 min. Twenty microliters of 0.25 mM H2O2 solution and 10µl of sample (liver or kidney homogenate diluted 100 times) were added to the assay mixture. A change in absorbance at 340 nm was monitored for 1 minute. A blank control with all the ingredients except the sample was also monitored. The specific activity was calculated as millimoles of NADPH consumed per minute per milligram of protein (i.e. U/mg protein). Proteins were estimated by the method of Lowry et al. (1957) [15] using the bovine serum albumin (BSA) as the standard protein.
GSH Content
In the organ’s homogenate, GSH content was estimated using a colorimetric technique, as mentioned by Ellman (1959) [16]. The assay is based on the development of a yellow color when DTNB [(5,5dithiobis-(2-nitrobenzoic acid)] is added. The absorbance at 412 nm was recorded and total GSH content was expressed as μmol GSH/g of liver or kidney.
Histological analyses
Classical procedures were used for histology. After fixation in Bouin solution, pieces of fixed tissue were embedded into paraffin, cut into 5µm slices and colored with hematoxyline-eosine.
Statistical analysis
For each group, data were presented as means ±SD of 6 animals. To compare between groups, we utilized one-way analysis of variance (ANOVA) followed by Fisher’s protected least significant difference (PLSD) test. Student unpaired t-test, used for comparison between 2 groups, was also utilized. Differences were considered significant at different levels (P <.05, P <.01, P <.001).