Effect of Aqueous Extract from Phaseolus vulgaris Pods on Lipid Peroxidation and Antioxidant Enzymes Activity in the Liver and Kidney of Diabetic Rats

Mariana Yuriivna Kyznetsova, Olha Mycholaivna Makieieva, Dariia Oleksandrivna Lavrovska, Maria Oleksandrivna Tymoshenko, Daryna Pavlivna Sheverova, Tetiana Ivanivna Halenova, Oleksiy Mycholayovych Savchuk, Lyudmila Ivanivna Ostapchenko Department of Biochemistry, Education and Scientific Center ‘Institute of Biology’, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine 2 Medical Center ‘Boris’, Kyiv, Ukraine. 3 Medical faculty 1, Bogomolets National Medical University, Kyiv, Ukraine.


INTRODUCTION
Diabetes mellitus (DM) is a metabolic disorder characterized by the elevated blood glucose level and subsequent pathological effects (Shafiee et al., 2012).It is well known that chronic hyperglycemia is associated with increased oxidative stress.Production of free radicals caused by elevated blood glucose level may occur via four different routes as: 1) enhanced glycolysis, 2) activation of the polyol pathway (also known as sorbitol-aldose reductase pathway), 3) glucose autoxidation and 4) non-enzymatic protein glycation (Ceriello et al., 1992;Williamson et al., 1993;Ceriello, 2000).Activation of these mechanisms leads to a number of other biochemical disorders including formation of highly active lipid peroxides and reactive oxygen species (ROS) which can directly damage cells.For these reasons, oxidative stress can be considered at the same time as a cause and as consequence of micro and macrovascular complications of diabetes (Goycheva et al., 2006;Yang et al., 2011).Nowadays, development of new effective therapeutic strategies for diabetes is task of extremely high importance.
The use of antioxidants for dealing with diabetes, and particularly for treatment of its complications, is being intensively studied.Plants, including herbs and spices, contain many phytochemicals, which are a potential source of natural antioxidants such as phenolic diterpenes, flavonoids, alkaloids, tannins and phenolic acids.Multiple studies have been made in order to find out the antioxidant activities of various herbs, fruits, vegetables, spices and their role in the prevention and treatment of diabetes complications (Shukia et al., 2000;Khan et al., 2012;Patel et al., 2012).
Phaseolus vulgaris, also known as kidney bean, is a common vegetable that possess plenty of curative and therapeutic properties.Various parts of this plant are extensively used in traditional medicine for the treatment of DM.P. vulgaris contains bioactive components with antihyperglycemic activity (Roman-Ramos et al., 1995;Pari and Venkateswaran, 2003).However, in order to understand more about the therapeutic values of this plant in prevention and treatment of DM further investigations are necessary.Purpose of the given study was to study the effect of the aqueous extract from P. vulgaris pods on the functional state of liver and kidney in rats under conditions of streptozotocin-induced diabetes.

Preparation of plant extract
The aqueous extract was prepared by boiling 132 g of dried powdered P. vulgaris pods in 1 liter of distilled water for 20 min.After boiling, extract was left overnight to infuse (Venkateswaran and Pari, 2002).In order to remove plant debris obtained extract was filtered and centrifuged at 1000×g for 10 min.Supernatant was lyophilized by incubation at -20˚C in a deep freezer for 8 h followed by drying in a freeze-dryer (The Telstar LyoQuest, Spain) at -56˚C for 24 h under pressure of 0.05 mbar.Dry extract (8 g) was stored at -20˚C.Right before use, required doses were taken and resuspended in 2 ml of distilled water.

Experimental animals
White non-linear rats of both sexes, each in the weight range of 100-120g, were obtained from the Animal house of Taras Shevchenko National University of Kyiv, Ukraine.All experimental protocols were approved by the Ethical Committee for Conduction of Animal Studies at the Educational and Scientific Center 'Institute of Biology' of Taras Shevchenko National University of Kyiv, Ukraine.Experimental DM was induced by single intraperitoneal injection of 45 mg/kg b.w.streptozotocin (STZ; Sigma, USA) (Zafar and Naqvi, 2010) dissolved in 0.5 ml of 0.01 M citrate buffer, pH 4.5.Control group received 0.5 ml of buffer that was used for dissolving of STZ.Two days after STZ injection, fasting animals with glycemic values more than 25 mM were chosen for the experiment.

Experimental design
The rats were weighed, tagged and randomly divided into four groups of ten animals each as followed."Control" and "Diabetes" (the untreated diabetic rats) were given by gavage deionized water (2 ml/day); "Control + Extract" and "Diabetes + Extract" were treated with P. vulgaris aqueous extract (200mg/kg b.w. per day) dissolved in 2 ml of deionized water and applied orally.The experiment was conducted for 28 days (Hernández-Saavedra et al., 2013).During the experiment animals were kept under standard conditions (temperature, humidity, 12 hour dark-light cycle and were fed with standard commercial food and water available ad libitum).

Analytical methods
After 28 days, the animals were deprived from food overnight and killed by decapitation.Blood was collected and used for the estimation of blood glucose levels and glycosylated hemoglobin.Serum was separated by centrifugation at 2500×g for 25 min and stored at -20˚C until used for biochemical analysis.Blood glucose level was evaluated using glucometer «Hlyukofot II» (Norma, Ukraine) and level of glycosylated hemoglobin was measured spectrophotometrically using commercial kit (ERBA-Lachema, Czech Republic).
Liver and kidney function markers as serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), creatinine, uric acid and urea were estimated by biochemical analyzer Microlab 300 (Elitech, France) and commercial kits from Elitech diagnostic (France) according to the standard protocols provided by manufacturers.
To estimate lipid peroxidation and enzymes activity liver and kidney were excised, rinsed in ice-cold physiological saline and homogenized with teflon pestle at 4˚C in 5 volumes of 50 mM Tris-HCl buffer (150 mM NaCl, 1 mM ЕDТА, 250 mМ sucrose) pH 7.4.Homogenates were first centrifuged at 600×g, 4°C for 10 min and then at 15000×g for 10 min and supernatants were used for biochemical analysis.

Statistical analysis
The data of biochemical estimations were reported as mean ± SEM for ten animals in each group.Statistical analyses were performed using one-way analysis of variance (ANOVA).Differences were considered to be statistically significant when P was < 0.05.All statistical analyses were performed with statistically available software (SPSS 16 for WINDOWS).

RESULTS AND DISCUSSION
The blood glucose and glycosylated hemoglobin concentrations in control and experimental groups of rats are represented in Fig 1 .Glucose concentration in blood of the untreated fasting diabetic animals was significantly higher than in the control group (Fig 1, A). Diabetic rats treated with aqueous extract from P. vulgaris pods had significantly lower blood glucose level compared to untreated diabetic rats.The blood glucose levels in control rats treated with plant extract and in untreated control animals were statistically comparable.Given data are mean ± SEM for ten animals in each group.Values are statistically significant at P<0.05.*Significantly different from the control rats; #significantly different from the diabetes control rats.A key pathogenic indicator of long-term hyperglycemia is the level of glycosylated hemoglobin (HbA 1c ) (Voǐtenko et al., 2013).Our results showed that both groups of diabetic animals had a significantly higher level of HbA 1c in comparison with the control group (Fig 1, B).However, HbA 1c content in rats treated with aqueous extract from P. vulgaris pods was decreased by 20% compared with untreated diabetic group.The level of HbA 1c in P. vulgaris treated control rats was found to be similar to that in the group of untreated control rats.Thus, it can be argued that P. vulgaris pods extract affects glucose metabolism only under conditions of high glucose concentration that occurs in diabetes.
Chronic hyperglycemia causes a number of metabolic disorders, which in turn can contribute to functional dysfunction of different organs.In our research we focused on the investigation of liver and kidney functional state as particularly important organs involved in development and progression of diabetes and diabetesrelated diseases.Liver is a central inner organ where the metabolic processes take place.Liver along with skeletal muscle and adipose tissue is a major consumer of insulin and play a key role in the metabolism of carbohydrates (Tolman et al., 2007).Liver dysfunction may cause or contribute to development of diabetes complications.One of the most prominent diabetic complications is nephropathy.This kidney condition occurs only in people with DM and results in progressive damage of glomeruli (small filtering units of kidney).This, eventually, leads to increased amount of protein in urine, high blood pressure and declining kidney function.Diabetic nephropathy is an important cause of kidney failure (Anil Kumar et al., 2014).
In order to examine the effect of P. vulgaris pods extract on the hepatic and renal functions serum levels of liver and kidney biochemical markers were determined (Table 1).There are three liver enzymes, which are commonly included in serum chemistry screening profiles: alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT).Measured levels of these enzymes in the serum of all experimental groups of rats are shown in Table 1.Significant elevation in levels of ALT, AST and GGT were observed in serum of the untreated diabetic rats compared to values in control group.The elevation of the serum enzyme levels attributed to liver dysfunction may result from rupture of hepatocytes, as far as it is well known that damage or destruction of liver cells leads to release of marker enzymes into the bloodstream, which subsequently leads to necrosis or changes in cell membrane permeability (Adeyemi et al., 2014).
Creatinine, blood urea level and uric acid are markers for routine analysis of the renal function.These parameters can be used as indicators of normal biological or pathologic processes in kidney as well as the way of monitoring renal disease progression.Data represented in Table 1 show that the value of creatinine was decreased while the value of uric acid was significantly increased in serum of untreated diabetes rats compared to control group.The level of blood urea was statistically the same for both groups.A high level of uric acid in the blood, or hyperuricemia, is associated with diabetes conditions and due to reduced excretion by the kidneys or other deficiencies of renal functions (Gowda et al., 2010).The decreased value of creatinine was also reported in kidney disease (Almdal et al., 1988).Above listed changes of studied biochemical parameters in diabetic animals bear evidence of renal failure development.
In the diabetic rats treated with P. vulgaris pods extract serum levels of ALT, AST, GGP and uric acid were significantly lower compared to untreated diabetic rats.However, it was observed that creatinine level in extract treated diabetic rats was similar to that in rats from untreated diabetic group when blood urea value was elevated by 43% compared with untreated diabetic group.Studied biochemical parameters of extract treated control rats were not statistically different from untreated control group with exception of GGT level that was decreased in 1.5 times compared with level of untreated control group of rats.Based on our results it is clear that the P. vulgaris pods extract had a positive influence on the blood biochemical parameters in diabetic rats.
One of the most important factors in the development of diabetic complications is increased lipid peroxidation level.Elevated endogenous peroxides may initiate uncontrolled lipid peroxidation, which leads to cellular infiltration and cell damage.Table 2 shows concentration of MDA, Schiff bases and conjugated dienes, parameters that characterize intensity of free radical production, in liver and kidney tissues of all experimental groups.There was a significant elevation in tissues MDA, conjugated dienes and Schiff bases in diabetes compared to the control group.In the diabetic rats treated with aqueous extract of P. vulgaris pods tissues levels of MDA, conjugated dienes and Schiff bases were significantly lower compared to those in the group of untreated diabetic rats, but some parameters (liver MDA, Schiff bases in liver and kidney) were higher than control data.
Free radical homeostasis in cells and tissues is ensured by balance between enzymatic and non-enzymatic systems of generating ROS and systems of their neutralization (Ahmed et al., 2005).Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of superoxide radical (O 2-) and thus protects membranes and other cellular structures from oxygen free radicals (Otitoju et al., 2008).Catalase is a hemoprotein that catalyzes the reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxyl radicals (Rjasekaran et al., 2005).Together, SOD and catalase are the two major enzymes that remove the toxic free radicals in vivo.
Table 3 illustrates the activities of SOD and catalase in liver and kidneys in examined groups of rats.We observed significant reduction in activity of SOD and catalase in untreated diabetic rats compared with control.Reduced activity of the antioxidant enzymes could be a result of their modification with glucose and ROS under DM conditions.Interestingly, catalase activity in kidney of diabetic rats was raised in 1.7 times compared with its activity in kidney of control rats.Catalase over activation under diabetes condition can be explained by compensation of decreased SOD activity.In the group of diabetic rats treated with aqueous P. vulgaris pods extract liver catalase activity and SOD activities in kidney were significantly higher whereas the kidney catalase activity was lower compared to untreated diabetic rats.Moreover, we found no significant differences in studied enzymes activities between extract treated diabetic group and control groups.
Another important player in regulation of free radical disposal is glutathione system.Reduced glutathione (GSH) is one of the main components of antioxidant defense system capable to react with the products of lipid peroxidation (Chugh et al., 1999).GSH maintaining carried out by several glutathione-dependent enzymes.
Glutathione peroxidase (GPx) eliminates products of lipid peroxidation catalyzing glutathione oxidation and subsequent hydrogen peroxide deactivation.The reverse glutathione reduction is ensured by glutathione reductase (GR).Glutathione-Stransferase (GST) carries out reduction of macromolecular hydrophobic hydrogen peroxides such as polyunsaturated fatty acids using reduced glutathione (Cnubben et al., 2001;Hayes and McLellan, 1999).
Activity of glutathione-dependent enzymes in liver and kidney are represented in Table 4.There was a significant reduction in activity of GPx and GST in liver of diabetic rats in comparison with control group of rats, but no significant difference was observed between liver GR activities of these experimental groups.Comparing the glutathione-dependent enzymes activities in kidney tissue of untreated diabetic and control animals we observed that the both GPx and GST activities were reduced in the group of diabetic rats.GR activity was higher in comparison with control.In the group of diabetic rats treated with P. vulgaris pods extract, activities of GPx and GST in kidney were significantly higher compared to untreated diabetic rats.It must be noted that kidney GST activity was significantly higher in extract treated diabetic rats compared to control values.We did not observe any difference between GR activity in liver as well as in kidney of treated diabetic rats in comparison with untreated diabetic rats.

CONCLUSION
Plants are extensively used by traditional medicine trough out the world for relieving diabetes mellitus symptoms.In Eastern European countries P. vulgaris due to its natural abundance in this geographic area has been used for same purpose since early times.Our experimental data in accordance with previous studies performed on animals (Román-Ramos et al., 1991) demonstrated that long-term administration of P. vulgaris pods extract decreases blood glucose level in fasting diabetic rats.We also showed that aqueous extract of this plant has a positive influence on the number of other blood biochemical parameters.Moreover, it was revealed that P. vulgaris pods extract modulates free radical production and activates antioxidant enzymes under diabetic conditions.This activity contributes to the protection against oxidative damage in streptozotocin-induced diabetes.Taking all together, one may suggest that long-term oral administration of P. vulgaris pods extract might have beneficial effect in treatment of diabetes.

Fig. 1 :
Fig. 1: Effect of Phaseolus vulgaris pods extract on (A) blood glucose and (B) glycosylated hemoglobin (HbA1c) levels.Given data are mean ± SEM for ten animals in each group: C -control rats; CE -control rats treated with extract; D -untreated diabetic rats; DE -diabetic rats treated with extract.Values are statistically significant at P<0.05.*Significantly different from the control rats; #significantly different from the diabetes control rats.

Table 1 :
Effect of Phaseolus vulgaris pods extract on serum levels of liver and enzyme markers.
*#Given data are mean ± SEM for ten animals in each group.Values are statistically significant at P<0.05.*Significantly different from the control rats; #significantly different from the diabetes control rats.

Table 2 :
Effect of Phaseolus vulgaris pods extract on the concentration of MDA, conjugated dienes and Schiff bases in liver and kidney.

Table 3 :
Effect of Phaseolus vulgaris pods extract on the activities of SOD and catalase in liver and kidney.

min -1 *mg prot. -1 ) Catalase (nmol H2O2*min -1 *mg prot. -1 )
Given data are mean ± SEM for ten animals in each group.Values are statistically significant at P<0.05.*Significantly different from the control rats; #significantly different from the diabetes control rats.

Table 4 :
Effect of Phaseolus vulgaris pods extract on the activities of the glutathione-dependent enzymes in liver and kidney.