Curcumin attenuates diabetic encephalopathy in rats: Behavioral and biochemical evidences

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European Journal of Pharmacology 576 (2007) 34 – 42 www.elsevier.com/locate/ejphar

Curcumin attenuates diabetic encephalopathy in rats: Behavioral and biochemical evidences Anurag Kuhad, Kanwaljit Chopra ⁎ Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC Center for Advanced Studies, Panjab University, Chandigarh-160 014, India Received 6 July 2007; received in revised form 31 July 2007; accepted 6 August 2007 Available online 14 August 2007

Abstract Emerging epidemiological data indicates that diabetes is a potential predisposing factor for neuropsychiatric deficits as stroke, cerebrovascular diseases, diabetic encephalopathy, depression and anxiety. Diabetic encephalopathy, characterized by impaired cognitive functions and neurochemical and structural abnormalities, involves direct neuronal damage caused by intracellular glucose. Curcumin, a well-established phenolic antioxidant and anti-inflammatory molecule, is capable of playing an important role against amyloid and dendritic pathology and thus has neuroprotective properties. The aim of the present study was to explore the effect of curcumin (60 mg/kg; p.o.) on cognitive functions, oxidative stress and inflammation in diabetic rats. Learning and memory behaviors were investigated using a spatial version of the Morris water maze test. Acetylcholinesterase activity, a marker of cholinergic dysfunction, was increased by 80% in the cerebral cortex of diabetic rats. There was 107% and 121% rise in thiobarbituric acid reactive substance levels in cerebral cortex and hippocampus of diabetic rats, respectively. Reduced glutathione level and enzymatic activities of superoxide dismutase and catalase were decreased in both cerebral cortex and hippocampal regions of diabetic rat brain. Nitrite levels in cerebral cortex and hippocampus were increased by 112% and 94% respectively. Serum TNF-α, a marker for inflammation, was found to increase by 1100% in diabetic rats. Chronic treatment with curcumin (60 mg/kg; p.o.) significantly attenuated cognitive deficit, cholinergic dysfunction, oxidative stress and inflammation in diabetic rats. The results emphasize the involvement of cholinergic dysfunction, oxidative stress and inflammation in the development of cognitive impairment in diabetic animals and point towards the potential of curcumin as an adjuvant therapy to conventional anti-hyperglycemic regimens for the prevention and treatment of diabetic encephalopathy. © 2007 Elsevier B.V. All rights reserved. Keywords: Acetylcholinesterase; Curcumin; Diabetes; Encephalopathy; Inflammation; Memory; Oxidative stress; TNF-α

1. Introduction Diabetes, a chronic metabolic disorder, has assumed pandemic proportions and its long-term complications can have devastating consequences. Besides the most common complications of the peripheral nervous system in diabetic patients (Bloomgarden, 2007; Dobretsov et al., 2007; Hoybergs and Meert, 2007), there are much evidences, which demonstrated that diabetes may also have negative impacts on the central nervous system (Biessels and Gispen, 2005; Biessels et al., 2006; Gispen and Biessels, 2000; Li et al., 2005; Mijnhout et al., 2006; Trudeaua et al., 2004;

⁎ Corresponding author. Tel.: +91 172 2534105; fax: +91 172 2541142. E-mail address: [email protected] (K. Chopra). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.08.001

Tuzcu and Baydas, 2006). Cognitive dysfunction in diabetic subjects has been recognized since the early 20th century (Miles and Root, 1922). Since then, a wealth of studies have described a series of neuropsychological and neurobehavioral changes in both type 1 and type 2 diabetic subjects, suggesting that ‘‘diabetic encephalopathy’’ should be recognized as a complication of diabetes (Sima et al., 2004). Several observations indicate that diabetes mellitus might be accompanied by a certain erosion of brain function. For example, impaired performance in global memory, attention, abstract reasoning and visual-motor tasks are recognized to be more frequent in the diabetic population (Franceschi et al., 1984; Ryan et al., 1985, 1993). Very recently, Mijnhout et al. (2006) proposed a new term—‘diabetesassociated cognitive decline’ (DACD) to facilitate research into this area and to increase recognition of the disorder. This term is

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not suggestive of a particular pathogenesis, but merely describes a state of mild to moderate cognitive impairment, in particular psychomotor slowing and reduced mental flexibility, not attributable to other causes. Diabetic encephalopathy, characterized by impaired cognitive functions and neurochemical and structural abnormalities, involves direct neuronal damage caused by intracellular glucose. Oxidative stress (Mastrocola et al., 2005) and inflammation (Somfai et al., 2006) plays a central role in diabetic tissue damage. Alongside enhanced reactive oxygen species levels, both nitric oxide levels and mitochondrial nitric oxide synthase expression were found to be increased in brain mitochondria, whereas glutathione peroxidase activity and manganese superoxide dismutase protein content were reduced (Mastrocola et al., 2005). Oxidative damage to various brain regions contributes to morphological abnormalities and memory impairment (Fukui et al., 2001). Antioxidants have been shown to protect neurons against a variety of experimental neurodegenerative conditions. Insulin (Biessels et al., 1998), melatonin and vitamin E (Tuzcu and Baydas, 2006) have been reported to prevent diabetesinduced learning and memory deficit. The polyphenolic flavonoid, curcumin, found in turmeric is a yellow curry spice with a long history of use in traditional Indian diets and herbal medicine (Ammon and Wahl, 1991; Kelloff et al., 1996). Curcumin (diferuloyl methane) from Curcuma longa has many pharmacological activities including anti-inflammatory properties (Srimal and Dhawan, 1973), powerful antioxidant activity (Masuda et al., 1999), anti-protease activity (Sui et al., 1993), and cancer preventive properties (Kim et al., 1998). Curcumin exerts anti-inflammatory and growth inhibitory effects in tumor necrosis factor (TNF)-alpha-treated HaCaT cells through inhibition of nuclear factor-κβ and mitogen activated protein kinase pathways (Cho et al., 2007). It has also been reported that curcumin is a more potent free radical scavenger than vitamin E (Zhao et al., 1989). Studies have shown that curcumin is a powerful scavenger of the superoxide anion, the hydroxyl radical, and nitrogen dioxide (Unnikrishnan and Rao, 1995) and that it also protects DNA against singlet-oxygen-induced strand breaks (Subrmanian et al., 1994) and lipids from peroxidation (Sreejayan and Rao, 1993). Oral administration of curcumin has been shown to be centrally neuroprotective (Rajakrishnan et al., 1999) and displays protective effects in diabetic neuropathy (Sharma et al., 2007). It also demonstrated neuroprotection against heavy metalinduced neurotoxicity (Dairam et al., 2007). In diabetes, curcumin can suppress blood glucose levels (Arun and Nalini, 2002), increase the antioxidant status of pancreatic β-cells (Srivivasan et al., 2003), and enhance the activation of PPAR-γ (Murugan and Pari, 2006). With above background, the present study was undertaken to investigate the effects of curcumin supplementation on diabetic encephalopathy in rats. 2. Materials and methods 2.1. Animals Male Wistar rats (250–280 g), bred in the Central Animal House Facility of the Panjab University, Chandigarh (India) were

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used. The animals were housed under standard laboratory conditions, maintained on a 12 h light and dark cycle and had free access to food (Hindustan Lever Products, Kolkata, India) and water. The experimental protocols were approved by the Institutional Animal Ethics Committee of the Panjab University, Chandigarh, and conducted according to the Indian National Science Academy Guidelines for the use and care of experimental animals. 2.2. Drugs Streptozotocin and curcumin was purchased from Sigma (St. Louis, MO, USA). A glucose oxidase peroxidase diagnostic enzyme kit was purchased from Span Diagnostic Chemicals, India. TNF-α ELISA kit was purchased from R & D Systems (USA). All other chemicals used for biochemical estimations were of analytical grade. 2.3. Induction and assessment of diabetes A single dose of 65 mg/kg streptozotocin prepared in citrate buffer (pH 4.4, 0.1 M) was injected intraperitoneally to induce diabetes. The age-matched control rats received an equal volume of citrate buffer. Diabetes was confirmed after 48 h of streptozotocin injection, the blood samples were collected through tail vein and plasma glucose levels were estimated by enzymatic GOD-PAP (glucose oxidase peroxidase) diagnostic kit method. The rats having fasting plasma glucose levels more than 250 mg/dL (Anjaneyulu and Chopra, 2004) were selected and used for the present study. Body weight and plasma glucose levels were measured before and at the end of the experiment to see the effect of curcumin on these parameters. 2.3.1. Treatment schedule Rats were randomly selected and divided in four groups of 8–10 animals each. First group consisted of control animals, second group was the diabetic control, third group consisted of diabetic animals treated with curcumin (60 mg/kg/day; p.o.) and fourth group was administered curcumin alone. Starting from the third day of experiment till 10th week, the control and diabetic control groups received vehicle of curcumin and other diabetic groups received suspension of curcumin (60 mg/kg; p.o.). Preliminary dose range (10–200 mg/kg; p.o.) studies of curcumin were carried out in our laboratory. The dose of curcumin (60 mg/kg; p.o.)was selected on the basis of our preliminary and published data (Sharma et al., 2006b). Curcumin was suspended in 0.5% w/v sodium carboxymethylcellulose immediately before administration in a constant volume of 5 ml/kg body weight. In tenth week, animals were tested for learning and memory task in Morris water maze for five consecutive days (Biessels et al., 1998; Tuzcu and Baydas, 2006). The animals were sacrificed under deep anesthesia, blood was collected by carotid bleeding and serum separated. Brains were rapidly removed, and the cerebral cortex and hippocampus were isolated. The samples were stored at − 80 °C until processed for biochemical estimations.

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2.4. Morris water maze test Animals were tested in a spatial version of Morris water maze test (Morris et al., 1982; Tuzcu and Baydas, 2006). The apparatus consisted of a circular water tank (180 cm in diameter and 60 cm high). A platform (12.5 cm in diameter and 38 cm high) invisible to the rats, was set inside the tank and filled with water maintained at approximately 28 ± 2 °C at a height of 40 cm. The tank was located in a large room where there were several brightly colored cues external to the maze; these were visible from the pool and could be used by the rats for spatial orientation. The position of the cues remained unchanged throughout the study. The water maze task was carried out for 5 consecutive days after 9th week. The rats received four consecutive daily training trials in the following 5 days, with each trial having a ceiling time of 90 s and a trial interval of approximately 30 s. For each trail, each rat was put into the water at one of four starting positions, the sequence of which being selected randomly. During test trials, rats were placed into the tank at the same starting point, with their heads facing the wall. The rat had to swim until it climbed onto the platform submerged underneath the water. After climbing onto the platform, the animal remained there for 20 s before the commencement of the next trial. The escape platform was kept in the same position relative to the distal cues. If the rat failed to reach the escape platform within the maximally allowed time of 90 s, it was gently placed on the platform and allowed to remain there for the same amount of time. The time to reach the platform (latency in seconds) was measured. 2.4.1. Memory consolidation test A probe trial was performed (Tuzcu and Baydas, 2006) wherein the extent of memory consolidation was assessed. The time spent in the target quadrant indicates the degree of memory consolidation that has taken place after learning. In the probe trial, the rat was placed into the pool as in the training trial, except that the hidden platform was removed from the pool. The time of crossing the former platform quadrant and the total time of crossing all quadrants were recorded for 1 min. 2.5. Acetylcholinesterase activity Cholinergic dysfunction was assessed by acetylcholinesterase activity. The quantitative measurement of acetylcholinesterase levels in cerebral cortex and hippocampus were performed according to the method of Ellman et al. (1961). The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01 M sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide and 0.10 ml 5,5, dithiobis (2-nitro benzoic acid) (Ellman reagent). The change in absorbance was measured at 412 nm for 5 min. Results were calculated using molar extinction coefficient of chromophore (1.36 × 104 M− 1 cm− 1) and expressed as percentage of control.

phosphate buffer (pH 7.4). The homogenates were centrifuged at 800 ×g for 5 min at 4 °C to separate the nuclear debris. The supernatant thus obtained was centrifuged at 10,500 ×g for 20 min at 4 °C to get the post mitochondrial supernatant, which was used to assay lipid perioxidation, reduced glutathione, catalase and superoxide dismutase activity. 2.6.1.1. Estimation of lipid peroxidation. The malondialdehyde content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid-reactive substances by the method of Wills (1965). Briefly, 0.5 ml of post mitochondrial supernatant and 0.5 ml of Tris HCl were incubated at 37 °C for 2 h. After incubation 1 ml of 10% trichloroacetic acid was added and centrifuged at 1000 ×g for 10 min. To 1 ml of supernatant, 1 ml of 0.67% thiobarbituric acid was added and the tubes were kept in boiling water for 10 min. After cooling 1 ml double distilled water was added and absorbance was measured at 532 nm. Thiobarbituric acid-reactive substances were quantified using an extinction coefficient of 1.56 × 105 M− 1 cm− 1 and expressed as nmol of malondialdehyde per mg protein. Tissue protein was estimated using the Biuret method and the brain malondialdehyde content expressed as nanomoles of malondialdehyde per milligram of protein. 2.6.1.2. Estimation of reduced glutathione. Reduced glutathione was assayed by the method of Jollow et al. (1974). Briefly, 1.0 ml of post mitochondrial supernatant (10%) was precipitated with 1.0 ml of sulphosalicylic acid (4%). The samples were kept at 4 °C for at least 1 h and then subjected to centrifugation at 1200 ×g for 15 min at 4 °C. The assay mixture contained 0.1 ml supernatant, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml 5,5, dithiobis (2-nitro benzoic acid) (Ellman's reagent, 0.1 mM, pH 8.0) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412 nm. 2.6.1.3. Estimation of superoxide dismutase. Cytosolic superoxide dismutase activity was assayed by the method of Kono (1978). The assay system consisted of 0.1 mM EDTA, 50 mM sodium carbonate and 96 mM of nitro blue tetrazolium (NBT). In the cuvette, 2 ml of above mixture was taken and to it 0.05 ml of post mitochondrial supernatant and 0.05 ml of hydroxylamine hydrochloride (adjusted to pH 6.0 with NaOH) were added. The auto-oxidation of hydroxylamine was observed by measuring the change in optical density at 560 nm for 2 min at 30/60 s intervals. 2.6.1.4. Estimation of catalase. Catalase activity was assayed by the method of Claiborne (1985). Briefly, the assay mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml post mitochondrial supernatant (10%) in a final volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated in terms of k min− 1 and expressed as mean ± S.E.M.

2.6. Assessment of oxidative stress 2.7. Nitrite estimation 2.6.1. Post mitochondrial supernatant preparation Cerebral cortex and hippocampus were rinsed with ice cold saline (0.9% sodium chloride) and homogenized in chilled

Nitrite was estimated in the cortex and hippocampus regions using the Greiss reagent and served as an indicator of nitric

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Table 1 Effect of curcumin on body weight and blood glucose levels (mean ± S.E.M.) in the four groups of rats at the onset and at the end of the experiment Treatment

Control Diabetic Diabetic + Curcumin Curcumin

Body weight (g)

Plasma glucose (mg/dl)

Onset of study

End of study

Onset of study

End of study

255 ± 1.21 267 ± 1.13 259 ± 0.93 263 ± 1.37

302 ± 0.84 149 ± 1.73 238 ± 1.02 293 ± 1.37

113 ± 3.96 111 ± 5.62 101 ± 6.43 84 ± 2.81

108 ± 2.19 589 ± 7.48 328 ± 10.5 91 ± 4.28

oxide production. 500 μl of Greiss reagent (1:1 solution of 1% sulphanilamide in 5% phosphoric acid and 0.1% napthaylamine diamine dihydrochloric acid in water) was added to 100 μl of post mitochondrial supernatant and absorbance was measured at 546 nm (Green et al., 1982). Nitrite concentration was calculated using a standard curve for sodium nitrite. Nitrite levels were expressed as percentage of control. 2.8. Estimation of tumor necrosis factor-alpha (TNF-α) Tumor necrosis factor-alpha (TNF-α) was estimated using rat TNF-α kit (R&D Systems). It is a solid phase sandwich enzyme linked immuno-sorbent assay (ELISA) using a microtitre plate reader at 450 nm. Concentrations of TNF-α were calculated from plotted standard curve. TNF-α levels were expressed as mean ± S.E.M. 2.9. Statistical analysis Results were expressed as mean ± S.E.M. The intergroup variation was measured by one way analysis of variance (ANOVA) followed by Tukey's test. Statistical significance was considered at P b 0.05. The statistical analysis was done using the Jandel Sigma Stat Statistical Software version 2.0. 3. Results 3.1. Effect of curcumin on body weight and blood glucose levels

Fig. 1. Effect of curcumin treatment on the performance of spatial memory acquisition phase in diabetic rats. Data are expressed as mean± S.E.M. ⁎ P b 0.05 and different from control and curcumin groups from 3rd day of the training sessions. CMN = curcumin.

between diabetic (68 ± 7.04) and control (46 ±8.60) animals. Chronic curcumin treatment significantly decreased mean transfer latency in diabetic animals (Fig. 1). Diabetic animals showed a lower ability to find the platform and learn its location in the 5th day of training. This poorer performance was prevented by the chronic treatment with curcumin and decreased latency to find the platform from 3rd day of training (P b 0.05). In the probe trial of the Morris water maze study, this measures how well the animals had learned and consolidated the platform location during the five days of training, animals showed significant difference (Fig. 2). The time spent in the target quadrant was significantly lower in diabetic animals as compared to the control group (P b 0.05). The rats chronically treated with curcumin spent more time in the target quadrant than the diabetic group in the probe test (P b 0.05). However, curcumin per se had no effect on cognition. 3.3. Effect of curcumin on diabetes-induced changes in acetylcholinesterase activity Acetylcholinesterase activity was increased by 80% (3.40 ± 3.06 and 1.89 ± 3.42 μ mol h− 1 mg− 1 protein for diabetic and control animals respectively) in cerebral cortex whereas the hippocampal (3.79 ± 2.73 and 3.47 ± 4.92 μ mol h− 1 mg− 1 protein

Ten weeks after streptozotocin injection, diabetic rat exhibit significantly increased (589 ± 7.48 mg/dl) plasma glucose levels as compared to the control rats (111 ± 5.62 mg/dl). There was marked decline in the body weights of streptozotocin-treated rats as compared to age matched control rats (Table 1). Chronic curcumin treatment significantly improved the blood glucose levels and body weights of diabetic rats. 3.2. Effect of curcumin on diabetes-induced cognitive deficit The cognitive function was assessed in the Morris water maze test. The mean escape latency for the trained rats decreased from 70 to 17 s over the course of the 20 learning trials. The mean escape latency did not differ between any of the groups on first and second days of testing in Morris water maze but from third day onwards there was significant difference in transfer latency

Fig. 2. Effects of curcumin on the mean percentage time spent in the target quadrant in which the platform had previously been located during acquisition. Curcumin significantly inhibited diabetes-induced memory deficits. Data are expressed as percentage control for 10 animals. ⁎ P b 0.05, different from control group; ⁎⁎ P b 0.05, different from diabetic groups. CMN = curcumin.

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Fig. 3. Effect of curcumin treatment on acetylcholinesterase activity in cerebral cortex and hippocampus of diabetic rats. Data are expressed as percentage control for 10 animals. ⁎ P b 0.05, different from control and ⁎⁎ P b 0.05, different from diabetic group. CMN = curcumin.

for diabetic and control animals respectively) acetylcholinesterase activity did not alter in the diabetic animals. Chronic curcumin treatment significantly prevented this rise in acetylcholinesterase activity (2.29 ± 4.13 μ mol h− 1 mg− 1 protein) in the cerebral cortex of streptozotocin-treated rats (Fig. 3). However, curcumin, per se had no effect on acetylcholinesterase activity. 3.4. Effect of curcumin on diabetes-induced changes in lipid peroxidation Thiobarbituric acid reactive substance levels were increased significantly in the cerebral cortex (107%) and hippocampus (121%) of diabetic rats as compared to control group. Chronic treatment with curcumin produced a significant reduction in thiobarbituric acid reactive substance levels in different brain areas of streptozotocin-treated rats. Curcumin per se did not alter thiobarbituric acid reactive substance (Table 2). 3.5. Effect of curcumin on diabetes-induced changes in the antioxidant profile The reduced glutathione levels and enzyme activity of superoxide dismutase and catalase significantly decreased in the Table 2 Effect of curcumin on lipid peroxide, reduced glutathione, superoxide dismutase and catalase levels (mean ± S.E.M.) Treatment

LPO (nmol/mg protein)

Control

1.01 ± 0.06 32.53 ± 3.21 8.89 ± 0.38 3.94 ± 0.31

Cerebral cortex Hippocampus Diabetic Cerebral cortex Hippocampus Diabetic + Cerebral CMN cortex Hippocampus CMN Cerebral cortex Hippocampus

GSH (mol)

SOD (units/mg protein)

Catalase (k/min)

cerebral cortex and hippocampus of diabetic rats as compared to control group (Table 2). This reduction was significantly improved by the treatment with curcumin in different brain areas of streptozotocin-treated rats. However, curcumin per se did not alter the endogenous antioxidant profile. 3.6. Effect of curcumin on diabetes-induced nitrosative stress Nitrite levels were significantly elevated in cerebral cortex (112%) and hippocampus (94%) of diabetic animals. Curcumin treatment significantly inhibited this increase in nitrite levels in different brain areas of streptozotocin-treated rats (Fig. 4). However, curcumin per se had no effect on brain nitrite levels. 3.7. Effect of curcumin on tumor necrosis factor-alpha (TNF-α) Serum TNF-α level was markedly increased (806.16 pg/ml) in diabetic rats as compared to control (73.25 pg/ml). Curcumin resulted in significant decreased serum TNF-α level (Fig. 5). However, curcumin per se had no effect on serum TNF-α levels. 4. Discussion Clinical evidence suggests that patients with diabetes have impaired cognitive functions but evidence from controlled studies is not clear. In a survey of 20 studies which included only Type 1 diabetic patients, at least one aspect of cognitive function

1.02 ± 0.10 31.43 ± 2.06 8.64 ± 0.26 3.76 ± 0.39 2.10 ± 0.07a 12.87 ± 2.19a 4.02 ± 0.28a 0.91 ± 0.08a 2.26 ± 0.09a 12.89 ± 3.41a 3.94 ± 0.35a 0.92 ± 0.06a 1.29 ± 0.09b 28.76 ± 1.83b 7.92 ± 0.46b 3.46 ± 0.20b 1.21 ± 0.12b 28.58 ± 1.06b 8.22 ± 1.01b 3.61 ± 0.09b 1.00 ± 0.05 34.59 ± 2.65 8.72 ± 0.16 3.96 ± 0.08 0.97 ± 0.09 33.62 ± 1.73 8.64 ± 0.30 4.01 ± 0.15

Different from control and different from diabetic (P b 0.05). CMN: curcumin; LPO: lipid peroxide, GSH: reduced glutathione, SOD: superoxide dismutase.

a

Fig. 4. Effect of curcumin treatment on nitrite levels in cerebral cortex and hippocampus of diabetic rats. Data are expressed as percentage of control for 10 animals. ⁎ P b 0.05, different from control group and ⁎⁎ P b 0.05, different from both diabetic group. CMN = curcumin.

b

Fig. 5. Effect of curcumin treatment on TNF-alpha release in diabetic rats. Data are expressed as mean ± S.E.M. ⁎ P b 0.05, different from control and ⁎⁎ P b 0.05, different from diabetic group. CMN = curcumin.

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was impaired in each study, but the effect sizes were relatively small. The potential mechanisms for this include direct effects of hypo- or hyperglycaemia and hypo- or hyperinsulima, and also indirect effects via cerebrovascular alterations (Brands et al., 2004; Lobnig et al., 2005). This study analyzed the role of curcumin on the behavioral and biochemical function of diabetic rats. Streptozotocin-induced diabetes produced marked impairment in cognitive function which was coupled with marked increase in acetylcholinesterase activity and increased oxidative stress in the brain. Chronic treatment with curcumin significantly ameliorated cognitive deficits and cholinergic dysfunction. In one of the earlier studies, curcumin prevented Aβinfusion induced spatial memory deficits in the Morris Water Maze and post-synaptic density (PSD)-95 loss and reduced Aβ deposits (Frautschy et al., 2001). Very recently, Dairam et al. (2007) have reported that curcumin reduced lead-induced memory deficit in rats. The direct glucose toxicity in the neurons is especially due to increased intracellular glucose oxidation (Nishikawa et al., 2000), which leads to an increase in reactive species production (Bonnefont-Rousselot, 2002; Evans et al., 2002): in both man and experimentally diabetic rats, oxidative stress seems to play a central role in brain damage (Arvanitakis et al., 2004). Recently, it has been reported that oxidative damage to rat synapses contributes to cognitive deficit (Tuzcu and Baydas, 2006). In the present study STZ-treatment produced significant increase in blood glucose level along with reduction in body weight. Curcumin treatment significantly prevented this rise in blood glucose level and maintained the body weight of diabetic animals. This effect was observed due to strong antioxidant (Srivivasan et al., 2003) and direct stimulatory action of curcumin on the pancreatic beta-cell that could contribute towards its hypoglycaemic activity (Arun and Nalini, 2002; Best et al., 2007). Increasing evidence indicates that factors such as oxidative and nitrosative stress, glutathione depletion, and impaired protein metabolism can interact in a vicious cycle which is central to pathogenesis of dementia. Our previous studies showed that diabetes induces lipid peroxidation in kidney (Sharma et al., 2006a,b). In the present study, lipid peroxidation levels were significantly increased whereas reduced glutathione, superoxide dismutase and catalase activities were markedly reduced in the cerebral cortex and hippocampus of diabetic rats. Treatment with curcumin returned the levels of lipid peroxides, reduced glutathione, superoxide dismutase and catalase towards their control values. These protective effects of curcumin against oxidative stress are in agreement with our previously published report (Sharma et al., 2006b). Alasubramanyam et al. (2003) reported curcumin-induced inhibition of cellular reactive oxygen species generation. Curcumin inhibited hydrogen peroxide-induced cell damage (Mahakunakorn et al., 2003). Boonchoong et al. (2004) have reported that curcumin manganese complex and acetylcurcumin manganese complex, low molecular weight synthetic compounds, showed much greater superoxide dismutase activity and an inhibitory effect on lipid peroxidation. Priyadarsini et al. (2003) have shown, by DPPH scavenging in vitro, that origin of the antioxidant activity

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of curcumin to be mainly from the phenolic hydroxyl group, although a small fraction may be due to the N CH2 site. In the present study curcumin significantly ameliorated the cognitive impairment in diabetic rats. Thus, curcumin may be preventing oxidative stress in hippocampal neurons and consequently may be improving synaptic plasticity. A transitory brain glutathione deficit results in selectively impaired in spatial memory (Cabungcal et al., 2007). As diabetes is chronic metabolic disorder associated with reduction in glutathione levels which finally leads to memory deficits in diabetic animals. Since, curcumin could prevent intracellular reduced glutathione depletion due to its antioxidant properties (our unpublished data), reversal of diabetic cognitive dysfunction could be observed. Acetylcholine, a neurotransmitter associated with learning and memory, is degraded by the enzyme acetylcholinesterase, terminating the physiological action of the neurotransmitter. In addition to their role in cholinergic transmission, cholinesterases may also play a role during morphogenesis and neurodegenerative diseases (Layer et al., 1987; Reyes et al., 1997). The changes in cholinesterases activities might reflect impairment in biosynthesis, degradation, or insertion into the plasma membrane. In this regard the diabetic state is known to cause membrane alterations that can affect the kinetic properties of the membrane-bound cholinesterases (Khandkar et al., 1995; Suhail and Rizvi, 1989). Sanchez-Chavez and Salceda (2000) reported that the activity of acetylcholinesterase was increased in serum and cerebral cortex of diabetic rats but no change was observed in hippocampal acetylcholinesterase activity of hyperglycemic rats. In the present study, diabetes produced significant rise in acetylcholinestrase activity in the cerebral cortex, which was attenuated by chronic treatment with curcumin. Besides the enhanced level of reactive oxygen species, nitric oxide levels are also increased, and expression of mitochondrial nitric oxide synthase appears to be significantly increased in the brain mitochondria of diabetic rats. In diabetic rat brain mitochondria, Mastrocola et al. (2005) reported that nitric oxide synthase activity increases, with consequent nitric oxide hyperproduction: in those conditions, nitric oxide inhibits cytochrome c oxidase activity but might also act on other mitochondrial components, inhibiting other respiratory chain complexes by nitrosylation or oxidizing protein thiols (Clancy et al., 1994). Beside its effects on the respiratory chain, nitric oxide may also contribute to cell damage in diabetes, modulating glucose entry into the cells. Indeed, nitric oxide has been shown to upregulate glucose transporters in neurons (Bolanos et al., 2004). This activity has been regarded as protective in conditions (such as cerebral ischemia) in which the glucose supply to the brain is reduced (Friberg et al., 2002). Conversely, the upregulation of glucose transporters by nitric oxide might be detrimental in conditions characterized by excessive glucose supply, such as diabetes, in which increased intracellular glucose leads to an oversupply of electrons in the mitochondrial transfer chain, resulting in mitochondrial membrane hyperpolarization and a further increase in free-radical production. On the other hand, altered nitric oxide synthase expression and activity increase peroxynitrite production, which overwhelms the detoxifying reactions so that the effects mediated by nitric oxide-derived

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reactive species prevail. Indeed, peroxynitrite, a harmful oxidant formed by reaction between superoxide and nitric oxide, reacts with a variety of molecules, including protein and nonprotein-thiols, unsaturated fatty acids and DNA, thus affecting energy conservation mechanisms and oxidative post-translation modification of protein, and ultimately causing neuronal cell death (Murray et al., 2003). Our results showed an increase in nitrite levels in the cortex and hippocampus of diabetic rats and chronic treatment with curcumin significantly decreased brain nitrite levels because of its potential to inhibit expression of inducible nitric oxide synthase (Camacho-Barquero et al., 2007) and nitric oxide scavenging effects (Nanji et al., 2003). Pro-inflammatory cytokines are known to be elevated in several neuropathological states that are associated with learning and memory. Experimental studies reported that the inhibition of long-term potentiation in the dentate gyrus region of the rat hippocampus, by TNF-α, represents a biphasic response, an early phase dependent on p38 mitogen activated protein kinase activation and a later phase, possibly dependent on protein synthesis. TNF-α inhibition of long-term potentiation is dependent upon the activation of tumor necrosis factor receptor 1 and mGlu5receptors along with involvement of ryanodine-sensitive intracellular Ca2+ stores (Cumiskey et al., 2007). Moreover, in addition to oxidative and nitrosative stress, hyperglycemia is also associated with enhanced inflammatory response (Fukuhara et al., 2007; Kowluru and Kanwar, 2007). Under chronic hyperglycemia, endogenous TNF-α production is accelerated in microvascular and neural tissues, which may cause increased microvascular permeability, hypercoagulability and nerve damage, thus initiating and promoting the development of characteristic lesions of diabetic microangiopathy, polyneuropathy and encephalopathy (Brands et al., 2004; Chan, 1995; Satoh et al., 2003). Recently, we have reported that uncontrolled diabetes significantly enhanced TNF-α level (Sharma et al., 2007). A significant inhibition of TNF-α levels by curcumin observed in our study is indicative of the fact that curcumin contributes to beneficial effects seen in diabetic encephalopathy. With respect to inflammation, in vitro, along with inhibiting the activation of free radical-activated transcription factors, such as nuclear factorκβ, curcumin reduces the production of pro-inflammatory cytokines such as TNF-α, interleukin-1beta (IL-1β) and interleukin-8 (IL-8) (Chan, 1995; Cho et al., 2007). Thus, curcumin treatment ameliorated cognitive deficit, cholinergic dysfunction, reduced oxidative stress, nitric oxide and TNF-α in the diabetic rats. Due to its low side-effect profile and long history of safe use, curcumin may find clinical application in treating neuronal disturbances in the diabetic patients. Moreover, the findings of the study also suggested that the principle mechanisms involved in the antidiabetic and neuroprotective effect of curcumin are its strong antioxidant and anti-inflammatory potential. Acknowledgements The Senior Research Fellowship (Anurag Kuhad) of the Indian Council of Medical Research (ICMR), New Delhi, is gratefully acknowledged.

References Alasubramanyam, M., Koteswari, A., Kumar, R., Monickaraj, S., Maheswari, J., Ohan, V., 2003. Curcumin induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications. J. Biosci. 28, 715–721. Ammon, H.P.T., Wahl, M.A., 1991. Pharmacology of Curcuma longa. Planta Med. 57, 1–7. Anjaneyulu, M., Chopra, K., 2004. Effect of irbesartan on the antioxidant defence system and nitric oxide release in diabetic rat kidney. Am. J. Nephrol. 24, 488–496. Arun, N., Nalini, N., 2002. Efficacy of turmeric on blood sugar and polyol pathway in diabetic albino rats. Plant Foods Hum. Nutr. 57, 41–52. Arvanitakis, Z., Wilson, R.S., Bienias, J.L., Evans, D.A., Bennett, D.A., 2004. Diabetes mellitus and risk of Alzheimer disease and decline in cognitive function. Arch. Neurol. 61, 661–666. Best, L., Elliott, A.C., Brown, P.D., 2007. Curcumin induces electrical activity in rat pancreatic beta-cells by activating the volume-regulated anion channel. Biochem. Pharmacol. 73, 1768–1775. Biessels, G.J., Gispen, W.H., 2005. The impact of diabetes on cognition: what can be learned from rodent models? Neurobiol. Aging 26, 36–41. Biessels, G.J., Kamal, A., Urban, I.J.A., Spruijt, B.M., Erkelens, D.W., Gispen, W.H., 1998. Water maze learning and hippocampal synaptic plasticity in streptozotocin-diabetic rats: effects of insulin treatment. Brain Res. 800, 125–135. Biessels, G.J., Staekenborg, S., Brunner, E., Brayne, C., Scheltens, P., 2006. Risk of dementia in diabetes mellitus: a systematic review. Lancet Neurol. 5, 64–74. Bloomgarden, Z.T., 2007. Diabetic Neuropathy. Diabetes Care 30, 1027–1032. Bolanos, J.P., Cidad, P., Garçia-Nogales, P., Delgado-Esteban, M., Fernandez, E., Almeida, A., 2004. Regulation of glucose metabolism by nitrosative stress in neural cells. Mol. Aspects Med. 25, 61–73. Bonnefont-Rousselot, D., 2002. Glucose and reactive oxygen species. Curr. Opin. Clin. Nutr. Metab. Care 5, 561–568. Boonchoong, P., Vajragupta, O., Berliner, L., 2004. Manganese complexes of curcumin analogues: evaluation of hydroxyl radical scavenging ability, superoxide dismutase activity and stability towards hydrolysis. Free Radic. Res. 38, 303–314. Brands, A.M., Kessels, R.P., de Haan, E.H., Kappelle, L.J., Biessels, G.J., 2004. Cerebral dysfunction in type 1 diabetes: effects of insulin, vascular risk factors and blood-glucose levels. Eur. J. Pharmacol. 490, 159–168. Cabungcal, J.H., Preissmann, D., Delseth, C., Cuenod, M., Do, K.Q., Schenk, F., 2007. Transitory glutathione deficit during brain development induces cognitive impairment in juvenile and adult rats: Relevance to schizophrenia. Neurobiol. Dis. 26, 634–645. Camacho-Barquero, L., Villegas, I., Sanchez-Calvo, J.M., Talero, E., SanchezFidalgo, S., Motilva, V., Alarcon de la Lastra, C., 2007. Curcumin, a Curcuma longa constituent, acts on MAPK p38 pathway modulating COX-2 and iNOS expression in chronic experimental colitis. Int. Immunopharmacol. 7, 333–342. Chan, M.M., 1995. Inhibition of TNF-α by curcumin, a phyochemical. Biochem. Pharmacol. 49, 1551–1556. Cho, J.W., Lee, K.S., Kim, C.W., 2007. Curcumin attenuates the expression of IL-1beta, IL-6, and TNF-alpha as well as cyclin E in TNF-alpha-treated HaCaT cells; NF-kappaB and MAPKs as potential upstream targets. Int. J. Mol. Med. 19, 469–474. Claiborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), Handbook of methods for oxygen radical research. CRC Press, Boca Raton, BR, pp. 283–284. Clancy, R.M., Levartosky, D., Leszczynska-Piziak, J., Yegudin, J., Abramson, S., 1994. Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary. PNAS 91, 3680–3684. Cumiskey, D., Butler, M.P., Moynagh, P.N., O'connor, J.J., 2007. Evidence for a role for the group I metabotropic glutamate receptor in the inhibitory effect of tumor necrosis factor-alpha on long-term potentiation. Brain Res. 1136, 13–19. Dairam, A., Limson, J.L., Watkins, G.M., Antunes, E., Daya, S., 2007. Curcuminoids, curcumin, and demethoxycurcumin reduce lead-induced memory deficits in male Wistar rats. J. Agric. Food Chem. 55, 1039–1044.

A. Kuhad, K. Chopra / European Journal of Pharmacology 576 (2007) 34–42 Dobretsov, M., Romanovsky, D., Stimers, J.R., 2007. Early diabetic neuropathy: triggers and mechanisms. World J. Gastroenterol. 13, 175–191. Ellman, G.L., Courtney, D.K., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Evans, J.L., Goldfine, I.D., Maddux, B.A., Grodsky, G.M., 2002. Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr. Rev. 23, 599–622. Franceschi, M., Cecchetto, R., Minicucci, F., Smizne, S., Baio, G., Canal, N., 1984. Cognitive processes in insulin-dependent diabetes. Diabetes Care 7, 228–231. Frautschy, S.A., Hu, W., Kim, P., Miller, S.A., Chu, T., Harris-White, M.E., Cole, G.M., 2001. Phenolic anti-inflammatory antioxidant reversal of Aβinduced cognitive deficits and neuropathology. Neurobiol. Aging 22, 993–1005. Friberg, H., Wieloch, T., Castilho, R.F., 2002. Mitochondrial oxidative stress after global brain ischemia in rats. Neurosci. Lett. 334, 111–114. Fukuhara, M., Matsumura, K., Wakisaka, M., Takata, Y., Sonoki, K., Fujisawa, K., Ansai, T., Akifusa, S., Fujii, K., Iida, M., Takehara, T., 2007. Hyperglycemia promotes microinflammation as evaluated by C-reactive protein in the very elderly. Intern. Med. 46, 207–212. Fukui, K., Onodera, K., Shinkai, T., Suzuki, S., Urano, S., 2001. Impairment of learning and memory in rats caused by oxidative stress and aging, and changes in antioxidative defense systems. Ann. N. Y. Acad. Sci. 928, 168–175. Gispen, W.H., Biessels, G.J., 2000. Cognition and synaptic plasticity in diabetes mellitus. Trends Neurosci. 23, 542–549. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131–138. Hoybergs, Y.M.J.J., Meert, T.F., 2007. The effect of low-dose insulin on mechanical sensitivity and allodynia in type I diabetes neuropathy. Neurosci. Lett. 417, 149–154. Jollow, D.J., Mitchell, J.R., Zampaglione, N., Gillette, J.R., 1974. Bromobenze induced liver necrosis: protective role of glutathione and evidence for 3,4bromobenzenoxide as the hepatotoxic intermediate. Pharmacology 11, 151–169. Kelloff, G.J., Crowell, J.A., Hawk, E.T., Steele, V.E., Lubet, R.A., Boone, C.W., Covey, J.M., Doody, L.A., Omenn, G.S., Greenwald, P., Hong, W.K., Parkinson, D.R., Bagheri, D., Baxter, G.T., Blunden, M., Doeltz, M.K., Eisenhauer, K.M., Johnson, K., Knapp, G.G., Longfellow, D.G., Malone, W.F., Nayfield, S.G., Seifried, H.E., Swall, L.M., Sigman, C.C., 1996. Strategy and planning for chemopreventative drug development:clinical development plans II: curcumin. J. Cell. Biochem. 26, 54–71. Khandkar, M.A., Mukherjee, E., Parmar, D.P., Katyare, S.S., 1995. Aloxan-diabetes alters kinetic properties of the membrane-bound form, but not of the soluble form, of acetylcholinesteras e in rat brain. Biochem. J. 307, 647–649. Kim, J.M., Araki, S., Kim, D.J., Park, C.B., Takasuka, N., Baba-Toriyama, H., Ota, T., Nir, Z., Khachik, F., Shimidzu, N., Tanaka, Y., Osawa, T., Uraji, T., Murakoshi, M., Nishino, H., Tsuda, H., 1998. Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2dimethylhydrazine initiation. Carcinogenesis 19, 81. Kono, Y., 1978. Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch. Biochem. Biophys. 186, 189–195. Kowluru, R.A., Kanwar, M., 2007. Effects of curcumin on retinal oxidative stress and inflammation in diabetes. Nutr. Metab. 16, 4–8. Layer, P.G., Alber, R., Sporns, O., 1987. Quantitative development and molecular forms of acetylcholinesteras e and butyrylcholinesteras e during morphogenesi s and synaptogenesi s of chick brain and retina. J. Neurochem. 49, 175–182. Li, Z., Zhanga, W., Sima, A.A.F., 2005. The role of impaired insulin/IGF action in primary diabetic encephalopathy. Brain Res. 1037, 12–24. Lobnig, B.M., Krömeke, O., Optenhostert-Porst, C., Wolf, O.T., 2005. Hippocampal volume and cognitive performance in long-standing Type 1 diabetic patients without macrovascular complications. Diabet. Med. 23, 32–39. Mahakunakorn, P., Tohda, M., Murakami, Y., Matsumoto, K., Watanabe, H., Vajaragupta, O., 2003. Cytoprotective and cytotoxic effects of curcumin: dual

41

action on H2O2-induced oxidative cell damage in NG108-15 cells. Biol. Pharm. Bull. 26, 725–728. Mastrocola, R., Restivo, F., Vercellinatto, I., Danni, O., Brignardello, E., Aragno, M., Boccuzzi, G., 2005. Oxidative and nitrosative stress in brain mitochondria of diabetic rats. J. Endocrinol. 187, 37–44. Masuda, T., Hidaka, K., Shimohara, A., Maekawa, T., Takeda, Y., Yamaguchi, H., 1999. Chemical studies on antioxidant mechanism of curcuminoid: analysis of radical reaction products from curcumin. J. Agric. Food Chem. 47, 71. Mijnhout, G.S., Scheltens, P., Diamant, M., Biessels, G.J., Wessels, A.M., Simsek, S., Snoek, F.J., Heine, R.J., 2006. Diabetic encephalopathy: a concept in need of a definition. Diabetologia 49, 1447–1448. Miles, W.R., Root, H.F., 1922. Psychologic tests applied to diabetic patients. Arch. Int. Med. 30, 767–777. Morris, R.G., Garrud, P., Rawlins, J.N., O'Keefe, J., 1982. Place navigation impaired in rats with hippocampal lesions. Nature 297, 681–683. Murray, J., Taylor, S.W., Zhang, B., Ghosh, S., 2003. Oxidative damage to mitochondrial complex I due to peroxynitrite. J. Biol. Chem. 278, 37223–37230. Murugan, P., Pari, L., 2006. Antioxidant effect of tetrahydrocurcumin in streptozotocin-nicotinamide induced diabetic rats. Life Sci. 79, 1720–1728. Nanji, A.A., Jokelainen, K., Tipoe, G.L., Rahemtulla, A., Thomas, P., Dannenberg, A.J., 2003. Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-kappa B-dependent genes. Am. J. Physiol.: Gasterointest. Liver Physiol. 284, G321–G327. Nishikawa, T., Edelstein, D., Du, X.L., Yamagishi, S., Matsumura, T., Kaneda, Y., Yorek, A., Beebe, D., Oates, P.J., Hammes, H.P., Giardino, I., Brownlee, M., 2000. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404, 787–790. Priyadarsini, K., Maity, D., Naik, G., Kumar, M., Unnikrishnan, M., Satav, J., Mohan, H., 2003. Role of phenolic O–H and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin. Free Radic. Biol. Med. 35, 475–484. Rajakrishnan, V., Viswanathan, P., Rajasekharan, K.N., Menon, V.P., 1999. Neuroprotective role of curcumin from curcuma longa on ethanol-induced brain damage. Phythother. Res. 13, 571–574. Reyes, A.E., Perez, D.R., Alvarez, A., Garrido, J., Gentry, M.K., Doctor, B.P., Inestrosa, N.C., 1997. A monoclonal antibody against acetylcholinesterase inhibits the formation of amyloid. brils induced by the enzyme. Biochem. Biophys. Res. Commun. 232, 652–655. Ryan, C., Vega, A., Drash, A., 1985. Cognitive deficits in adolescents who developed diabetes early in life. Pediatric 75, 921–927. Ryan, C.M., Williams, T.M., Finegold, D.N., Orchard, T.J., 1993. Cognitive dysfunction in adults with type 1 (insulin-dependent) diabetes mellitus of long duration: effects of recurrent hypoglycaemia and other chronic complications. Diabetologia 36, 329–334. Sanchez-Chavez, G., Salceda, R., 2000. Effect of streptozotocin-induced diabetes on activities of cholinesterases in the rat retina. IUBMB Life 49, 283–287. Satoh, J., Yagihashi, S., Toyota, T., 2003. The possible role of tumor necrosis factor-alpha in diabetic polyneuropathy. Exp. Diabesity Res. 4, 65–71. Sharma, S., Anjaneyulu, M., Kulkarni, S.K., Chopra, K., 2006a. Resveratrol, a polyphenolic phytoalexin, attenuates diabetic nephropathy in rats. Pharmacology 76, 69–75. Sharma, S., Kulkarni, S.K., Chopra, K., 2006b. Curcumin, the active principle of turmeric (Curcuma longa), ameliorates diabetic nephropathy in rats. Clin. Exp. Pharmacol. Physiol. 33, 940–945. Sharma, S., Chopra, K., Kulkarni, S.K., 2007. Effect of insulin and its combination with resveratrol or curcumin in attenuation of diabetic neuropathic pain: participation of nitric oxide and TNF-alpha. Phytother. Res. 21, 278–283. Sima, A.A.F., Kamiya, H., Lia, Z.G., 2004. Insulin, C-peptide, hyperglycemia, and central nervous system complications in diabetes. Eur. J. Pharmacol. 490, 187–197. Somfai, G.M., Knippel, B., Ruzicska, E., Stadler, K., Toth, M., Salacz, G., Magyar, K., Somogyi, A., 2006. Soluble semicarbazide-sensitive amine oxidase (SSAO) activity is related to oxidative stress and subchronic inflammation in streptozotocin-induced diabetic rats. Neurochem. Int. 48, 746–752.

42

A. Kuhad, K. Chopra / European Journal of Pharmacology 576 (2007) 34–42

Sreejayan, N., Rao, M.N.A., 1993. Curcumin inhibits iron dependent lipid peroxidation. Int. J. Pharm. 100, 93–97. Srimal, R.C., Dhawan, B.N., 1973. Pharmacology of diferuloyl methane (curcumin), a non-steroidal anti-inflammatory agent. J. Pharm. Pharmacol. 25, 447–452. Srivivasan, A., Menon, V.P., Periaswamy, V., Rajasekaran, K.N., 2003. Protection of pancreatic beta-cell by the potential antioxidant bis-ohydroxycinnamoyl methane, analogue of natural curcuminoid in experimental diabetes. J. Pharm. Pharm. Sci. 6, 327–333. Subrmanian, M., Sreejayan, N., Rao, M.N.A., Devasagayam, T.P.A., Singh, B.B., 1994. Diminution of singlet oxygen-induced DNA damage by curcumin and related antioxidants. Mutat. Res. 311, 249–255. Suhail, M., Rizvi, S.I., 1989. Erythrocyte membrane acetylcholinesterase in type I (insulin dependent) diabetes mellitus. Biochem. J. 259, 897–899. Sui, Z., Salto, R., Li, J., Craik, C., De Montellano, P.R.O., 1993. Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes. Bioorg. Med. Chem. 1, 415.

Trudeaua, F., Gagnonb, S., Massicotte, G., 2004. Hippocampal synaptic plasticity and glutamate receptor regulation: influences of diabetes mellitus. Eur. J. Pharmacol. 490, 177–186. Tuzcu, M., Baydas, G., 2006. Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. Eur. J. Pharmacol. 537, 106–110. Unnikrishnan, M.K., Rao, M.N.A., 1995. Curcumin inhibits nitrogen dioxide induced oxidation of haemoglobin. Mol. Cell. Biochem. 146, 35–37. Wills, E.D., 1965. Mechanism of lipid peroxide formation in animals. Biochem. J. 99, 667–676. Zhao, B.L., Li, X.J., He, R.G., Cheng, S.J., Xin, W.J., 1989. Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals. Cell Biophys. 14, 175–185.