The (-)-epigallocatechin gallate (EGCG) ameliorates hyperglycemia and hyperlipidemia - mediated oxidative stress in liver and kidney of alloxan induced diabetic mice


 Background: Diabetes mellitus has become a serious problem associated with health complications, such as metabolism disorders and liver-kidney dysfunctions. The inadequacies associated with conventional medicines have led to a determined search for alternative natural therapeutic agents. The present study is conducted to evaluate the hypoglycemic, antilipidemic and antioxidant effects of EGCG in surviving diabetic mice.Methods: Alloxan diabetic mice were injected with EGCG. Their bloods were collected and submitted to various biochemical measurements, including blood glucose, cholesterol, triglycerides, urea, creatinine and transaminases. Their livers and kidneys were isolated to perform histological analysis.Results: Both EGCG and insulin treatments of diabetic mice resulted in a significant reduction in fasting blood glucose level. EGCG supplementation also ameliorated hepatic as well as renal toxicity indices. Moreover, diabetic mice injected by EGCG exhibited significant changes in antioxidant enzyme activities in the liver and kidney. Histological analyses also showed that it exerted an ameliorative action on these organs and efficiently protected the liver-kidney functions of diabetic mice.Conclusion: The findings revealed that EGCG administration induced attractive curative effects on diabetic mice, particularly in terms of liver-kidney functions. EGCG can, therefore, be considered as a potential strong candidate for future application to treat and prevent diabetes.


Introduction
Diabetes is one of the leading chronic diseases related to oxidative stress. This disease is characterized by chronic hyperglycaemia resulting from a defect in the secretion or action of insulin. It is associated in the long term with macroangiopathic and microangiopathic complications. The classic symptoms of diabetes are defined as: hunger and thirst with increased urine volume, thinness or obesity and risk of coma. Some studies have shown that in the case of type 1 diabetes, oxidative stress causes insular destruction of the pancreas, either by necrosis or apoptosis of the beta cell [1]. Indeed, four main molecular mechanisms have been implicated in this oxidative stress-induced tissue damage: activation of protein kinase C (PKC) isoforms, increased flow of the hexosaminase pathway, glycation. There has been an explosive trend in antidiabetic herbal medicine that is currently experiencing considerable growth because of the discovery of a large number of antidiabetic and antioxidant plant extracts.
In fact, numerous studies have made it possible to isolate bioactive molecules from plants that could protect the body against diabetes mellitus and the associated oxidative stress, namely polyphenols, which are widespread in nature and present in plants. Fruits, vegetables, wine [2] and especially medicinal plants namely walnut, green tea and mallow [3], which are considered among the most important exogenous natural antioxidants. In addition, flavonoids are known for their antioxidant, antibacterial, antidiabetic, antiviral and anti-inflammatory properties [4]. Appreciated for ages, green tea, a widespread drink in Tunisia, from the plant Camellia sinensis, contains a host of active ingredients, including polyphenols, namely catechins, which have attracted the attention of scientists. The scientific community's interest in the beneficial effects of tea is more directed towards the most abundant catechin in green tea, epigallocatechin-3-gallate (EGCG).
The present study was designed to evaluate "in vitro" the antioxidant power of the EGCG molecule through the application of total antioxidant activity, DPPH, FRAP, NO• and the antihyperglycemic and antihyperlipidemic powers of EGCG by studying the activities of α-amylase and lipase. To study "in vivo" the impact of an exposure to alloxan associated or not with EGCG by the assay of the various parameters of the oxidative status and this during 15 days. To study the histopathological profile of the liver and kidney in order to confirm the protective effect of EGCG against the glycaemic status as well as the oxidative and enzymatic processes following the injection of alloxan.

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.
A 0 is absorbance of blank at 517 nm and A 1 is the absorbance of the sample in the presence of the extract. IC 50 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% H 3 PO 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:

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.

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 acidreactive 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 H 2 O 2 and 20µl (about 1-1.5 mg of protein) of liver homogenate. H 2 O 2 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 H 2 O 2 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 reducednicotinamide adenine dinucleotide phosphate (NADPH), 20 mM glutathione (GSH), 10 mM sodium azide (NaN 3 ), and 23 mU of oxidized glutathione (GSSG) reductase was incubated at 37°C for 5 min. Twenty microliters of 0.25 mM H 2 O 2 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 oneway 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).

Results of in vitro antioxidant assays
In this study, the antioxidant activity of EGCG was evaluated in vitro by using four antioxidant tests. The results of in vitro assays showed that EGCG had strong DPPH radical scavenging activity (IC 50 = 4,64 µg/mL), moderate total antioxidant capacity (IC 50 = 811.74 mg AAE g -1 DW) and relatively good ferrous iron chelating activity (IC 50 = 0,645 µg/mL), and strong NO • Chelation activity (IC 50 = 0.12 mg/mL) ( Table 1).

Results of laboratory animals Blood glucose levels in diabetic and treated mice
The blood glucose level in the diabetic group was significantly higher than that in the control group (p <0.001). In diabetic mice treated with EGCG or insulin, about 68% and 82% decrease in glucose levels was observed respectively ( Table 2).

Amylase inhibition assay
Our results show that EGCG has a glucose-lowering effect demonstrated by the decreased amylase activity by 47,7% in the presence of the molecule compared to the control.

Effects of insulin or EGCG on liver and kidney weights
According to the results, alloxan-treated mice exhibited significant liver weight loss over the 2 week period (p <0.05). EGCG and insulin treatment also cause a variation in the liver weight but the difference was not statistically significant ( Table 3).

Effects of insulin or EGCG on liver function
In alloxan-treated animals, the serum levels of AST, ALT, LDH and Bilirubin (p <0.001) were significantly higher than control animals by respectively (137%, 72%, 17% and 70%) and treatment with insulin or EGCG significantly decreased elevation of these enzymes induced by alloxane ( Table 4). Administration of EGCG in normal mice did not significantly change AST, ALT, LDH and Bilirubin levels compared to the control group (Data not schown).

Effects of insulin or EGCG on renal toxicity markers
Serum parameters of kidney function (urea and creatinine) in healthy, diabetic mice were significantly higher than control group ( Table 4). Administration of EGCG significantly inhibited this elevation of renal markers levels induced by alloxan. EGCG showed no statistically significant differences compared to the control group ( Table 4).

Effects of EGCG on lipid profile of alloxan-induced diabetic mice
As shown in table 5, the plasma TG, TC and HDL-C levels increased significantly in diabetic mice as compared to the control group. Administration of EGCG to diabetic mice significantly decreased these levels when compared to the diabetic group (p <0.05, Table 3).

Lipase inhibition assay
Our results show that EGCG has an anti-obesity effect demonstrated by the decreased lipase activity by 30,6% in the presence of the molecule compared to the control.

Effects of insulin or EGCG on oxidative stress parameters
The liver and kidney tissues of alloxane-treated animals showed the significant elevation in the TBARS and NO . levels in comparison with the control group (p <0.001). However, administration of EGCG could significantly inhibit elevation of TBARS (liver and kidney; p <0.01) level induced by alloxane. Furthermore, treatment with EGCG significantly reduced the NO . level in liver and kidney tissues of alloxane-treated mice. Moreover, administration of EGCG by itself did not significantly change the level of TBARS and NO . when compared to the control group (data not showen) (Figure 1).

Effects of insulin or EGCG on GSH level and antioxidant enzymes activities
The significant reduction was observed in the content of GSH and the activity of GPx, SOD and CAT in kidney and liver tissues of diabetic animals when compared to the control group (p <0.001). On treatment with EGCG marked increase was observed in the level of GSH compared to the alloxan-treated group. Results indicated that the GSH content and antioxidant enzymes activities did not significantly change in tissues of normal animals administrated with EGCG (Figure 2 and 3).

Effects of insulin or EGCG on histological changes in liver and kidney tissues
Histological examinations of liver tissues showed that compared to the control group (A), the liver tissue ofalloxane-treated mice (B) showed severe damages which were observable in the form of focal degeneration of hepatocytes, dilation of liver sinusoids and high inflammatory cells diffusion around portal triad areas. (C) Insulin treated diabetic mice also had mild degeneration of hepatic tissues with existence of binucleated hepatocytes which as indication of remedy progress against diabetes induce liver pathology. (D) EGCG treated diabetic mice had normal hepatic architecture, although blood vessels congestion was noticed. Overall, protective effects of EGCG treatment on the mice' liver histopathology were general better than that of insulin mice (Figure 4).
Renal tissue treated with alloxane revealed severe renal injury and all types of kidney cells such as mesangial cells, podocytes and tubulo-interstive cells were affected and glomerular and tubular hypertrophy was also noticeable in addition to tubular dilatation (B'). Insulin treatment of diabetic mice (C') reduced diabetes-induced dilation of the proximal tubules. The group of mice treated with EGCG (D') exhibited normal tubular and glomerular integrity with insignificant cellular infiltration ( Figure 5).

Discussion
Currently, Diabetes mellitus is a major cause of disability and hospitalization. Available therapeutic options for non-insulin-dependent diabetes mellitus such as dietary modification, oral hypoglycemics and insulin have limitations of their own [17]. The study of such medicines might offer a natural key to unlock a diabetologist's pharmacy for the future. A few reports are available on the hypoglycemic effects of EGCG.
Epigallocatechin-3-gallate (EGCG) is the main catechin flavonoid found in green tea (Camellia sinensis).. It is a powerful antioxidant capable of neutralizing reactive oxygen species and free radicals involved in aging and chronic degenerative diseases. Indeed, EGCG is characterized by antitumor properties [18], antidiabetic agents [5], anti-inflammatory, and especially by antioxidant properties [19]. During this work, we tested in vivo the antioxidant and antidiabetic properties of EGCG and we made an attempt to study the in vitro activity of EGCG given that polyphenols can stabilize excess free radicals, preventing the establishment of oxidative stress. Therefore, our in vitro study have focused on evaluating antiradical and antioxidant activities (DPPH, FRAP, NO, and the total antioxidant capacity) of epigallocatechin-3-gallate (EGCG) shows the powerful antioxidant effect of this bioactive molecule. EGCG may interact directly with ROS or act indirectly by inhibiting ROS-generating enzymes or chelating potentially pro-oxidizing metal ions [20]. The antioxidant activity of EGCG was also confirmed by the trapping power of the DPPH radical [21]. Indeed, it is known that flavonoids are natural antioxidants that increase the expression of antioxidant enzymes (SOD, catalase and GSH-Px) at the transcriptional level [22]. Previous scientific studies have also demonstrated that the particular structure of polyphenols, whose flavonoids allow them to rapidly reduce superoxide radicals, peroxyl (ROO •), alkoxyl (RO •) and hydroxyl by hydrogen transfer. They also decrease the production of xanthine oxidase, a source of free radicals [23] (Table 1).
In the present study, alloxan treatment resulted in increase in blood glucose level of animals ( Table 2). Both insulin and EGCG injections significantly reduced the blood glucose level.
On the other hand, the liver and kidney showed significant decrease in absolute weight and tissue damage in diabetic mice, whereas tissue regeneration and normal growth were found after treatment with EGCG. Such hepato and nephrocellular damage observed in diabetic mice likely is the result of oxidative stress [24]. Assessment of liver and kidney damage was done by measuring marker indices (i.e. ALT, AST, and LDH, Bilirubine, urea and creatinin), which leak into blood stream upon hepatic and kidney injuries [24][25]. In the present study, alloxan treatment caused severe liver and kidney damage, as evidenced by the significant increase in serum ALT, AST and LDH activities as well as elevated total and direct bilirubin levels in the plasma of diabetic mice. Urea and creatinine levels shows also a significant elevation in the diabetic mice.
Conversely, treatment with EGCG showed a significant decrease in serum AST, ALT and ALT, indicating a protective role for this molecule against liver dysfunctions (Table 3). Similar findings were reported earlier upon EGCG treatment against STZ-induced hepatotoxicity [26].
Diabetes mellitus may be accompanied by other metabolic perturbations such as those affecting lipid profiles; the most common are hypertriglyceridemia and hypercholesterolemia [27]. Accordingly, we observed a significant increase in TG, TC and LDL-Ch levels in alloxan-induced diabetic mice, suggesting lipid breakdown and mobilization of free fatty acids from the peripheral stores [27]. Similar observations were reported in diabetic patients where insulin deficiency caused a variety of perturbations in metabolic and regulatory processes, leading to the accumulation of total cholesterol and triglycerides. Conversely, we found that insulin injection decreased TG, TC and LDL-Ch levels, confirming the hypolipidemic effect of insulin. Similar observations could be done upon EGCG supplementation, reinforcing the notion that consumption of the catechins prevents hypercholesterolemia [28].
Oxidative stress has been known to induce the production of ROS scavenging enzymes [29]. These enzymes are supposed to respond in a compensatory response, increasing enzyme activity in diabetic mice, in an attempt to maintain homeostasis. Our findings on disturbed ROS enzymatic levels due to hyperglycaemia and their amelioration by plant extract are congruent with previous reports [30][31].
Indeed, a significant decrease in antioxidant enzymes activity in kidney and liver was found. This can be due to inhibition of the enzymes by increased production of free radicals during diabetes. SOD protects tissues against oxygen free radicals by catalyzing the removal of superoxide radical (O 2− ), which damages the membrane and biological structures (Table 4- Intraperitoneal administration of EGCG produces a statistically significant decrease in serum glucose concentration only in alloxan-induced diabetic fasted mice but not in healthy fasted mice. Therefore, the catechin did not affect insulin releasing from the pancreas of both animal groups. On the other hand, intraperitoneal administration of EGCG does not change serum glucose. Administration of EGCG results in the activation of SOD, CAT and GPx and therefore restoring near normal antioxidant enzyme levels. No histological changes was determined (Figure 2-3).
In conclusion, our results have shown that EGCG possess an hypoglycaemic effect on alloxan-induced hyperglycaemic mice intraperitoneally as well as being good inhibitors of the α-amylase an lipase enzymes. This study presented new findings about the biological and pharmacological potential of EGCG, contributing to the understanding of the action and efficacy of the use of this molecule in the digestive enzyme inhibitions and in antioxidant processe. Thus, these results open possibilities for further studies and controlled clinical trials using this catechin in order to be validated and used as an antidiabetic agent. This will be required to confirm its hypoglycaemic action and general safety.

Figure 4
Histological examinations of liver were conducted to understand the effect of different treatments on liver tissue