Protective effects of Pycnogenol® on hyperglycemia-induced oxidative damage in the liver of type 2 diabetic rats

Chemico-Biological Interactions 186 (2010) 219–227

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Protective effects of Pycnogenol® on hyperglycemia-induced oxidative damage in the liver of type 2 diabetic rats Kehkashan Parveen a , Mohd. Rashid Khan a , Mohd. Mujeeb b , Waseem A. Siddiqui a,∗ a b

Department of Biochemistry, Lipid Metabolism Laboratory, Jamia Hamdard (Hamdard University), New Delhi 110062, India Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110062, India

a r t i c l e

i n f o

Article history: Received 4 February 2010 Received in revised form 18 April 2010 Accepted 20 April 2010 Available online 28 April 2010 Keywords: Type 2 diabetes mellitus Pycnogenol® Streptozotocin Oxidative stress Hyperglycemia

a b s t r a c t Abnormal regulation of glucose and impaired carbohydrate utilization that result from a defective or deficient insulin are the key pathogenic events in type 2 diabetes mellitus (T2DM). Experimental and clinical studies have shown the antidiabetic effects of Pycnogenol® (PYC). However, the protective effects of PYC on the liver, a major metabolic organ which primarily involves in glucose metabolism and maintains the normal blood glucose level in T2DM model have not been studied. The present study evaluated the beneficial effect of PYC, French maritime pine bark extract, on hyperglycemia and oxidative damage in normal and diabetic rats. Diabetes was induced by feeding rats with a high-fat diet (HFD; 40%) for 2 weeks followed by an intraperitoneal (IP) injection of streptozotocin (STZ; 40 mg/kg; body weight). An IP dose of 10 mg/kg PYC was given continually for 4 weeks after diabetes induction. At the end of the 4-week period, blood was drawn and the rats were then sacrificed, and their livers dissected for biochemical and histopathological assays. In the HFD/STZ group, levels of glycosylated hemoglobin (HbA1c), significantly increased, while hepatic glycogen level decreased. PYC supplementation significantly reversed these parameters. Moreover, supplementation with PYC significantly ameliorated thiobarbituric reactive substances, malonaldehyde, protein carbonyl, glutathione and antioxidant enzymes [glutathione-Stransferase, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase] in the liver of HFD/STZ rats. These results were supported with histopathological examinations. Although detailed studies are required for the evaluation of the exact protective mechanism of PYC against diabetic complications, these preliminary experimental findings demonstrate that PYC exhibits antidiabetic effects in a rat model of type 2 DM by potentiating the antioxidant defense system. These finding supports the efficacy of PYC for diabetes management. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Type 2 diabetes mellitus (T2DM), one of the fastest-growing epidemics of our time, is a chronic metabolic disorder characterized by insulin resistance in the liver, muscle, and adipose tissue, as well as progressive ␤-cell dysfunction leading to hyperglycemia [1,2]. It usually occurs in obese individuals, and is associated with hypertension and dyslipidaemia [3–5]. Treatment of T2DM remains a challenging issue. During the last decade, there has been much interest in the development of antidiabetic drugs [6–8]. However, clinical trials have been disappointing as, so far, they all have failed due to therapeutic limitations. The search for a safe and effective drug which can reduce the many harmful effects of T2DM, including

∗ Corresponding author Tel.: +91 11 2605 9688x5522; fax: +91 11 2605 9663. E-mail addresses: [email protected] (K. Parveen), [email protected] (W.A. Siddiqui). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.04.023

hyperglycemia, hyperlipidaemia, oxidative stress, inflammation, and arthrosclerosis, among others, is thus urgently needed. Although the exact mechanisms which contribute to diabetic complications remain unclear, much attention has been paid to the role of oxidative stress, widely believed to contribute to the acceleration of diabetes and its complications [9–11]. Oxidative stress is defined as imbalance in homeostatic phenomenon between oxidants and antioxidants, leads to generation of free radicals [12]. Oxidative stress is increased in diabetes because of multiple factors. Hyperglycemia itself generates ROS, which in turn cause lipid peroxidation (LPO) and membrane damage via auto-oxidation of glucose [13,14]. Furthermore, alteration of the glutathione (GSH) redox state by activation of nicotinamide adenine dinucleotide phosphate reduced form (NADPH)-dependent aldose reductase (polyol pathway) and non-enzymatic glycosylation of protein have also been shown to be a source of free radicals [15]. The detoxification of free radicals is important because free radicals cause oxidative damage to membrane lipids (LPO), proteins (protein carbonyl [PC] formation), and nucleic acid molecules

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[16]. The concerted action of various body antioxidants such as GSH, glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) keeps the concentration of these free radicals relatively low. Experimental and clinical evidence suggests an important role for antioxidants in the management of T2DM [17–19]. French maritime pine (Pinus maritima) bark extract, Pycnogenol® (PYC), a patented combination of bioflavonoids, has a high antioxidant potential [20–22]. A concentrate of polyphenols, mainly phenolic acids, and procyanidins, the extract is used extensively as a dietary supplement [22–24]. PYC also has the ability to enhance the status of the antioxidant, including Vitamin E, GSH and antioxidant enzymes [20,21,25] and protect them from oxidative damage [20,21,26]. PYC has diverse beneficial effects on a wide range of medical conditions, including cancer, atherosclerosis, diabetes, inflammation, asthma, hypertension, atherosclerosis, immune disease, and others [22,24,27]. A number of experimental and clinical studies have shown hypolipidemic, antioxidant, and other beneficial effects of PYC in diabetic conditions [11,17,28–32]. Despite these pharmacotherapeutic properties of PYC, its beneficial effects against type 2 diabetes using a high-fat diet model have not been explored. The present study evaluated the protective effects of PYC on hyperglycemia and oxidative damage in the liver of high-fat diet/streptozotocin (HFD/STZ)-induced diabetic rats. Our results support the efficacy of PYC in reducing glucose and improving diabetic complications in a clinically relevant model of T2DM. 2. Experimental protocol 2.1. Chemicals and reagents Glutathione oxidized (GSSG) and reduced (GSH), glutathione reductase (GR), nicotinamide adenine dinucleotide phosphate reduced form (NADPH), 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB), thiobarbituric acid (TBA), and 2,4-dinitrophenyhydrazine (DNPH), streptozotocin (STZ) were purchased from Sigma–Aldrich, Chemicals Pvt. Ltd., India. Pycnogenol® was gifted by Horphag Research, Geneva, Switzerland. Other chemicals were of analytical reagent grade. 2.2. Animals Male Wistar rats weighing 160–200 g were used in the study. They were kept in the Central Animal House of Jamia Hamdard (Hamdard University) in colony cages at an ambient temperature of 25 ± 2 ◦ C and relative humidity of 45–55% with 12 h light/dark cycles. They had free access to standard rodent pelleted diet (Hindustan Lever Ltd., Bombay, India) and water ad libitum, prior to the dietary manipulation. All procedures using animals were reviewed and approved by the Institutional Animal Ethical Committee that is fully accredited by the Committee for Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Chennai, India. 2.3. Experimental design and induction of the HFD/STZ rat model of type 2 diabetes Thirty-two rats were divided into four groups of eight animals each: group I (control group) rats were fed standard diet (12% calories as fat) throughout the experiment; group II (control + PYC) rats were fed standard diet throughout the experiment and given PYC (10 mg/kg body wt., intraperitoneally (IP); in saline) for 4 weeks; group III (HFD/STZ group) rats were fed HFD (40% fat, 18% protein

and 41% carbohydrate, as a percentage of total kcal) for 2 weeks and then injected with STZ (40 mg/kg body wt., IP, in citrate buffer; pH 4.5) as described previously [33]; group IV (HFD/STZ + PYC) rats were fed HFD for 2 weeks and then injected with STZ and then supplemented with PYC (10 mg/kg body wt.; IP; in saline) for 4 weeks. During PYC treatment all the animals were fed with normal pellet diet. The development of hyperglycemia in rats was confirmed by fasting blood glucose estimation after 6 days of STZ injection. The animals that maintained fasting blood glucose higher than 140 mg/dl were considered diabetic and selected for studies. The PYC treatment was started after diabetes was confirmed, and dose was determined from previous studies [11,34]. 2.4. Tissue preparation At the end of experiment, rats were anesthetized by ether inhalation and blood was collected from the dorsal aorta. Approximately 0.2 ml of whole blood was taken in EDTA containing microtubes from the total blood of each animal, and immediately preserved in the refrigerator for subsequent analysis of glycated hemoglobin (HbA1c). Serum was separated by centrifugation at 1200 × g for 10 min and stored at −80 ◦ C before analysis. Rats were then sacrificed, and their liver were excised immediately and perfused with ice-cold saline. To prevent auto-oxidation or ex vivo oxidation of the tissue, homogenization was carried out at 4 ◦ C with 10 times (w/v) in 0.1 M phosphate-buffer (pH 7.4) containing protease inhibitors: 5 mM leupeptin, 1.5 mM aprotinin, 2 mM phenylethylsulfonylfluoride (PMSF), 3 mM peptastatin A, and 10 mM EDTA, 0.1 mM EGTA, 1 mM benzamidine and 0.04% butylated hydroxytoluene. The homogenate was centrifuged at 800 × g for 5 min at 4 ◦ C to separate the nuclear debris and was used for estimation of thiobarbituric reactive substances (TBARS). The supernatant was further centrifuged at 10,000 × g for 20 min at 4 ◦ C to get the post-mitochondrial supernatant (PMS), which was used for various biochemical assays. 2.5. Analytical procedures 2.5.1. Oral Glucose Tolerance Test In the last week of experiment, the Oral Glucose Tolerance Test (OGTT) was performed to assess the glucose tolerance. For this purpose, overnight fasted rats were given orally 2 g/kg body wt. glucose. Blood was collected at 0, 30, 60, and 120 min intervals from orbital sinus for glucose estimation. Animals were not anesthetized for this procedure. 2.5.2. Determination of HbA1c level HbA1c was assayed by cation-exchange method using a diagnostic kit from Crest Biosystem, Goa, India. 2.5.3. Estimation of liver glycogen content Liver glycogen content was determined using the anthrone reagent method [35]. Immediately after excision from the animal, approximately 1 g of liver tissue is dropped in 3 ml of 30% KOH. This tube was placed in a boiling water bath for 30 min, following this digest was cooled, transferred to a 50-ml volumetric flask and diluted to mark with water. Slowly, 5 ml of solution from the previous step was added to a test tube containing 10 ml of anthrone reagent, which was placed in cold water to prevent excessive heating. After thorough mixing, the tube was placed in a boiling water bath for exactly 10 min for the development of color and cooled with running tap water. The optical density was read within 2 h in a spectrophotometer at 620 nm against a blank that was prepared by subjecting 5 ml of distilled water instead of sample to the same procedure.

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2.6. Biochemical parameters in liver 2.6.1. TBARS content The method of Utley et al. [36] with some modification was used to estimate the rate of LPO. Homogenate (0.25 ml) was pipetted into 15 mm × 100 mm test tubes and incubated at 37 ◦ C in a metabolic shaker for 1 h. An equal volume of homogenate was pipetted into a centrifuge tube, placed at 0 ◦ C and marked at 0 h incubation. After 1 h of incubation, 0.5 ml of 5% (w/v) chilled trichloroacetic acid (TCA), followed by 0.5 ml of 0.67% TBA (w/v) was added to each test tube and centrifuge tube, and centrifuged at 1000 × g for 15 min. Thereafter, the supernatant was transferred to other test tubes and was placed in a boiling water bath for 10 min. The absorbance of pink color produced was measured at 535 nm in a spectrophotometer (Shimadzu-1601, Japan). The TBARS content was calculated by using a molar extinction coefficient of 1.56 × 105 M−1 cm−1 and expressed as nmol of TBARS formed min−1 mg−1 of protein. 2.6.2. Assay for malonaldehyde (MDA) MDA which is a measure of the end product of lipid peroxidation was measured as described by Ohkawa et al. [37]. Briefly, the reagents acetic acid 1.5 ml [20%] pH 3.5, 1.5 ml TBA [0.8%], and 0.2 ml sodium dodecyl sulfate [8.1%] were added to 0.1 ml of PMS sample. The mixture was then heated at 100 ◦ C for 1 h. The mixture was cooled with tap water and 5 ml of n-butanol:pyridine [15:1%, v/v] 1 ml of distilled water was added. The mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was withdrawn, and absorbance was measured at 532 nm using a spectrophotometer (Shimadzu-1601, Japan). The amounts of MDA formed in each of the samples were expressed as the nmol MDA formed/min/mg protein by using a molar extinction coefficient of 1.56 × 105 M−1 cm−1 . 2.6.3. Assay for PC PC level was measured by the method of Levine et al. [38]. The PMS (0.5 ml) was treated with an equal volume of 20% TCA for protein precipitation. After centrifugation, the pellet was resuspended in 0.5 ml of 10 mM DNPH in 2 M HCl and vortexed repeatedly at 10 min intervals for 1 h in dark. This mixture was treated with 0.5 ml of 20% TCA. After centrifugation at 10,000 × g at 4 ◦ C for 3 min, the precipitate was extracted three times with 0.5 ml of 10% TCA and dissolved in 2.0 ml of NaOH at 37 ◦ C. Absorbance was recorded at 360 nm in a spectrophotometer (Shimadzu-1601, Japan). PC level was expressed as nmol carbonyl mg−1 protein, using a molar extinction coefficient of 22 × 104 M−1 cm−1 . 2.6.4. Assay for GSH Reduced GSH content was determined by the method of Jollow et al. [39], with slight modification. PMS was mixed with 4.0% sulfosalicylic acid (w/v) in a 1:1 ratio (v/v). The samples were incubated at 4 ◦ C for 1 h, and later centrifuged at 1200 × g for 15 min at 4 ◦ C. The assay mixture contained 0.1 ml of supernatant, 1.0 mM DTNB, and 0.1 M PB (pH 7.4) in a total volume of 1.0 ml. The yellow color that developed was read immediately at 412 nm in a spectrophotometer (Shimadzu-1601, Japan). The GSH content was calculated as mmol GSH mg−1 of protein, using a molar extinction coefficient of 13.6 × 103 M−1 cm−1 . 2.6.5. Assay for GST The activity of GST was measured by the method of Habig et al. [40]. The reaction mixture consisted of 1.0 mM GSH, 1.0 mM CDNB, 0.1 M phosphate buffer (pH 7.4) and 0.1 ml of PMS in a total volume of 3.0 ml. The change in absorbance was recorded at 340 nm and enzyme activity was calculated as nmol of CDNB conjugate formed min−1 mg−1 protein using molar extinction coefficient of 9.6 × 103 M−1 cm−1 .

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2.6.6. Assay for CAT CAT activity was assayed by the method of Claiborne [41]. Briefly, the assay mixture consisted of 0.05 M phosphate-buffer (pH 7.0), 0.019 M hydrogen peroxide (H2 O2 ), and 0.05 ml PMS in a total volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. CAT activity was expressed as nmol H2 O2 consumed min−1 mg−1 protein. 2.6.7. Assay for SOD SOD activity was measured as described by Martin et al. [42] with some modification The reaction mixture contained 0.8 ml of 50 mmol/l glycine buffer (pH 10.4), and 0.2 ml supernatant. The reaction was initiated by the addition of 0.02 ml of a 20 mg/ml solution of (−)-epinephrine. Absorbance was recorded at 480 nm in a spectrophotometer (Shimadzu-1601, Japan). SOD activity was expressed as nmol of (−)-epinephrine protected from oxidation by the sample compared with the corresponding readings in the blank cuvette. The molar extinction coefficient of 4.02 × 103 M−1 cm−1 was used for calculations. 2.6.8. Assay for GPx GPx (EC 1.11.1.9) activity was measured by the coupled assay method of Wheeler et al. [43] in which oxidation of GSH was coupled to NADPH oxidation, catalyzed by GR. The reaction mixture consisted of 0.2 mM H2 O2 , 1 mM GSH, 1.4 unit of GR, 1.43 mM NADPH, 1 mM sodium azide, PMS (0.1 ml) and PB (0.1 M) in total volume of 2.0 ml. GPx activity was defined as nmol NADPH oxidized min−1 mg−1 protein, using a molar extinction coefficient of 6.22 × 103 M−1 cm−1 . 2.6.9. Assay for GR GR (EC 1.6.4.2) activity was measured by the method of Carlberg and Mannerviek [44]. The assay system consisted of 0.1 M PB (pH 7.6), 0.5 mM EDTA, 1 mM GSSH, 0.1 mM NADPH and PMS (0.1 ml) in a total volume of 2.0 ml. The enzyme activity was quantitated at 25 ◦ C by measuring the disappearance of NADPH at 340 nm, and calculated as nmol NADPH oxidized min−1 mg−1 protein, using a molar extinction coefficient of 6.22 × 103 M−1 cm−1 . 2.6.10. Protein content Protein content was determined by the method of Lowry et al. [45], using bovine serum albumin (BSA) as a standard. 2.7. Histological examinations For histological examinations, liver sections from different groups were stained with hematoxylin and eosin (H and E). Briefly, at end of experiment, the rats were anesthetized with ether and perfused transcardially with saline. Livers were removed quickly and postfixed in buffered formalin (10%) for 24 h. After fixation was completed, slices (3–4 mm) of these tissues were dehydrated and embedded in paraffin. At least four cross-sections were taken from each tissue in 5-␮m thickness and stained with H and E. Following two changes xylene washes of 2 min each tissue sections were mounted with DPX mountant. The slides were observed for histopathological changes and microphotographs were taken using an Olympus BX50 microscope system (Olympus, Japan). 2.8. Statistical analysis Results are expressed as mean ± S.E.M. (n = 8). Statistical analysis of the data was obtained via analysis of variance (ANOVA), followed by Tukey’s test. P < 0.05 was considered statistically significant.

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no significant change in the level of HbA1c in the control + PYCtreated group compared to the control. 3.3. PYC treatment augmented hepatic glycogen content in the HFD/STZ-induced rat model of diabetes Table 1 shows the effect of PYC treatment on the hepatic glycogen content in the HFD/STZ group. The HFD/STZ group showed a significantly (P < 0.05) decreased in glycogen content compared to the control. PYC supplementation significantly (P < 0.05) increased glycogen content in the HFD/STZ + PYC group compared to the HFD/STZ group. The results obtained for the glycogen content indicates that there was no significant difference in glycogen content between the control and the control + PYC group. 3.4. PYC treatment modulated TBARS and MDA contents in the HFD/STZ-induced rat model of diabetes

Fig. 1. Effect of PYC supplementation on oral glucose tolerance (OGTT). Values are expressed as mean ± S.E.M. (n = 8). The HFD/STZ group showed the increase in blood glucose level at 60 min and remained high over next 60 min after oral glucose administration compared to the control group (a P < 0.05 HFD/STZ vs. control group). PYC supplementation decreased the blood glucose level at 60 and 120 min in the HFD/STZ + PYC group compared to the HFD/STZ group (b P < 0.05 HFD/STZ + PYC vs. HFD/STZ group).

3. Result 3.1. PYC treatment ameliorated OGTT in the HFD/STZ-induced rat model of diabetes Fig. 1 Shows the blood glucose levels of the control, HFD/STZ group and the HFD/STZ + PYC groups after oral administration of glucose (2 g/kg). In the HFD/STZ group, the peak increase in blood glucose level was observed after 60 min. Its level remained high over next 60 min. The HFD/STZ + PYC group showed significant (P < 0.05) decrease in blood glucose level at 60 and 120 min compared to the HFD/STZ group. 3.2. PYC treatment restored HbA1c level in the HFD/STZ-induced rat model of diabetes Table 1 shows the effect of PYC on HbA1c level. A significant (P < 0.05) increase in HbA1c level was observed in HFD/STZ group compared to the control. PYC treatment significantly (P < 0.05) decreased the HbA1c level in the HFD/STZ + PYC group. There was Table 1 Effect of PYC treatment on HbA1c and hepatic glycogen level in the control and experimental groups. Parameters/groups

GHbA1c (%)

Control Control + PYC HFD/STZ PYC + HFD/STZ

5.58 5.63 10.90 8.88

± ± ± ±

0.07 0.13 (+0.89%) 0.24a (+95.34%) 0.27b (−18.53%)

Hepatic glycogen (␮g) 357.00 361.14 173.10 241.19

± ± ± ±

3.67 2.4 (+1.15%) 2.0a (−51.51%) 2.4b (+39.33%)

Values are expressed as mean ± S.E.M. (n = 8). HFD/STZ group showed a significant increase in HbA1c with the significant decrease in hepatic glycogen in the HFD/STZ group compared to the control group (a P < 0.05 HFD vs. control group). PYC treatment significantly ameliorated these parameters in the HFD/STZ + PYC group compared to the HFD/STZ group (b P < 0.05 HFD/STZ vs. HFD/STZ + PYC group). Values in parentheses indicate percentage increase (+) or decrease (−) as compared with the control or HFD/STZ group.

There were no significant changes in TBARS and MDA contents in control + PYC-treated group compared to the control group. These parameters were significantly (P < 0.05) increased in the HFD/STZ group compared to the control group. Levels of TBARS and MDA decreased significantly (P < 0.05) with PYC supplementation in the HFD/STZ + PYC group compared to the HFD/STZ group (Fig. 2A and B). 3.5. PYC treatment attenuated PC in the HFD/STZ-induced rat model of diabetes PC content did not change by PYC supplementation in the control + PYC group compared to the control group. PC content was significantly (P < 0.05) increased in the HFD/STZ group compared to the control alone. PYC supplementation significantly (P < 0.05) decreased PC content in the HFD/STZ + PYC group compared to HFD/STZ group alone (Fig. 3). 3.6. PYC treatment restored GSH in the HFD/STZ-induced rat model of diabetes Level of GSH did not affect by PYC supplementation in the control + PYC-treated group compared to the control group. However, a significant (P < 0.05) depletion in GSH was observed in the HFD/STZ group compared to the control group. PYC supplementation significantly (P < 0.05) restored GSH level in the HFD/STZ + PYC group compared to the HFD/STZ group (Fig. 4). 3.7. PYC treatment increased GST, CAT, SOD, GPx and GR activity in the HFD/STZ-induced rat model of diabetes PYC treatment increased antioxidant enzymes activity in the HFD/STZ group (Table 2). The activity of antioxidant enzymes in control + PYC group was not increased significantly compared to the control. On the other hand, the activity of these antioxidant enzymes was depleted significantly (P < 0.05) in the HFD/STZ group compared to the control group. PYC supplementation significantly (P < 0.05) increased the activity of these enzymes in the HFD/STZ + PYC group compared to the HFD/STZ group. 3.8. Histopathological findings 3.8.1. Effect of PYC treatment on H and E staining in the HFD/STZ-induced rat model of diabetes H and E staining is used to visualize and differentiate between tissue components in normal and pathological conditions. The histological examination of the H and E-stained control liver section

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Fig. 2. (A) Effect of PYC supplementation on TBARS levels in the liver. Values are expressed as mean ± S.E.M. (n = 8). The HFD/STZ group showed significant increase in TBARS levels compared to the control group (a P < 0.05 HFD/STZ vs. control group). PYC supplementation significantly decreased TBARS levels in the HFD/STZ + PYC group compared to the HFD/STZ group (b P < 0.05 HFD/STZ + PYC vs. HFD/STZ group). (B) Effect of PYC supplementation on MDA level in the liver. Values are expressed as mean ± S.E.M. (n = 8). The HFD/STZ group showed significant increase in MDA level compared to the control group (a P < 0.05 HFD/STZ vs. control group). PYC supplementation significantly decreased MDA level in the HFD/STZ + PYC group compared to the HFD/STZ group (b P < 0.05 HFD/STZ + PYC vs. HFD/STZ group).

showed normal architecture of hepatocytes (Fig. 5). Liver section of HFD/STZ-induced diabetic group showed vacuolization of hepatocytes in the centrizonal area with variation of nuclear size and dilated sinusoidal spaces. The severity of degenerative changes was lessened by PYC supplementation in the HFD/STZ + PYC group compared to the HFD/STZ group. PYC supplementation did not show any remarkable effects in the group treated with PYC alone compared with the vehicle control group (data not shown).

4. Discussion

Fig. 3. Effect of PYC supplementation on PC content in the liver. Values are expressed as mean ± S.E.M. (n = 8). The HFD/STZ group showed significant increase in PC content compared to the control group (a P < 0.05 HFD/STZ vs. control group). PYC supplementation significantly decreased PC content in the HFD/STZ + PYC group compared to the HFD/STZ group (b P < 0.05 HFD/STZ + PYC vs. HFD/STZ group).

Fig. 4. Effect of PYC supplementation on GSH content in the liver. Values are expressed as mean ± S.E.M. (n = 8). The HFD/STZ group showed significant decrease in GSH content compared to the control group (a P < 0.05 HFD/STZ vs. control group). PYC supplementation significantly increased GSH content in the HFD/STZ + PYC group compared to the HFD/STZ group (b P < 0.05 HFD/STZ + PYC vs. HFD/STZ group).

The present study shows that HFD/STZ-induced hyperglycemia accompanied by the presence of oxidative damage in the liver of rats. Moreover, treatment with PYC, by virtue of its antioxidant capacity, modulated the HFD/STZ-induced biochemical and morphological alterations. These beneficial effects of PYC provide additional support to finding from previous studies that PYC is free radicals scavenger with powerful antioxidant [21,22,27,31] and antidiabetic potentials [11,17,28–32]. Recently, we have also demonstrated the antioxidant potential of PYC on potassium dichromate-induced renal toxicity in rats [34]. PYC is known to bind the proteins, thereby affecting both structural and functional characteristics of key enzymes and other proteins involved in metabolism [46]. During oxidative stress auto-oxidation and the presence of an excess of hydroxyl radical damaged to carbohydrates. This reaction is well evidenced by the production of thiobarbituric acid-reactive material, reactive carbonyl compound and malonaldehyde [47,48]. These effects are regarded as an important risk factor in the acceleration of chronic diseases including diabetes [9,14,49]. Overproduction of free radicals in diabetes could be due to due to a persistent chronic increase in blood glucose levels [9]. In the present study, the HFD/STZ showed a significant increase in the blood glucose level at 60 min and was maintained for 120 min following glucose administration in OGTT. PYC supplementation improved glucose tolerance at both time points. Because it significantly decreased blood glucose in glucose-loaded rats, we hypothesize that PYC may enhance glucose utilization by peripheral tissues, and increase the glycogen stores in the liver by restoring delayed insulin response. In diabetes, there is an increased glycosylation of a number of proteins, including hemoglobin and ␤-crystalline of the eye lens [50]. Estimation of HbA1c has been found to be particularly useful in monitoring the effectiveness of therapy in diabetes [51]. In our

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Fig. 5. Effect of PYC supplementation on H and E staining in the liver sections. Photograph from the control group at low power – 100×. showing normal liver architecture. PT: portal triad; CV: central vein, at high power – 400×. showing details with normal portal triad. PV: portal vein, BD: bile duct, HA: hepatic artery. The HFD/STZ group at low power – 100×. showing mild sinusoidal dilatation in the centrizonal area, at high power – 400×. showing vacuolated hepatocytes, variation in nuclear size and mild sinusoidal dilatation in the centrizonal area. PYC supplementation in the HFD/STZ + PYC group at low power – 100×. showing normal hepatic parenchyma with normal PT and CV, at high power – 400×. showing a normal PV and BD.

study, the HbA1c level was found to be significantly increased in the HFD/STZ group. Agents with antioxidant or free radical scavenging properties may inhibit oxidative reactions associated with protein glycation [52]. Administration of PYC reduced the glycosylation of hemoglobin by means of its free radical scavenging property and thus decreased the level of HbA1c. A decrease in blood glucose level might also contribute to the decreased level of glycated hemoglobin in the PYC-treated group. The liver is primarily responsible for maintaining normal blood glucose concentrations via its ability to store glucose as glycogen and its ability to produce glu-

cose from glycogen breakdown or from gluconeogenic precursors. It has been previously demonstrated that glycogen deposition from glucose is impaired in diabetic animals [53]. In our study, the hepatic glycogen content in the HFD/STZ group decreased compared to the HFD/STZ + PYC group. This antidiabetic potential of PYC is consistent with previous studies which showed hypoglycemic and glycosylated hemoglobin (HbA1C) lowering effects of PYC in the patients with type 2 DM [11,31,32]. Lipids and proteins, the major structural and functional components of the cell membrane, are the target of oxidative modification

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Table 2 Effect of PYC treatment on the activity of antioxidant enzymes in the liver of control and experimental groups. Parameters/groups

GST*

Control (C) C + PYC HFD/STZ HFD/STZ + PYC

240.23 238.56 188.36 218.39

CAT** ± ± ± ±

5.8 5.9 (−0.69%) 3.2a (−21.59%) 4.6b (+15.94%)

55.57 53.75 27.23 41.25

SOD*** ± ± ± ±

1.25 2.12 (−3.27%) 1.58a (−50.99%) 2.2b (+51.48%)

23.14 21.57 11.89 18.68

GPx# ± ± ± ±

0.86 0.84 (−6.78%) 0.42a (−48.61%) 0.53b (+57.10%)

41.06 45.05 21.21 33.71

GR# ± ± ± ±

0.96 1.2 (+9.71%) 0.75a (−48.34%) 0.65b (+58.93%)

65.13 67.96 31.35 51.31

± ± ± ±

1.37 1.44 (+4.34%) 1.12a (−51.86%) 1.39b (+63.66%)

Values are expressed as mean ± S.E. (n = 8). HFD/STZ lead to significant alterations on the activities of antioxidant enzymes (GST, CAT, SOD GPx and GR) in HFD/STZ group as compared to control group (a P < 0.05 HFD/STZ vs. control group). Administration of PYC significantly attenuated activity of these enzymes in HFD/STZ + PYC group as compared to HFD/STZ group (b P < 0.05 HFD/STZ vs. HFD/STZ + PYC group). Values in percentage increase (+) or decrease (−) as compared to control or HFD/STZ group. * nmol CDNB conjugate formed × min−1 mg−1 protein. ** nmol H2 O2 consumed × min−1 mg−1 protein. *** nmol epinephrine/mg protein. # nmol NADPH oxidized min−1 mg−1 protein.

by free radicals. There is extensive evidence that lipid peroxidation and protein oxidation lead to loss of membrane integrity, an important factor in acceleration of DM [9,11,54]. LPO is frequently used as an index of tissue oxidative stress, in which oxygen interacts with polyunsaturated fatty acids and leads to the formation of lipid products such as MDA and 4-HNE, and increase generation of free radicals, which then leads to damage to membrane components of the cell, cell necrosis and inflammation [55]. MDA is end product of lipid peroxidation and the increased MDA production plays an important role in the progression of diabetes [56] by altering the transbilayer fluidity gradient, which could hamper the activities of membrane-bound enzymes and receptors. Increased generation of free radicals can also lead to the formation of protein–protein crosslinkages, oxidation of protein backbones resulting in protein fragmentation, and modification of amino acid side chains, which includes oxidation of sulphydryl moieties and formation of PC [57]. LPO may be enhanced due to the depletion of GSH content, which is often considered the first line of defense as an endogenous antioxidant. The liver plays a major role in glutathione homeostasis and is the main export organ for glutathione [58]. Increased oxidative stress can change the redox potential of GSH [59] due, it has been proposed, to oxidation of its thiol group [60]. Decreased levels of GSH in the liver of diabetic rats may increase susceptibility to oxidative damage. The depletion of GSH content also may lower antioxidant enzyme GST activity, because GSH is required as a substrate for GST activity. CAT and SOD are the two major scavenging enzymes that remove radicals in vivo. A decrease in the activity of these antioxidants can lead to an excess availability of superoxide anion (O2 • −) and hydrogen peroxide (H2 O2 ), which in turn generate hydroxyl radicals (• OH), resulting in initiation and propagation of lipid peroxidation. SOD can catalyze dismutation of O2 • − into H2 O2 , which is then deactivated to H2 O by CAT or glutathione peroxidase [61,62]. GPx catalyzes the reaction of hydroperoxides with GSH to form glutathione disulphide. GPx uses GSH as a proton donor, converts H2 O2 to water and molecular oxygen; in this process GSH is oxidized to GSSG, which is reconverted to GSH by the action of enzyme GR, thus maintaining the pool of GSH. A significant decrease in GPx and GR activity could suggest inactivation by reactive oxygen species, [63], which are increased in diabetic rats. The decrease may also be due to the decreased availability of its substrate, GSH, which has been shown to be depleted during diabetes [64,65]. It has been demonstrated that the activity of these antioxidant enzymes (GST, CAT, SOD, GPx and GR) decrease in diabetic rats [66–68]. In the present study, we observed the increased levels of TBARS (as an active index of rate of LPO, MDA (a final product of LPO) and PC with a concomitant decrease in GSH and antioxidant enzyme (GST, CAT, SOD, GPx and GR) activity in the liver of HFD/STZ rats. Treatment with PYC significantly prevented lipid and protein from oxidation by enhancing the level of GSH and the status of antioxidant enzymes in the HFD/STZ + PYC group. These effects indicate that

PYC may scavenge or inhibit free radical formation and participate in stabilizing the endogenous antioxidant network, including GSH, and concomitantly decrease LPO in various conditions caused by free radicals, consistent with previous studies [11,21–24,31]. PYC has been shown to alter the expression of genes [69], enzymes [70] and other molecules [71] which are involved in antioxidant defenses. Our findings are also supported by histopathological observations. Sections from the liver of the HFD/STZ group showed vacuolization of hepatocytes in the centrizonal area, with variation of nuclear size and dilated sinusoidal spaces. All these degenerative changes were ameliorated with PYC supplementation, indicating hepatoprotective effect of PYC as a potent antioxidant. Other than its antioxidant effects, plant inhibited the expression of nitric oxide synthase and helped in correcting the secretary defects in diabetes [72,73]. The one possible mechanism of action of PYC might be that it stimulated the beta islets to secrete insulin by inhibiting the expression of nitric oxide synthase [46]. This prevents depletion of glycogen in the liver possibly due to stimulation of insulin release from ␤-cells [74]. Elevated insulin production by PYC treatment may be responsible for the alteration in glycogen content of the liver. PYC also has been found to stimulate glucose transporter 4 (Glut4) expression by regulating the kinases (p38 MAPK and phosphoinositol 3-kinase) which induce insulin resistance by affecting expression of Glut4 and insulin signaling [75]. Conclusively PYC stimulate glucose uptake due to presence of insulin-like effects in insulin sensitive cells that could help to explain their anti-hyperglycemic effect in vivo. Furthermore studies have suggested hyperglycemia and oxidative stress is linked to nuclear factor kappa-B (NF␬B) activation [76,76]. Furthermore NF␬B is involved in regulations of COX-2 and iNOS expressions [77], which also play a role in hyperglycemia, and oxidative stress [78]. PYC administration can counteract cytokine-induced activation of NF␬B [79]. Other possible mechanisms of action by which PYC show glucose lowering effect is due to presence of its constituent oligomeric procyanidin, monomeric epicatechin and catechin,. These forms act through different mechanisms: procyanidin inhibit alphaglucosidase activity [80], epicatechin induces pancreatic ␤-cell regeneration [81], catechin inhibits intestinal glucose absorption [82]. In conclusion, our results showed that PYC through its antioxidant properties enhanced endogenous GSH levels and thereby conferred protection against HFD/STZ-induced T2DM in rats. Further, study suggests that PYC is effective in preventing diabetic complications such as hyperglycemia and oxidative damage. Thus, PYC may be considered as a potential candidate in the armamentarium of drugs for prophylactic treatment in diabetic patients, pending further investigations to trace out the exact mechanistic pathways.

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