Key words: Diabetes mellitus; Streptozotocin; Pancreas; Oxidative stress; Tualang honey; Glibenclamide

Transcription

1 International Journal of Applied Research in Natural Products Vol. 4 (2), pp. 1-10, June-July 2011 Directory of Open Access Journals IJARNP-HS Publications Original Report Effect of Glibenclamide alone versus Glibenclamide and Honey on Oxidative Stress in Pancreas of Streptozotocin-Induced Diabetic Rats Erejuwa OO 1, Sulaiman SA 1*, Wahab MS 1, Sirajudeen KNS 2, Salleh MS 3, Gurtu S 4 1 Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia. 2 Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia. 3 Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia. 4 Monash University Sunway Campus, Jeffrey Cheah School of Medicine and Health Sciences, Jalan Lagoon Selatan, Bandar Sunway, Selangor, Malaysia. Summary: Diabetes mellitus is a public health problem with increasing global prevalence. In spite of its management, both microvascular and macrovascular complications partly linked to oxidative stress are not efficiently prevented. This study compared the effect of glibenclamide alone with that of combined glibenclamide and honey on pancreatic oxidative stress in streptozotocin-induced diabetic rats. Diabetes was induced by streptozotocin (60 mg/kg; ip). Rats were randomly divided into four groups and treated as follows: non-diabetic rats received distilled water (0.5ml/day), diabetic rats administered distilled water (0.5ml/day), glibenclamide (600 µg/kg/day) or a combination of glibenclamide (600 µg/kg/day) and honey (1.0 g/kg/day). The animals were treated orally once daily for four weeks. Fasting blood glucose was significantly increased in diabetic rats. The diabetic pancreas showed increased levels of malondialdehyde (MDA), reduced activity of catalase (CAT) and increased activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Treatment of diabetic rats with glibenclamide reduced hyperglycemia but produced no significant effects on the MDA levels, activities of SOD, GPx, and CAT. In contrast, the pancreas of diabetic rats that received combination of glibenclamide and honey showed increased CAT activity, reduced MDA levels and GPx activity while blood glucose was also reduced. The results of glibenclamide alone suggest that decreased hyperglycemia does not necessarily translate to reduced oxidative stress. These data demonstrate the beneficial effects of combining honey with glibenclamide on oxidative stress parameters in pancreas of diabetic rats. Industrial relevance: Diabetes mellitus is one of the five leading causes of death globally. The increasing prevalence of this disorder not only poses severe medical implications but also has financial consequences due to the costs of managing this disorder and its associated complications. Considering that diabetes mellitus is a heterogeneous disorder with multiple causes, the combination of therapeutic agents aimed at specific patho-biological pathways of diabetes and its complications may result in a better and more effective management of this disorder. Even though the currently available drugs may be valuable in the management of diabetes mellitus, these drugs have limitations due to undesirable adverse effects such as hypoglycemia, weight gain, secondary failure, and inability to arrest pancreas degeneration or diabetic complications which have been linked to oxidative stress. Since many natural products have reduced toxicity, the hypoglycemic effect of tualang honey coupled with its protective effects on pancreas and kidney against oxidative damage would appear to make it a suitable candidate for such adjunctive use. Key words: Diabetes mellitus; Streptozotocin; Pancreas; Oxidative stress; Tualang honey; Glibenclamide Introduction Diabetes mellitus remains a burdensome and public health problem with increasing global incidence (Shaw et al., 2010). The current global prevalence of diabetes mellitus, 285 million people in 2010, is estimated to be 439 *Corresponding Author: sbsamrah@kb.usm.my Tel: Available online

2 Erejuwa et al. million by 2030 (Shaw et al., 2010). In spite of the availability of several oral hypoglycemic drugs, diabetes is still one of the main causes of many micro- and macro-vascular complications such as retinopathy leading to blindness, end stage renal disease and cardiovascular complications (Danaei et al., 2006; Kokil et al., 2010; Roglic et al., 2010). It is characterized by defects in insulin secretion and/or insulin action resulting in impaired metabolism of glucose, lipid and protein (Tripathy and Chavez, 2010; Chan and Watts, 2011). In diabetes, inability of excess glucose to enter insulin dependent tissues, such as skeletal muscles and adipocytes due to deficiency of or insensitivity to insulin, results in accumulation of glucose in the blood and other non-insulin dependent tissues such as pancreas and brain (Czech et al., 2010; Rizza et al., 2010). Besides enhanced ROSgenerating enzymes, hyperglycemia increases oxidative stress through several mechanisms such may glucose autoxidation, glycation reactions with proteins and lipoproteins or glucose entering the polyol pathway leading to its conversion to sorbitol as well as other mechanisms (Chung et al., 2003; Yang et al., 2009; Tsuruta et al., 2010; Bravard et al., 2011). Normally, the levels of free radicals are maintained within physiological concentrations by the antioxidant enzymes, mainly superoxide dismutase, catalase, glutathione peroxidase and other antioxidants such as reduced glutathione (Dröge, 2002). However, hyperglycemia in diabetes mellitus causes a depletion of the cellular antioxidant defenses and increases the levels of free radicals (Sharma et al., 2010; Tsuruta et al., 2010). A number of studies have implicated a role of oxidative stress in the onset and progression of diabetes mellitus and its related complications (Jay et al., 2006; Wei et al., 2009; Giacco and Brownlee, 2010). In both experimental and clinical models of diabetes, antioxidants have been reported to reduce markers of oxidative stress (Johansen et al., 2005; Fenercioglu et al., 2010; Neri et al., 2010). Besides, some studies have showed that antioxidants are effective and cheaper than conventional therapy in management of some diseases (Berkson, 1999; Trevithick et al., 2004). Therefore, antioxidants or nutrients with high antioxidant capacity may offer additional health benefits with potential for limiting the progression of diabetes and its related complications (Maritim et al., 2003; Johansen et al., 2005; Fenercioglu et al., 2010). Until now, the possible beneficial effect of honey for the control of progression of diabetes has not been given much serious attention. Glibenclamide is one of the most frequently prescribed oral hypoglycemic agents (Nathan et al., 2009). It stimulates insulin secretion and also reduces hepatic glucose production resulting in reduced blood glucose (Rendell, 2004). However, the use of glibenclamide is limited due to prolonged hypoglycemia, high secondary failure rate and other adverse events (Harrower, 1994; Mukai et al., 2007). Tualang honey is produced by wild Asian bees (Apis dorsata). The honey derives its name from the tualang tree (Koompasia excelsa) which houses the honey combs of the bees. Koompasia excelsa is a gigantic tree which can be found growing in the Southeast Asian rainforests of peninsular Malaysia, southern Thailand, northeastern Sumatra, Borneo, and Palawan. In our previous study, we have reported that tualang honey ameliorates oxidative stress in kidney (Erejuwa et al., 2010a). This study was a follow up to our previous study which showed the protective effect of tualang honey on pancreas of streptozotocin-induced diabetic rats (Erejuwa et al., 2010b). The main aim of this study was to investigate the hypothesis that a combination of tualang honey and glibenclamide could be more effective in ameliorating oxidative stress in pancreas than glibenclamide administered alone. Materials and methods Animals: Male Sprague-Dawley rats aged weeks ( g) were purchased from Laboratory Animal Research Unit of Universiti Sains Malaysia, Health Campus, Kelantan, Malaysia. The animals were caged individually in an animal room with 12-h dark/light cycles at ambient temperature of 25±2 0 C. The experimental protocol was approved by the Animal Ethics Committee of Universiti Sains Malaysia [USM / Animal Ethics Approval / 2007 / (28) (095)]. The animals were handled in accordance with the Institutional Guidelines for the Care and Use of Animals for Experimental Purposes. The rats were provided standard food and tap water ad libitum. Chemicals: Streptozotocin, thiobarbituric acid and reduced glutathione were purchased from Sigma Aldrich (St. Louis, MO, USA). Superoxide dismutase and glutathione peroxidase assay kits were purchased from Cayman (MI, USA). Bio-Rad protein assay kit was purchased from Bio-Rad (USA). All other chemicals used were of analytical grade. Preparation of glibenclamide and tualang honey: Glibenclamide was procured from Sigma Aldrich (St. Louis, MO, USA). The drug was dissolved in dimethyl sulfoxide (DMSO) before it was administered. The dose of glibenclamide (600 µg/kg body weight) was chosen on the basis of previously published data (Pari and Umamaheswari, 2000; Eliza et al., 2009). Tualang honey (AgroMas, Malaysia) was provided by FAMA (Federal Agricultural Marketing Authority), Kedah, Malaysia. The honey was previously evaporated at 40 0 C to achieve 20% water content. The honey has the following composition: total reducing sugar (67.5%) [fructose (29.6 %), glucose (30.0 %), maltose (7.9 %); fructose/glucose ratio (0.99)], sucrose (0.6 %) and water (20.0 %). It was freshly prepared by diluting in distilled water prior to administration to the animals. 2

3 Antidiabetic properties of Honey Induction of diabetes: Diabetes was induced by intraperitoneal administration of streptozotocin (60 mg/kg body weight dissolved in 0.1M citrate buffer, ph 4.5) in rats fasted for 16 hours. Another group of rats was injected with citrate buffer alone without streptozotocin. This group served as control. Two days after streptozotocin injection, development of diabetes was confirmed by measuring blood glucose levels in blood samples taken from tail vein. Rats with blood glucose concentrations of 12 mmol/l or higher were considered to be diabetic. Blood glucose levels of the control rats remained normal (< 4.2 mmol/l). Glucose measurement was performed with an Accu-Chek glucometer (Roche, Germany). Animal treatment: The animals were randomly allotted to groups and treated as follows: Non-diabetic control rats received distilled water (0.5 ml), diabetic control rats received distilled water (0.5 ml), diabetic rats administered glibenclamide (600 µg/kg body weight), diabetic rats administered glibenclamide (600 µg/kg body weight) and tualang honey (1.0 g/kg body weight). Glibenclamide, tualang honey and distilled water were administered orally once daily for 4 weeks. The fasting blood glucose was measured weekly. At the end of the experimental period, the rats were fasted overnight for at least 16 hours and sacrificed by decapitation. Pancreata were rapidly excised and washed in ice cold normal saline (0.9% NaCl), blotted dry and then frozen in liquid nitrogen and stored at 80 o C till use. Sample preparation: 10% homogenates (w/v) of the pancreatic tissues were made in Tris-HCl (0.1M, ph 7.4) using an ice-chilled glass homogenizing vessel in a homogenizer fitted with Teflon pestle (Glas-Col, USA) at 900 rpm. The homogenates were centrifuged at 1000 x g for 10 min at 4 C. The supernatant was used for the assays of total protein, concentrations of malondialdehyde (MDA) and activities of free radical scavenging enzymes. Protein assay: Protein concentration was estimated using Bio-Rad protein assay kit based on the principle of Bradford (Bradford, 1976). The procedure is a dye-binding technique in which a differential colour change of a dye occurs according to the concentrations of protein in the samples. The colour change, which has a maximum absorbance at 595 nm, is measured spectrophotometrically. Lipid peroxidation assay: The levels of lipid peroxidation in the pancreatic tissues were measured as malondialdehyde (MDA) according to the method of Ohkawa et al. (Ohkawa et al., 1979). To 0.1 ml pancreatic homogenate, 0.2 ml of 8.1% (w/v) sodium dodecyl sulphate (SDS), 1.5 ml of 20% (v/v) glacial acetic acid (ph 3.5) and 1.5 ml of 0.8% (w/v) thiobarbituric acid (TBA) were added. The mixture was made up to 4 ml with the addition of 0.7ml of distilled water. The test tubes were heated at 95ºC for an hour with a marble on top of each test tube. After incubation, the test tubes were cooled and then centrifuged at 3000 g for 10 minutes. After centrifugation, the concentrations of MDA were measured spectrophotometrically at 532 nm. 1,1,3,3- tetraethoxypropane (TEP) was used as standard. The MDA levels were expressed as nmol per mg protein. Superoxide dismutase assay: Assay of superoxide dismutase (SOD) was performed using Cayman assay kit according to manufacturer s instructions. SOD activity was assessed by measuring the dismutation of superoxide radicals generated by xanthine oxidase and hypoxanthine in a convenient 96 well format. A standard curve was generated using a quality-controlled SOD standard. The activity of the SOD was accurately quantified from the standard curve. Glutathione peroxidase (GPx) assay: GPx activity was assayed using Cayman assay kit according to manufacturer s instructions. This kit measures GPx activity indirectly by a coupled reaction with glutathione reductase (GR). Oxidized glutathione (GSSG), produced upon reduction of organic hydroperoxide by GPx, is recycled to its reduced state by GR and NADPH. The oxidation of NADPH to NADP + is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in absorbance is directly proportional to the GPx activity in the sample. Catalase (CAT) assay: The activity of CAT was measured spectrophotometrically as described by Goth (Gott, 1991). Briefly, the procedure entailed incubating 100µl of pancreatic homogenate with 500µl of hydrogen peroxide at 37ºC for a minute. The addition of 500µl of ammonium molybdate solution stopped the reaction with a formation of a yellow complex. The absorbance was then measured at a wavelength of 405nm using a spectrophotometer. Glutathione reductase (GR) assay: GR activity was assayed according to the method of Goldberg and Spooner (Goldberg and Spooner, 1983). This method utilizes oxidized glutathione (GSSG) as a substrate. GR catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). The hydrogen ion used by GR for the reduction of GSSG to GSH is provided by NADPH. In the process, NADPH is oxidized to NADP +. Briefly, 40µl of pancreatic homogenate was incubated with 1000µl of 2.728mM GSSG at 37ºC for 5 minutes. After incubation, the reaction was initiated by addition of 200µl of 1.054mM NADPH solution. The decrease in absorbance was measured by a spectrophotometer at 340nm every 30 seconds for 5 minutes. Glutathione-S-transferase (GST) assay: GST activity was measured according to the method of Habig et al (Habig et al., 1974). Briefly, 100µl of pancreatic homogenate was pipetted into a test tube containing 2ml of 0.3M potassium phosphate buffer (ph 6.35), 75µl of 30mM CDNB solution and 725µl of distilled water. The test tube was vortexed and incubated at 37ºC for 10 minutes. The reaction was then initiated by addition of 3

4 Erejuwa et al. 100µl of 30mM reduced glutathione (GSH) solution. The decrease in absorbance was measured spectrophotometrically at 340nm every 30 seconds for 4 minutes. Reduced glutathione (GSH) assay: GSH was estimated using assay kit (Calbiochem, US) according to the manufacturer's instructions. Briefly, the kidney homogenates were deproteinized in 5% metaphosphoric acid, centrifuged and the glutathione contents of the supernatants were measured by the rate of colorimetric change of 5,5 -dithiobis(nitrobenzoic acid) at 412 nm in the presence of glutathione reductase and NADPH. Statistical analysis: Data were analyzed using SPSS The data are expressed as median (interquartile range). Groups were compared by Kruskal-Wallis H test. Differences between two groups were identified by Mann-Whitney U test followed by Bonferonni s correction. Results Blood glucose: Before induction of diabetes, all the rats exhibited blood glucose levels similar to control rats (results not shown). However, 48 hours after the STZ injection, STZ-treated rats showed increased blood glucose concentrations which remained significantly (p < 0.05) elevated till the end of the treatment period (Table 1). Administration of glibenclamide alone or in combination with tualang honey significantly (p < 0.05) reduced the elevated blood glucose levels in diabetic rats (Table 1). Body weight: At the end of the treatment period, STZ-treated rats had significantly (p < 0.05) reduced body weight (Table 1). Glibenclamide did not improve body weight in diabetic rats. In contrast, combination of glibenclamide with tualang honey significantly improved body weight compared with both the diabetic control and glibenclamide-treated diabetic rats. Lipid peroxidation: The levels of lipid peroxidation, measured as malondialdehyde (MDA), were significantly (p 0.016) elevated in diabetic pancreas compared to non-diabetic pancreas (Table 1). MDA levels in glibenclamide-treated diabetic rats did not differ from the diabetic control rats. On the other hand, the diabetic rats that received a combination of glibenclamide and honey exhibited a significant (p 0.016) decrease in MDA concentrations compared to diabetic control rats. Reduced glutathione: The concentrations of reduced glutathione (GSH) were not significantly different in all the groups compared with the diabetic control rats (Table 1). Free Radical Scavenging Enzymes: No significant differences in the activities of superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-s-transferase (GST) were observed in pancreas of normal and STZ-treated rats (Table 1). There was a significant (p 0.016) decrease in catalase (CAT) activity in pancreas during diabetes compared to the corresponding non-diabetic control group. Glibenclamide did not increase CAT activity in diabetic rats. On the contrary, the diabetic rats administered glibenclamide in combination with honey showed significantly (p 0.016) increased CAT activity (Table 1). The glutathione peroxidase (GPx) activity was significantly (p 0.016) increased in diabetic control rats compared to nondiabetic control group. Glibenclamide did not reduced GPx activity in diabetic rats. In contrast, the diabetic rats administered glibenclamide in combination with honey showed significantly (p 0.016) reduced GPx activity (Table 1). Discussion In diabetes, hyperglycemia enhances the generation of reactive oxygen/nitrogen species (ROS/RNS) and reduces antioxidant potential, thus causing oxidative stress which is implicated in β-cell dysfunction and other diabetic complications (Yang et al., 2009; Tsuruta et al., 2010). Treatment of diabetic rats with glibenclamide alone or in combination with tualang honey significantly decreased elevated blood glucose. Although glibenclamide is a common drug of choice in the treatment of type 2 diabetes, its hypoglycemic effect in druginduced diabetes, a type 1 diabetic model, is also well documented (Pari and Umamaheswari, 2000; Eliza et al., 2009; Sunil et al., 2009). The dose of 1.0 g/kg body weight was chosen based on the results of our previous study which showed that tualang honey at a dose of 0.2 g/kg body weight, the human dose as consumed locally in Malaysia, produced less effects on blood glucose, body weight and renal morphological structures in streptozotocin-induced diabetic rats compared to other doses investigated (Erejuwa et al., 2010a). The mechanisms by which honey reduces blood glucose still remain unclear (Al-Waili, 2003; Erejuwa et al. 2010a; Erejuwa et al. 2010b). Considering that honey consists of predominantly glucose and fructose, its hypoglycemic effect might be connected to the high levels of fructose in honey. This is based on findings from a number of in vitro and in vivo studies which showed that fructose stimulated an insulin response (Malaisse et al., 1967; Ashcroft et al., 1972; Dunnigan and Ford, 1975; Lawrence et al., 1980). These studies seem to corroborate our findings in which tualang honey did not exhibit any effect on blood glucose in non-diabetic control rats whereas it produced hypoglycemic response in diabetic rats (Erejuwa et al. 2010a; Erejuwa et al. 2010b). In addition, studies have documented the roles of copper and zinc in diabetes mellitus. These mineral ions, which are also present in honey, might be involved in the hypoglycemic effect of honey (Abdul-Ghani et al., 1996; Tobia et al., 1998; Bogdanov et al., 2008). Besides, 4

5 Antidiabetic properties of Honey Table 1. Effects of glibenclamide or its combination with honey on fasting blood glucose, body weight and markers of oxidative stress in kidney of STZ-induced diabetic rats Parameters Non-diabetic control Diabetic control Diabetic + Glibenclamide Diabetic + Glibenclamide + Honey Kruskal- Wallis (P value) Fasting Blood glucose (mmol/l) 4.0 (0.9) 17.9 (2.6) * 13.5 (10.4) 14.2 (6.9) Body weight (g) (40.5) (19.0) * (20.5) (41.5), MDA (nmol/mg protein) GSH (nmol/mg protein) SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein) GR (U/mg protein) GST (U/mg protein) 0.28 (0.17) 0.58 (0.49) * 0.47 (0.13) 0.30 (0.09) (0.039) (0.028) (0.049) (0.026) (0.46) 1.89 (0.64) 1.63 (0.92) 1.73 (0.66) (8.3) 15.0 (6.4) * 22.4 (13.1) 23.3 (18.5) (41.4) (123.8) * (254.7) (151.4) (25.4) 63.9 (21.8) 80.5 (59.7) 72.9 (6.9) (38.4) 68.8 (49.1) 96.6 (60.3) 98.1 (29.3) Data are expressed as median (interquartile range). Each group consisted of 5 7 rats. Groups were compared by Kruskal-Wallis H test. Differences between two groups were identified by Mann-Whitney U test followed by Bonferonni s correction. Values are statistically significant at * p compared to non-diabetic control group; p compared to diabetic control group. MDA, malondialdehyde; GSH, reduced glutathione. One unit of SOD (superoxide dismutase) is the amount of enzyme required to exhibit 50% dismutation of superoxide radical. One unit of CAT (catalase) is the amount of enzyme that catalyzes the decomposition of 1 µmol of H 2O 2 per minute. One unit of GPx (glutathione peroxidase) or GR (glutathione reductase) is the amount of enzyme that catalyzes the oxidation of 1 nmol of NADPH per minute. One unit of GST (glutathione-s-transferase) is the amount of enzyme that catalyzes the conjugation of 1 nmol of GSH- CDNB per minute. many plant extracts and natural products are reported to produce hypoglycemic effect by promoting regeneration of beta cells or by protecting the pancreas against oxidative stress and damage (Jadhav, 2009). Thus, it is possible that tualang honey might also exhibit similar effects in exerting its hypoglycemic effect. Our data showed an increased pancreatic activity of superoxide dismutase (SOD) in STZ-induced diabetic rats. Other researchers have reported increased pancreatic SOD activity (Balasubramanian et al., 2004) while some reported reduced SOD activity in pancreas of diabetic rats (Sivakumar and Subramanian, 2009). SOD metabolizes superoxide radical (O 2 ) to hydrogen peroxide (H 2 O 2 ) (Fridovich, 1995). Since the non-diabetic rats had lower SOD activity, enhanced SOD activity in diabetic pancreas might indicate high levels of O 2 being generated as result of chronic hyperglycemia. Therefore, it might suggest that increased SOD activity is an adaptive mechanism to protect the pancreatic β-cells from the excessive and toxic levels of O 2. This view is corroborated by a study which showed that transgenic mice with over-expression of Cu/Zn SOD are more resistant to oxidative stress (Kubisch et al., 1997). In this study, the activity of glutathione peroxidase (GPx) in the diabetic control pancreas was enhanced. GPx is an antioxidant enzyme involved in the detoxification of hydrogen and lipid peroxides (Brigelius-Flohe et al., 2003) and also acts as a peroxynitrite reductase (Sies et al., 1997). A study by Shull and his colleagues reported an increase in GPx mrna at higher concentrations of hydrogen peroxide (Shull et al., 1991). Some recent studies now suggest a protective role for GPx in the diabetes mellitus-associated atherogenesis (Blankenberg et al., 2003). Therefore, the increased activity of GPx might be a protective mechanism in response to increased concentrations of H 2 O 2 and other lipid peroxides in 5

6 Erejuwa et al. the diabetic pancreas. These findings may imply that GPx is a key enzyme for the protection of cells against atherogenesis and oxidative stress especially in the highly pro-oxidant diabetic environment. Our results revealed that the pancreas of the STZ-induced diabetic rats had reduced activity of catalase (CAT). The reduced activity of CAT observed in the diabetic rat pancreas might be a consequence of elevated O 2 since increased O 2 is known to inactivate catalase activity (Kono and Fridovich, 1983). CAT catalyzes the reduction of hydrogen peroxide to molecular oxygen and water. Our results showed that glibenclamide produced no significant effect on the activities of SOD, CAT and GPx in diabetic pancreas. Thus, these data together with the elevated levels of MDA in glibenclamide-treated diabetic rats suggest that glibenclamide may not protect the pancreas against oxidative stress and damage. On the other hand, the groups of diabetic rats treated with a combination of glibenclamide and tualang honey showed attenuation of GPx and CAT activities. Thus, these results together with reduced MDA levels showed that tualang honey offered synergistic antioxidant protection to the pancreas of glibenclamide-treated diabetic rats. In our study, the levels of lipid peroxidation marker (MDA) were significantly increased in the pancreas of STZ-treated diabetic rats similar to what had been previously reported (Coskun et al., 2005; Erejuwa et al., 2010b). The diabetic rats that received glibenclamide in combination with tualang honey showed significantly reduced lipid peroxidation (MDA) while those that received glibenclamide alone showed slightly but insignificant reduction in MDA. This seems to indicate that glibenclamide alone may not offer protection against lipid peroxidative damage in pancreas. These data appear to corroborate previously reported findings which showed that diabetes mellitus cannot be effectively managed with monotherapy but will require multiple drugs (Turner et al., 1999). It has been reported that, in spite of optimum therapy, the worsening glycemic control observed in diabetics is due to the deterioration of pancreatic β-cell function (Cook et al., 2005; Robertson and Harmon, 2006) which is believed to be associated with oxidative stress (Kajimoto et al., 2004). Our data showed that, in spite of its hypoglycemic effect, glibenclamide did not ameliorate oxidative stress in pancreas of diabetic rats. The reason for this still remains unclear. However, normalization of hyperglycemia is known to be ineffective in preventing the development of macrovascular but microvascular complications (DCCT, 1993; Nathan et al., 2005), which is linked to oxidative stress (King and Loeken, 2004; Giacco and Brownlee, 2010). To explain this concept, investigators have introduced the phrase glycemic or metabolic memory, a phenomenon whereby early glycemic milieu is remembered in many target organs such as heart, eye, kidney, pancreas, etc (Yan et al., 2004; Ihnat et al., 2007; Kowluru et al., 2007). In all these studies, oxidative stress is identified as a unifying mechanism that plays a prominent role in glycemic or metabolic memory (Yan et al., 2004; Ihnat et al., 2007; Kowluru et al., 2007). Consequently, glycemic or metabolic memory might be responsible for the inability of glibenclamide to attenuate antioxidant enzymes and prevent lipid peroxidative damage in diabetic pancreas in spite of its hypoglycemic effect. It is also worth mentioning that despite reduced hyperglycemia, the levels of blood glucose in the glibenclamide-treated diabetic rats were still higher than those of the non-diabetic control rats. This suggests that the pancreas of glibenclamide-treated diabetic rats was still susceptible to oxidative stress as evident from the increased MDA levels. The failure of glibenclamide to ameliorate oxidative stress in pancreas of diabetic rats despite its hypoglycemic effect seems to corroborate findings from other previous studies which reported elevated levels of MDA in diabetic patients with poor glycemic control and normalization of MDA in diabetic patients with good glycemic control compared with the healthy controls (Griesmacher et al., 1995 and Wierusz- Wysocka et al., 1995). A study reported lower levels of oxidative stress markers in type 2 diabetic patients after than before glycemic control (Sharma et al., 2000). The study showed higher MDA and lower total glutathione concentrations in glycemic control diabetic patients than in healthy controls, suggesting that complete normalization of oxidative stress indices was not attained (Sharma et al., 2000). Supplementation with vitamin E in these patients further reduced the levels of oxidative stress, which indicate the beneficial effect of vitamin E supplementation in reducing oxidative damage in glycemic control diabetic patients (Sharma et al., 2000). Another study which investigated the effect of vitamin C combined with insulin on oxidative stress and endothelial dysfunction showed that, despite normalized glycemic control in type 1 diabetic patients, chronic hyperglycemia induces long-term endothelial dysfunction by elevating oxidative stress (Ceriello et al., 2007). Similar to our findings, their study indicated that a combination of hypoglycemic agent (insulin) and antioxidant (vitamin C) significantly reduced oxidative stress and normalized endothelial dysfunction in type 1 diabetic patients (Ceriello et al., 2007). The authors also reported that, even though insulin reduced hyperglycemia, neither insulin nor vitamin C was able to ameliorate oxidative stress or normalize endothelial dysfunction. Therefore, comparable to our findings, their study clearly shows the beneficial effect of an antioxidant in combination with a hypoglycemic agent. Conclusions This study confirms previous studies that pancreatic tissues of diabetic rats are susceptible to oxidative stress. Our data indicate that glibenclamide alone may not effectively ameliorate oxidative stress in diabetic pancreas. Besides, our results show that glibenclamide administered together with tualang honey can still ameliorate 6

7 Antidiabetic properties of Honey oxidative stress without reducing the antioxidant effect of tualang honey. Hence, a combination of the two agents seems justifiable in diabetes. This is because, with the combination, glibenclamide will correct hyperglycemia while tualang honey will protect pancreatic tissues against oxidative stress and damage, thus possibly slowing down the progression of diabetes and related complications. Thus, this study establishes a basis for the need of antioxidative / antioxidant therapy in combination with hypoglycemic drugs in the management of diabetes mellitus. A similar study in human subjects is desirable to determine if these results can be appropriately extrapolated to human diabetes. Acknowledgements This study was supported by Research University grant (1001/PPSP/ ) from Universiti Sains Malaysia (USM). The first author acknowledges USM Fellowship. We would like to thank Federal Agricultural Marketing Authority (FAMA), Ministry of agriculture and Agro based Industry Malaysia for supplying tualang honey. References Abdul-Ghani AS, Abu-Hijleh AL, Nahas N, Amin R Hypoglycemic effect of copper (II) acetate imidazole complexes. Biol Trace Elem Res 54: Akinola OB, Caxton-Martins EA, Dini L Chronic treatment with ethanolic extract of the leaves of Azadirachta indica ameliorates lesions of pancreatic islets in streptozotocin diabetes. Int J Morphol 28(1): Al-Waili N Intrapulmonary administration of natural honey solution, hyperosmolar dextrose or hypoosmolar distill water to normal individuals and to patients with type-2 diabetes mellitus or hypertension: their effects on blood glucose level, plasma insulin and C-peptide, blood pressure and peaked expiratory flow rate. Eur J Med Res 8(7): Ashcroft SJH, Bassett JM, Randle PJ Insulin secretion mechanisms and glucose metabolism in isolated islets. Diabetes 21(2): Balashova TS, Golega EN, Rud'ko IA, Balabolkin MI, Kubatiev AA Effect of biosynthetic insulin on lipid peroxidation in erythrocyte membranes in patients with type I diabetes mellitus. Probl Endokrinol (Mosk) 40(3): Balasubramanian R, Kasiappan R, Vengidusamy N, Muthusamy K, Sorimuthu S Protective effect of macrocyclic binuclear oxovanadium complex on oxidative stress in pancreas of streptozotocin induced diabetic rats. Chem-Biol Interact 149:9-21. Berkson BM A conservative triple antioxidant approach to the treatment of hepatitis C. Combination of alpha lipoic acid (thioctic acid), silymarin, and selenium: three case histories. Med Klin 94(3): Blankenberg S, Rupprecht HJ, Bickel C, Torzewski M, Hafner G, Tiret L, Smieja M, Cambien F, Meyer J, Lackner KJ Glutathione peroxidase 1 activity and cardiovascular events in patients with coronary artery disease. New Eng J Med 349: Bogdanov S, Jurendic T, Sieber R, Gallmann P Honey for nutrition and health: a review. J Am Coll Nutr, 27(6): Bradford MA Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem 72: Bravard A, Bonnard C, Durand A, Chauvin MA, Favier R, Vidal H, Rieusset J Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice. American journal of physiology. Endocrinology and metabolism, 300 (3), E581- E591. Brigelius-Flohe R, Banning A, Schnurr K Selenium-dependent enzymes in endothelial cell function. Antioxid Redox Signal 5: Ceriello A, Kumar S, Piconi L, Esposito K, Giugliano D Simultaneous control of hyperglycemia and oxidative stress normalizes endothelial function in type 1 diabetes. Diabetes Care 30: Chan DC, Watts GF Dyslipidaemia in the metabolic syndrome and type 2 diabetes: pathogenesis, priorities, pharmacotherapies. Expert Opin Pharmacother 12(1): Chung SS, Ho EC, Chung KS Contribution of polyol pathway to diabetes-induced oxidative stress. J Am Soc Nephrol 14(8):S233-S236. Cook MN, Girman CJ, Stein PP, Alexander CM, Holman RR UK Prospective Diabetes Study (UKPDS) Group. Glycemic control continues to deteriorate after sulfonylureas are added to metformin among patients with type 2 diabetes. Diabetes Care 28: Coskun O, Kanter M, Korkmaz A, Oter S Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and beta-cell damage in rat pancreas. Pharmacol Res 51(2): Czech A, Taton J, Piatkiewicz P Cellular glucose transport disturbances in the pathogenesis and therapy of type 2 diabetes mellitus. Endokrynol Pol. 61(3):

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