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Antioxidant activity of costunolide and eremanthin isolated from Costus speciosus (Koen ex. Retz) Sm.
Chemico-Biological Interactions 188 (2010) 467–472
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Antioxidant activity of costunolide and eremanthin isolated from Costus speciosus (Koen ex. Retz) Sm. J. Eliza a , P. Daisy a , S. Ignacimuthu b,∗ a b
Department of Biotechnology, Holy Cross College, Trichy 620002, Tamil Nadu, India Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Nungambakkam, Chennai 600034, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 24 May 2010 Received in revised form 5 August 2010 Accepted 9 August 2010 Available online 13 August 2010 Keywords: Costunolide Eremanthin Oxidative stress Experimental diabetes Enzymatic activities Non-enzymatic antioxidants Protective effect
a b s t r a c t Antioxidant properties of many medicinal plants have been widely recognized and some of them have been commercially exploited. Plant derived antioxidants play a very important role in alleviating problems related to oxidative stress. The present study was aimed at assessing the antioxidant property of costunolide and eremanthin isolated from a medicinal plant Costus speciosus (Koen ex. Retz) Sm. rhizome. Experimental diabetes was induced by a single dose of STZ (60 mg/kg, i.p.) injection. The oxidative stress was measured by tissue thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) content and enzymatic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in brain, liver, heart, kidney and pancreas. An increase in TBARS level, a significant reduction in GSH content along with decreased enzymatic activities of SOD, CAT, and GPx were seen in untreated diabetic rats. Administration of either costunolide (20 mg/kg day) or eremanthin (20 mg/kg day) for 60 days caused a significant reduction in TBARS level and a significant increase in GSH content along with increased enzymatic activities of SOD, CAT and GPx in the treated rats when compared to untreated diabetic rats. Acute toxicity test revealed the non-toxic nature of the compounds. The results indicated for the first time the protective effect of costunolide and eremanthin from oxidative stress, thus opening the way for their use in medication. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a serious complex chronic condition. It is a major source of ill-health worldwide. This metabolic disorder is characterized by hyperglycemia and disturbances of carbohydrate, protein and fat metabolisms, secondary to an absolute or relative lack of insulin [1]. Chronic oxidative stress due to hyperglycemia may, therefore, play an important role in progression of -cell dysfunction in both types of diabetes. Studies have demonstrated that this can be inhibited by antioxidants [2]. Hyperglycemia induced higher lipid peroxidation in the tissues and glycosylation of protein, resulting in formation of free radicals of oxygen (ROS) and nitrogen (RNS) [3]. The antioxidant pool is highly aggravated in hyperglycemia due to persistent challenge by ROS [4,5]. Findings have suggested that the generation of ROS/RNS leads to oxidation of lipids and proteins, which potentiate diabetes related complications [6]. Oxidative environment in cells is also created by the impairment in functioning of endogenous antioxidant enzymes namely superoxide dismutase (SOD), glutathione
∗ Corresponding author. Tel.: +91 044 2817 8348; fax: +91 044 2817 5566. E-mail address: [email protected] (S. Ignacimuthu). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.08.002
peroxidase (GPx) and catalase (CAT). GPx, CAT, SOD and glutathione reductase (GR) are known to be inhibited in diabetes mellitus as a result of non-enzymatic glycosylation and oxidation [7]. There has been an upsurge of interest in the therapeutic potential of medicinal plants as antioxidants in reducing free radical-induced tissue injury [8]. Besides, well known and traditionally used natural antioxidants from teas, wines, fruits, vegetables and spices, some natural antioxidants (e.g. from rosemary and sage) are already exploited commercially either as antioxidant additives or as nutritional supplements [9]. Costunolide and eremanthin are sesquiterpene compounds. Costunolide has been previously isolated from Saussurea radix and the dried root of S. lappa. It is reported to possess various biological actions [10–12]. Eremanthin is reported to be present in Pterodon pubescens, Eremanthus elaeagnus [13], and n-hexane extract of Larus nobilis leaves [14]. We have already reported the antidiabetic and hypolipidemic action of costunolide and eremanthin isolated from Costus speciosus [15,16]. There are no detailed reports available for the antioxidant activity of costunolide and eremanthin on various organs. So the present study was aimed at evaluating the antioxidant activity of costunolide and eremanthin in various organs such as brain, pancreas, liver, heart and kidneys which were damaged by oxidative stress due to diabetes induced by streptozotocin.
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2. Materials and methods 2.1. Chemicals Streptozotocin was procured from Sigma Chemical Co., St. Louis, MO, USA. All other chemicals were of analytical grade.
tube for 60 days. After 60 days of treatment rats were sacrificed by decapitation; their blood was collected without the addition of anticoagulant, kept at 4 ◦ C for 30 min and serum was obtained immediately by centrifugation. Organs such as brain, heart, liver, pancreas, and kidneys were removed, dried on tissue paper, and stored at −80 ◦ C for determining TBARS and enzymatic and nonenzymatic antioxidants.
2.2. Plant compounds Costunolide and eremanthin were isolated from the rhizome of C. speciosus (Koen ex. Retz) Sm. The isolation and structural elucidation have already been described [15,16]. 2.3. Experimental animals Male Wistar strain rats weighing about 190–200 g bred in the Laboratory of King’s Institute for preventive medicine, Guindy, Chennai, Tamil Nadu, India were used. All the animals were maintained under laboratory conditions of temperature (22 ± 2 ◦ C), relative humidity (45 ± 5%) and 12 h day:12 h night cycle and were allowed to feed (standard pellet diet) and water ad libitum. The experimental protocol was approved by the Institutional Animal’s Ethics Committee and by the regulatory body of the government (Reg. No. 585/05/A/CPCSEA). 2.4. Acute toxicity test An experiment was performed to know whether toxic effects were produced by eremanthin and costunolide. Rats fasted for 12 h were treated with either eremanthin or costunolide (10, 20, 40, 80 and 160 mg/kg bw) dissolved in 0.1 M citrate buffer. Rats in the control groups received only the vehicle (0.1 M citrate buffer; 1 ml/kg bw). The rats were allowed access to feed and water. Behavioural changes like abnormal locomotion, respiratory distress, uncoordinated muscle movements, weight loss and mortality were observed for 10 days. 2.5. Induction of diabetes Diabetes mellitus was induced by single intra-peritoneal injection of freshly prepared STZ (60 mg/kg bw) in 0.1 M citrate buffer (pH −4.5) in a volume of 1 ml/kg bw. Diabetes was stabilized in these STZ-treated rats over a period of 7 days. The control animals were treated with citrate buffer (pH −4.5). After 7 days the blood was collected by sinocular puncture and the plasma glucose level of each rat was determined. Rats with a fasting plasma glucose range of 280–350 mg/dl were considered diabetic and included in the study.
2.6.1. Estimation of plasma glucose and TBARS Fasting plasma glucose was estimated using glucose oxidase–peroxidase method [17]. The concentration of TBARS in the tissues was estimated by the method of Nichans and Samuelson [18]. In this method, malondialdehyde and other thiobarbituric acid reactive substances (TBARS) reacted with thiobarbituric acid in an acidic condition to generate a pink color chromophore which was read at 535 nm. 2.6.2. Oral glucose tolerance test Oral glucose tolerance test was conducted after the 60th day just prior to the animal sacrifice. After overnight fasting, plasma glucose level was estimated for 0 min in normal and experimental rats by sinocular puncture. Immediately the compounds costunolide, eremanthin (20 mg/kg bw) and insulin (3 IU/kg bw) were given to the respective experimental group rats. After 30 min glucose solution (2 g/kg bw) was administered by gavage to all the experimental rats and blood samples were taken at 30, 60, 120 and 180 min after treatment. Plasma glucose was estimated in all the experimental groups. 2.7. Non-enzymatic antioxidant 2.7.1. Estimation of reduced glutathione (GSH) Reduced glutathione in the tissues was estimated by the method of Ellman [19]. Briefly, 1.0 ml of the supernatant was treated with 0.5 ml Ellman reagent and 3.00 ml 0.2 M sodium phosphate buffer, pH 8.0, and absorbance was read at 412 nm. Reduced GSH activity is reported as nmol/g wet tissue. 2.8. Enzymatic antioxidants 2.8.1. Assay of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (EC 1.11.1.19) Superoxide dismutase in the tissues was assayed by the method of Kakkar et al. [20]. The activity of catalase in the tissues was determined by the method of Sinha [21]. The activity of GPx in the tissues was measured by the method of Rotruck et al. [22].
2.6. Experimental design and treatment schedule
2.9. Statistics
In the experiment, a total of 42 rats (18 normal; 24 STZ-induced diabetic rats) were used. The effective dose of the compounds was determined through our earlier experiments [15,16]. The rats were divided into seven groups of six each. Group 1 consisted of normal rats treated with vehicle alone (dimethysulfoxide, DMSO) 0.5%; 1 ml/kg body weight; Group 2 consisted of normal rats treated with costunolide (20 mg/kg bw); Group 3 consisted of normal rats treated with eremanthin (20 mg/kg bw); Group 4 consisted of STZ-induced diabetic rats treated with vehicle alone. Group 5 consisted of STZ-induced diabetic rats treated with costunolide (20 mg/kg bw). Group 6 consisted of STZ-induced diabetic rats treated with eremanthin (20 mg/kg bw). Group 7 consisted of STZ-induced diabetic rats treated with insulin (3 IU/kg bw). Single dose of costunolide and eremanthin was suspended in vehicle solution and was administered every day orally using intra-gastric
Statistical analysis was performed using SPSS software package Version 16.0. The values were analysed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) [23]. All the results were expressed as mean ± SD for six rats in each group. P-values < 0.05 were considered significant. 3. Results 3.1. Acute toxicity test There were no behavioural changes on the rats. Mortality was not observed during acute toxicity test. Both the compounds had not shown any behavioural changes or toxic effects up to 160 mg/kg bw.
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Table 1 Effect of costunolide and eremanthin on plasma glucose levels in normal and streptozotocin-induced diabetic male Wistar rats. Groups
Plasma glucose levels (mg/dl) 0 day
Normal Normal + costunolide (20 mg/kg bw) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
91.4 86.5 88.43 415.5 385.6 408.5 420.7
15th day ± ± ± ± ± ± ±
9.4a 14.5a 13.5a 9.4 11.5 9.3 13.5
89.6 88.4 90.11 410.41 245.6 257.65 95.67
± ± ± ± ± ± ±
30th day 11.6a 9.32a 9.7a 11.4 7.4 9.4 7.4a
90.5 85.8 89.76 422.8 184.45 196.56 90.45
± ± ± ± ± ± ±
60th day 16.7a 8.78a 11.5a 13.6 11.2a 7.7a 8.3a
86.7 90.12 85.78 427.8 137.4 140.34 87.54
± ± ± ± ± ± ±
9.23a 11.2a 17.34a 15.4 6.8a 10.23a 9.6a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats.
3.2. Estimation of plasma glucose and TBARS Changes in blood glucose levels of normal and diabetic rats treated discretely with costunolide and eremanthin are presented in Table 1. Normal untreated and normal rats treated with the compounds discretely, did not show any significant change in blood glucose levels during 60 days of treatment. However blood glucose level increased significantly in diabetic rats. The diabetic rats treated with costunolide and eremanthin at the dose of 20 mg/kg bw for 60 days showed decrease in blood glucose in a time dependent manner. 3.3. Oral glucose tolerance test The effect of costunolide and eremanthin on oral glucose tolerance test in normal and diabetic rats is given in Table 2. In STZ-treated diabetic rats, an increased plasma glucose level was observed throughout the experimental period. But the rats treated with costunolide and eremanthin showed significant (P < 0.05) decrease in plasma glucose level after 120 and 180 min. The TBARS levels of various tissues are presented in Table 3. There was a significant increase in TBARS levels in the brain, heart, liver, pancreas and kidney tissues of STZ-induced diabetic rats. Oral administration of costunolide and eremanthin for 60 days showed a significant (P < 0.05) decrease in the tissue TBARS levels, and these results were comparable with that of insulin. 3.4. Estimation of reduced glutathione (GSH) and antioxidant enzyme activities The non-enzymatic antioxidant GSH levels in liver, kidney, heart, brain and pancreas of normal, diabetic and treated groups are given in Table 4. There was a significant decrease in GSH level in brain, heart, liver, pancreas and kidneys of diabetic group when compared with normal rats. However after the administration of costunolide and eremanthin (20 mg/kg bw), there was an increase in GSH level of brain, heart, liver, pancreas and kidneys. The normal
rats treated with costunolide and eremanthin for 60 days did not exhibit any difference. The activities of glutathione peroxidase, superoxide dismutase, and catalase in the tissues of liver, kidneys, heart, brain and pancreas of normal, diabetic and treated rats are presented in Tables 5–7. The enzymatic antioxidant activities were decreased significantly in STZ-induced diabetic rats when compared with the normal untreated rats. However treatment with costunolide and eremanthin restored the enzymatic antioxidant activities in the tissues of liver, kidneys, heart, brain and pancreas almost to near normal. These results were significant (P < 0.05) and comparable with that of insulin. The normal rats treated with costunolide and eremanthin also showed normal activities of these enzymatic antioxidants. 4. Discussion Demand for natural antioxidant has been increasing due to concerns about safety of synthetic antioxidants. The present study with natural antioxidants, costunolide and eremanthin isolated from C. speciosus, is a new report for the prevention of lipid peroxidation. Oxidative stress, defined as an imbalance between oxidants and antioxidants, leads to many biochemical changes and acts as the causative factor for many diseases like diabetes, atherosclerosis, cardiovascular problems etc. [24]. Further hyperglycemia results in the generation of free radicals [25], reduced total antioxidants status (TAS) of serum [26] and oxidative damage to membranes and lipid peroxidation. The lipid peroxidation is accelerated when free radicals are formed as the result of losing a hydrogen atom from the double bond in the structure of unsaturated fatty acids. Scavenging of free radicals is one of the major antioxidation mechanisms to inhibit the chain reaction of the lipid peroxidation. Scientists have been trying to alleviate this problem by using antioxidants. Plant derived antioxidants play an important role in this. Costunolide and eremanthin isolated from the rhizome of C. speciosus were used in this study. Acute toxicity test showed that there was no marked changes in feed intake. Behavioural changes like irritation, restlessness, respi-
Table 2 Effect of costunolide and eremanthin on oral glucose tolerance in normal and streptozotocin-induced diabetic male Wistar rats after 60 days. Groups
Plasma glucose levels (mg/dl) 0 min
Normal Normal + costunolide (20 mg/kg bw) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
70.72 72.11 75.62 318.28 90.45 95.62 92.77
30 min ± ± ± ± ± ± ±
10.51a 12.71a 11.63a 14.77 9.86 10.15 12.29a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats.
108.12 105.62 110.75 355.17 138.11 146.12 102.71
60 min ± ± ± ± ± ± ±
8.72a 15.1a 13.6a 15.25 8.18a 10.11a 9.62a
135.81 128.19 138.65 420.25 188.72 196.81 120.65
120 min ± ± ± ± ± ± ±
12.82a 13.62a 14.71a 16.22 12.72 10.81 14.62a
92.33 90.16 98.21 438.32 145.46 140.71 88.19
± ± ± ± ± ± ±
180 min 11.77a 11.65a 15.81a 15.81 11.78a 9.82a 8.95a
80.75 87.6 85.82 445.55 107.63 110.72 83.85
± ± ± ± ± ± ±
10.55a 12.19a 9.52a 12.33 10.45a 12.64a 9.86a
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Table 3 Effect of costunolide and eremanthin on tissue TBARS in normal and streptozotocin-induced diabetic male Wistar rats after 60 days. Groups
Tissue TBARS (nmol/100 g tissue) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
1.59 1.53 1.56 2.85 1.62 1.70 1.64
Heart ± ± ± ± ± ± ±
0.04a 0.02a 0.04a 0.07 0.02a 0.03a 0.03a
Liver
± ± ± ± ± ± ±
0.51 0.50 0.52 0.92 0.58 0.62 0.55
.02a .03a .04a .04 .06a .05a .02a
1.36 1.27 1.33 2.8 1.42 1.52 1.8
Pancreas ± ± ± ± ± ± ±
0.06a 0.06a 0.06a 0.08 0.06a 0.04a 0.03a
± ± ± ± ± ± ±
38.85 34.40 37.18 72.00 45.79 51.05 49.19
Kidney 2.58a 2.20a 2.59a 3.63 2.97a 2.7a 2.42a
1.49 1.41 1.45 2.50 1.58 1.67 1.58
± ± ± ± ± ± ±
0.02a 0.03a 0.05a 0.02 0.05a 0.02a 0.06a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats. Table 4 Effect of costunolide and eremanthin on tissue reduced glutathione in normal and streptozotocin-induced diabetic male Wistar rats after 60 days of treatment. Groups
Reduced glutathione (nmol/g wet tissue) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
Heart
11.59 10.49 9.47 4.89 9.79 8.69 7.55
± ± ± ± ± ± ±
1.42a 1.28a .74a 1.01 1.75a 1.57a 1.22a
Liver ± ± ± ± ± ± ±
12.35 12.04 11.40 5.13 11.20 10.46 7.61
1.46a 1.64a 1.57a 1.13 1.54a 1.3a 1.18a
Pancreas
22.61 23.03 22.83 14.46 21.11 19.45 17.22
± ± ± ± ± ± ±
1.98a 2.10a 2.20a 1.33 1.86a 1.9a 2.18a
± ± ± ± ± ± ±
21.85 21.12 20.25 11.83 20.91 19.75 17.56
Kidney 1.81a 1.13a 1.46a 1.52 1.80a 2.01a 1.74a
20.68 22.51 21.40 11.00 22.26 20.68 19.34
± ± ± ± ± ± ±
1.48a 2.24a 2.39a 1.76 2.26a 1.48a 1.73a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats. Table 5 Effect of costunolide and eremanthin on tissue glutathione peroxidase in normal and streptozotocin-induced diabetic male Wistar rats after 60 days of treatment. Groups
GPx (g of GSH consumed/min/mg protein) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
Heart ± ± ± ± ± ± ±
6.93 6.71 6.55 2.19 5.34 5.16 4.11
a
0.44 0.63a 0.51a 0.29 0.36a 0.23a 0.43a
0.99 0.96 0.94 0.56 0.93 0.89 0.85
Liver
± ± ± ± ± ± ±
a
0.04 0.10a 0.04a 0.07 0.03a 0.02a 0.06a
9.15 9.22 9.14 5.00 8.00 7.79 6.59
Pancreas ± ± ± ± ± ± ±
a
0.53 0.64a 0.45a 0.25 0.33a 0.44a 0.37a
28.77 27.83 28.14 14.73 26.91 27.10 22.62
± ± ± ± ± ± ±
Kidney a
1.63 2.11a 2.23a 0.84 1.94a 2.66a 1.77a
6.51 7.13 6.92 3.17 5.96 5.14 4.26
± ± ± ± ± ± ±
0.41a 0.71a 0.62a 0.24 0.35a 0.81a 0.44a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats. Table 6 Effect of costunolide and eremanthin on superoxide dismutase in normal and streptozotocin-induced diabetic male Wistar rats after 60 days of treatment. Groups
SOD (U/mg protein) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
7.91 7.53 7.75 3.18 6.88 6.65 4.12
Heart ± ± ± ± ± ± ±
a
0.62 0.51a 0.29a 0.36 0.49a 0.55a 0.37a
9.72 8.91 8.23 4.25 8.82 8.60 7.00
Liver
± ± ± ± ± ± ±
a
0.33 0.52a 0.41a 0.72 0.61a 0.59a 0.17a
8.95 8.18 8.09 4.19 7.11 7.52 5.89
Pancreas ± ± ± ± ± ± ±
a
0.51 0.33a 0.45a 0.22 0.55a 0.43a 0.71a
4.94 4.61 4.76 2.11 4.49 4.66 3.11
± ± ± ± ± ± ±
Kidney a
0.54 0.38a 0.29a 0.11 0.30a 0.22a 0.35a
14.71 14.23 13.82 6.71 12.59 12.43 9.26
± ± ± ± ± ± ±
0.73a 0.94a 1.24a 0.11 1.17a 0.92a 0.78a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats. Table 7 Effect of costunolide and eremanthin on tissue catalase in normal and streptozotocin-induced diabetic male Wistar rats after 60 days of treatment. Groups
CAT (mol of H2 O2 consumed/min/mg protein) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
2.85 3.19 3.28 0.78 2.31 2.25 1.65
Heart ± ± ± ± ± ± ±
0.39a 0.24a 0.33a 0.04 0.19a 0.41a 0.21a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats.
8.77 8.09 8.56 4.00 7.52 7.18 5.78
± ± ± ± ± ± ±
Liver 0.51a 0.68a 0.43a 0.33 0.59a 0.47a 0.55a
80.13 79.72 79.19 46.33 73.69 74.19 62.88
Pancreas ± ± ± ± ± ± ±
5.19a 5.06a 4.63a 2.18 4.11a 3.58a 3.17a
17.18 16.78 16.95 6.96 13.37 13.25 10.87
± ± ± ± ± ± ±
Kidney 0.77a 0.64a 0.81a 0.49 0.47a 0.91a 0.68a
35.19 36.11 35.82 14.98 29.41 28.72 23.17
± ± ± ± ± ± ±
1.72a 3.21a 1.48a 2.33 2.19a 1.55a 0.98a
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ratory distress, abnormal locomotion and catalapsy over a period of 10 days were not observed. This revealed the non-toxic nature of both the compounds. Daily administration of these compounds decreased the plasma glucose level significantly. Oral administration of the compounds produced significant effect when glucose challenge was given to normal and diabetic rats. Decrease in the plasma glucose was observed at 120 min after the administration of costunolide and eremanthin. Dysidine, a sesquiterpene, effectively activated the insulin signaling pathways, promoted glucose uptake and showed insulin-sensitizing activities through the inhibition of protein tyrosine phospatases [27]. The glucose lowering effect of costunolide and eremanthin which are sesquiterpenes might be due to similar action. Our results are similar to the one in which Eriobotrya japonica seeds effectively improved glucose tolerance in the KK-Ay mice [28]. Hyperglycemia results in the generation of free radicals which can exhaust antioxidant defenses thus leading to the disruption of cellular functions, oxidative damage to membranes and enhanced susceptibility to lipid peroxidation [29,30]. It has been reported that increased oxidative stress may play a role in the pathogenesis and progression of diabetic tissue damage [31–33]. STZ has been reported to generate ROS [34]. In the present study a marked increase in the level of tissue TBARS in STZ-induced diabetic rats indicated enhanced lipid peroxidation leading to tissue injury and failure of the antioxidant defense mechanism to prevent the formation of excess free radicals. The diabetic rats treated with costunolide and eremanthin for 60 days showed very low levels of tissue damage compared to untreated diabetic rats. These results showed that costunolide and eremanthin protected the various organs from tissue damage by reducing the ROS production. Studies concluded by other investigators in diabetic patients and diabetic rats reported similar findings [35,36]. The water and ethanol extracts of Helichrysum plicatum ssp. and Plicatum capitulums produced significant decrease in kidney and liver MDA levels of diabetic rats [37]. GSH, a non-enzymatic antioxidant, effectively scavenges free radicals and other ROS directly and indirectly through enzymatic reactions [38]. Depletion of tissue GSH is one of the primary factors that permit lipid peroxidation [39]. It has been proposed that antioxidants that maintain the concentration of GSH may restore the cellular defense mechanisms, block lipid peroxidation and thus protect the tissue against oxidative damage [40]. In the present investigation the GSH concentration was low in the diabetic group. This is in accordance with the results of Tagami et al. [41] who demonstrated a reduced concentration of GSH in diabetic rabbits. In the present study, oral administration with costunolide and eremanthin significantly increased the GSH levels. Increase in GSH level in turn activated the GSH dependent enzymes such as glutathione peroxidase and glutathione-S-transferase by which it could reduce the tissue damage. Apart from the non-enzymatic antioxidants, enzymatic antioxidants such as SOD and CAT play an important role in preventing cells from being exposed to oxidative damage [42]. SOD is an enzymatic antioxidant which catalyzes the conversion of superoxide radical to hydrogen peroxide (not a free radical itself, but a reactive molecule) and molecular oxygen. Other enzymatic antioxidant CAT catalyzes the reduction of hydrogen peroxides and protects the tissues against reactive hydroxyl radicals. GPx was considered biologically essential in the reduction of hydrogen peroxide. In the present study, there was a reduction in the antioxidant enzyme activities of SOD, CAT and GPx in diabetic condition. Our results are in agreement with those of Mohamed et al. [43] who reported a decrease in antioxidant enzymes such as SOD, CAT and GPx in diabetes mellitus. Daily treatment with costunolide and eremanthin to STZ-induced diabetic rats showed increased level of enzymic
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antioxidants (SOD, CAT and GPx). These results depict the protective mechanism of costunolide and eremanthin by reducing the oxidative damage and strengthening the enzymic and non-enzymic antioxidant pool in the cell. Costunolide exhibited strong nitric oxide synthase inhibitory activity in the endotoxin-activated murine macrophage [44]. It also inhibited the inducible nitric oxide synthase (iNOS) gene in a human monocyte cell line THP-1 [45] and also inhibited nitric oxide production in lipopolysaccharide (Lps) activated murine macrophages [46]. In comparison to previous reports [5,8,14,27,45,46], our study clearly demonstrated the antioxidant nature of costunolide and eremanthin by significantly decreasing the TBARS levels, by increasing the levels of GSH and enzymatic antioxidants and by reducing the levels of lipid peroxidation markers. In conclusion it can be stated that the treatment of the STZinduced diabetic rats with costunolide and eremanthin isolated from C. speciosus alleviated the oxidative stress conditions, indicating the protective nature of these compounds. Conflict of interest None. References [1] K.G. Alberti, P.Z. Zimmet, New diagnostic criteria and classification of diabetesagain? Diabet. Med. 15 (1998) 535–536. [2] J.W. Baynes, S.R. Thrope, Role of oxidative stress in diabetic complications, Diabetes 48 (1999) 1. [3] A.P. Rolo, C.M. Palmeria, Diabetes and mitochondrial function: role of hyperglycemia and oxidative stress, Toxicol. Appl. Pharm. 212 (2006) 167–178. [4] P.E. Jennings, M. McLaren, N.A. Scott, A.R. Saniabadi, J.J.F. Belch, The relationship of oxidative stress to thrombotic tendency in type 1 diabetic patients with nephropathy, Diabet. Med. 8 (1991) 860–865. [5] H.F. Al-Azzawie, M.S.S. Alhamdani, Hypoglycemic and antioxidant effect of oleuropin in alloxan-diabetic rabbits, Life Sci. 78 (2006) 1371–1377. [6] P.M. Gallan, A. Carrascosa, M. Gussinye, C. Domínguez, Biomarkers of diabetesassociated oxidative stress and antioxidant status in young diabetic patients with or without subclinical complications, Free Radic. Biol. Med. 34 (2003) 1563–1574. [7] T.J. Lyons, Oxidised low density lipoproteins: a role in the pathogenesis of atherosclerosis in diabetes? Diabet. Med. 8 (1991) 411–419. [8] F. Pourmorad, S.J. Hosseinimehr, N. Shahabimajd, Antioxidant activity, phenols, flavanoid contents of selected Iranian medicinal plants, S. Afr. J. Biotechnol. 5 (2006) 1142–1145. [9] P. Schuler, Natural antioxidants exploited commercially, in: B.J.F. Hudson (Ed.), Food Antioxidants, Elsevier, London, 1990, p. 99. [10] J. Yamahara, M. Kobayashi, K. Miki, M. Kozuka, T. Sawada, H. Fujimura, Cholagogic and antiulcer effect of Saussureae radix and its active components, Chem. Pharm. Bull. 33 (1985) 1285–1288. [11] H. Chen, C. Chou, S. Lee, J. Wang, S. Yeh, Active compounds from Saussurea lappa clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells, Antivir. Res. 27 (1995) 99–109. [12] M. Taniguchi, T. Kataoka, H. Suzuki, M. Uramoto, M. Ando, J. Magae, T. Nishimura, N. Otake, K. Nagai, Costunolide and dehydrocostus lactone as inhibitors of killing function of cytotoxic T lymphocytes, Biosci. Biotechnol. Biochem. 59 (1995) 2064–2067. [13] L. Dias Fda, C.S. Takahashi, E. Sakamoto-Hojo, W. Vichnewski, S.J. Sarti, Genotoxicity of the natural cercaricides “sucupira” oil and eremanthine in mammalian cells in vitro and in vivo, Environ. Mol. Mutagen. 26 (1995) 338–344. [14] C. Filomena, S. Giancarlo, U. Dimitar, M. Francesco, Comparative chemical composition and antioxidant activities of wild and cultivated Laurus nobilis L. leaves and Foeniculum vulgare subsp. piperitum (Ucria) coutinho seeds, Biol. Pharm. Bull. 29 (2006) 2056. [15] J. Eliza, P. Daisy, S. Ignacimuthu, V. Duraipandiyan, Normo-glycemic, Hypolipidemic effect of costunolide isolated from Costus speciosus (Koen ex. Retz.) Sm. in streptozotocin induced diabetic rats, Chem. Biol. Interact. 179 (2008) 329–334. [16] J. Eliza, P. Daisy, S. Ignacimuthu, V. Duraipandiyan, Antidiabetic and antilipidemic effect of eremanthin from Costus speciosus (Koen.) Sm., in STZ induced diabetic rats, Chem. Biol. Interact. 182 (2009) 67–72. [17] P. Trinder, Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor, Ann. Clin. Biochem. 6 (1969) 24–27. [18] W.G. Nichans, B. Samuelson, Formation of malondialdehyde from phospholipid arachidonate during microsomal lipid peroxidation, Eur. J. Biochem. 6 (1968) 126–130. [19] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77.
472
J. Eliza et al. / Chemico-Biological Interactions 188 (2010) 467–472
[20] P. Kakkar, B. Das, P.N. Viswanathan, A modified spectrophotometric assay of superoxide dismutase, Indian J. Biochem. Biophys. 21 (1984) 130–132. [21] A.K. Sinha, Colorimetric assay of catalase, Anal. Biochem. 47 (1972) 389–394. [22] J.T. Rotruck, A.L. Pope, H.E. Ganther, Selenium: biochemical role as a component of glutathione peroxidase, Science 179 (1973) 588–590. [23] B.D. Duncan, Multiple range tests for correlated and heteroscedastic means, Biometrics 13 (1957) 359–364. [24] R.C. Evans, Flavonoids and isoflavones: absorption, metabolism, and bioactivity, Free Radic. Biol. Med. 36 (2004) 827–828. [25] I. Giardino, D. Edelstein, M. Brownlee, BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intra-cellular advanced glycation endproducts in bovine endothelial cells, J. Clin. Invest. 197 (1996) 1422–1428. [26] J. Valabhji, A.J. McColl, W. Richmond, M. Schachter, M.B. Rubens, R.S. Elkeles, Total antioxidant status and coronary artery calcification in type 1 diabetes, Diabetes Care 24 (2001) 1608–1613. [27] Y. Zhang, Y. Li, Y. Guo, H. Jiang, X. Shen, Asesquiterpene quinine, dysidine from the sponge Dysidea villosa activities the insulin pathway through inhibition of PT pages, Acta Pharmacol. Sin. 30 (2009) 333–345. [28] K. Tanaka, O.S. Nishizon, N. Makino, S. Tamaru, O. Terai, I. Ikeda, Hypoglycemic activity of Eriobotrya japonica seeds in type 2 diabetic rats and mice, Biosci. Biotechnol. Biochem. 72 (2008) 686–693. [29] D. Giugliano, A. Ceriello, G. Paolisso, Oxidative stress and diabetic vascular complications, Diabetes Care 19 (1996) 257–267. [30] P.S. Van Dam, B.S. Van Asbeck, D.W. Erkelens, J.J.M. Marx, W.H. Gispen, B. Bravenboer, The role of oxidative stress in neuropathy and other diabetic complications, Diabetes Metab. Rev. 11 (1995) 181–192. [31] K. Hiramatsu, S. Arimori, Increased superoxide production by mononuclear cells of patients with hypertriglyceridemia and diabetes, Diabetes 37 (1988) 832–837. [32] S.P. Wolff, Z.Y. Jang, V.J. Hunt, Protein glycation and oxidative stress in diabetes mellitus and ageing, Free Radic. Biol. Med. 10 (1991) 339–352. [33] R. Kakkar, J. Kalra, S.V. Manth, K. Parsad, Lipid peroxidation and activity of antioxidant enzymes in diabetic rats, Mol. Cell. Biochem. 151 (1995) 113– 119. [34] F.J. Bedoya, F. Solano, M. Lucas, N-monomethyl-arginine and micotinamicle prevent streptozotocin induced double strand DNA break formation in pancreatic rat islets, Experimentia 52 (1996) 344–347.
[35] J.C. Chang, M.C. Wu, I.M. Liu, J.T. Cheng, Increase of insulin sensitivity by stevioside in fructose-rich chow-fed rats, Horm. Metab. Res. 37 (2005) 610. [36] M. Mahboob, M.F. Rahman, P. Grover, Serum lipid peroxidation and antioxidant enzyme levels in male and female diabetic patients, Singapore Med. J. 46 (2005) 322–324. [37] M. Aslan, D.D. Orhan, N. Orhan, In vivo antidiabetic and antioxidant potential of Helichrysum plicatum ssp. plicatum capitulums in streptozotocin-induceddiabetic rats, J. Ethnopharmacol. 109 (2007) 54–59. [38] W. Guoyao, Z.Y. Fang, S. Yag, R.J. Lupton, D.N. Turner, Glutathione metabolism and implication for health, J. Nutr. 134 (2004) 489–492. [39] D. Konukoglu, O. Serin, D.G. Kemerli, E. Serin, A. Hayiroglu, B. Oner, A study on the carotid artery intima-media thickness and its association with lipid peroxidation, Clin. Chim. Acta 277 (1998) 91–98. [40] S.N. Chugh, R. Kakkar, S. Kalra, A. Sharma, An evaluation of oxidative stress in diabetes mellitus during uncontrolled and controlled state and after vitamin E supplementation, J. Assoc. Physicians India 47 (1999) 380–383. [41] S. Tagami, T. Kondo, K. Yoshida, J. Hirokawa, Y. Ohtsuka, Y. Kawakami, Effect of insulin on impaired antioxidant activities in aortic endothelial cells from diabetic rabbits, Metabolism 41 (1992) 1053–1058. [42] D. Sathishsekar, S. Subramanian, Beneficial effects of Momordica charantia seeds in the treatment of STZ-induced diabetes in experimental rats, Biol. Pharm. Bull. 28 (2005) 978–983. [43] A.A.M. Mohamed, A. Mayra, M.K.R. Thanaa, A.S. Nabila, A.S. Nashami, E.G. Joseph, Association of serum sialic acid with cardiovascular metabolic risk factors in Kuwaiti children and adolescents with type 1 diabetes, Metabolism 53 (2004) 638–643. [44] H.J. Park, W.T. Jung, P. Basnet, S. Kadota, T. Namba, Syringin 4-O-beta-glucoside, a new phenylpropanoid glycoside, and costunolide, a nitric oxide synthase inhibitor, from the stem bark of Magnolia sieboldii, J. Nat. Prod. 59 (1996) 1128–1130. [45] K. Fukuda, S. Akao, Y. Ohno, K. Yamashita, H. Fujiwara, Inhibition by costunolide of phorbol ester-induced transcriptional activation of inducible nitric oxide synthase gene in a human monocyte cell line THP-1, Cancer Lett. 164 (2001) 7–13. [46] S. De Marino, N. Borbone, F. Zollo, A. Ianaro, P. Di Meglio, M. Iorizzi, New sesquiterpene lactones from Laurus nobilis leaves as inhibitors of nitric oxide production, Planta Med. 71 (2005) 706–710.
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Antioxidant activity of costunolide and eremanthin isolated from Costus speciosus (Koen ex. Retz) Sm. J. Eliza a , P. Daisy a , S. Ignacimuthu b,∗ a b
Department of Biotechnology, Holy Cross College, Trichy 620002, Tamil Nadu, India Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Nungambakkam, Chennai 600034, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 24 May 2010 Received in revised form 5 August 2010 Accepted 9 August 2010 Available online 13 August 2010 Keywords: Costunolide Eremanthin Oxidative stress Experimental diabetes Enzymatic activities Non-enzymatic antioxidants Protective effect
a b s t r a c t Antioxidant properties of many medicinal plants have been widely recognized and some of them have been commercially exploited. Plant derived antioxidants play a very important role in alleviating problems related to oxidative stress. The present study was aimed at assessing the antioxidant property of costunolide and eremanthin isolated from a medicinal plant Costus speciosus (Koen ex. Retz) Sm. rhizome. Experimental diabetes was induced by a single dose of STZ (60 mg/kg, i.p.) injection. The oxidative stress was measured by tissue thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) content and enzymatic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in brain, liver, heart, kidney and pancreas. An increase in TBARS level, a significant reduction in GSH content along with decreased enzymatic activities of SOD, CAT, and GPx were seen in untreated diabetic rats. Administration of either costunolide (20 mg/kg day) or eremanthin (20 mg/kg day) for 60 days caused a significant reduction in TBARS level and a significant increase in GSH content along with increased enzymatic activities of SOD, CAT and GPx in the treated rats when compared to untreated diabetic rats. Acute toxicity test revealed the non-toxic nature of the compounds. The results indicated for the first time the protective effect of costunolide and eremanthin from oxidative stress, thus opening the way for their use in medication. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a serious complex chronic condition. It is a major source of ill-health worldwide. This metabolic disorder is characterized by hyperglycemia and disturbances of carbohydrate, protein and fat metabolisms, secondary to an absolute or relative lack of insulin [1]. Chronic oxidative stress due to hyperglycemia may, therefore, play an important role in progression of -cell dysfunction in both types of diabetes. Studies have demonstrated that this can be inhibited by antioxidants [2]. Hyperglycemia induced higher lipid peroxidation in the tissues and glycosylation of protein, resulting in formation of free radicals of oxygen (ROS) and nitrogen (RNS) [3]. The antioxidant pool is highly aggravated in hyperglycemia due to persistent challenge by ROS [4,5]. Findings have suggested that the generation of ROS/RNS leads to oxidation of lipids and proteins, which potentiate diabetes related complications [6]. Oxidative environment in cells is also created by the impairment in functioning of endogenous antioxidant enzymes namely superoxide dismutase (SOD), glutathione
∗ Corresponding author. Tel.: +91 044 2817 8348; fax: +91 044 2817 5566. E-mail address: [email protected] (S. Ignacimuthu). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.08.002
peroxidase (GPx) and catalase (CAT). GPx, CAT, SOD and glutathione reductase (GR) are known to be inhibited in diabetes mellitus as a result of non-enzymatic glycosylation and oxidation [7]. There has been an upsurge of interest in the therapeutic potential of medicinal plants as antioxidants in reducing free radical-induced tissue injury [8]. Besides, well known and traditionally used natural antioxidants from teas, wines, fruits, vegetables and spices, some natural antioxidants (e.g. from rosemary and sage) are already exploited commercially either as antioxidant additives or as nutritional supplements [9]. Costunolide and eremanthin are sesquiterpene compounds. Costunolide has been previously isolated from Saussurea radix and the dried root of S. lappa. It is reported to possess various biological actions [10–12]. Eremanthin is reported to be present in Pterodon pubescens, Eremanthus elaeagnus [13], and n-hexane extract of Larus nobilis leaves [14]. We have already reported the antidiabetic and hypolipidemic action of costunolide and eremanthin isolated from Costus speciosus [15,16]. There are no detailed reports available for the antioxidant activity of costunolide and eremanthin on various organs. So the present study was aimed at evaluating the antioxidant activity of costunolide and eremanthin in various organs such as brain, pancreas, liver, heart and kidneys which were damaged by oxidative stress due to diabetes induced by streptozotocin.
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2. Materials and methods 2.1. Chemicals Streptozotocin was procured from Sigma Chemical Co., St. Louis, MO, USA. All other chemicals were of analytical grade.
tube for 60 days. After 60 days of treatment rats were sacrificed by decapitation; their blood was collected without the addition of anticoagulant, kept at 4 ◦ C for 30 min and serum was obtained immediately by centrifugation. Organs such as brain, heart, liver, pancreas, and kidneys were removed, dried on tissue paper, and stored at −80 ◦ C for determining TBARS and enzymatic and nonenzymatic antioxidants.
2.2. Plant compounds Costunolide and eremanthin were isolated from the rhizome of C. speciosus (Koen ex. Retz) Sm. The isolation and structural elucidation have already been described [15,16]. 2.3. Experimental animals Male Wistar strain rats weighing about 190–200 g bred in the Laboratory of King’s Institute for preventive medicine, Guindy, Chennai, Tamil Nadu, India were used. All the animals were maintained under laboratory conditions of temperature (22 ± 2 ◦ C), relative humidity (45 ± 5%) and 12 h day:12 h night cycle and were allowed to feed (standard pellet diet) and water ad libitum. The experimental protocol was approved by the Institutional Animal’s Ethics Committee and by the regulatory body of the government (Reg. No. 585/05/A/CPCSEA). 2.4. Acute toxicity test An experiment was performed to know whether toxic effects were produced by eremanthin and costunolide. Rats fasted for 12 h were treated with either eremanthin or costunolide (10, 20, 40, 80 and 160 mg/kg bw) dissolved in 0.1 M citrate buffer. Rats in the control groups received only the vehicle (0.1 M citrate buffer; 1 ml/kg bw). The rats were allowed access to feed and water. Behavioural changes like abnormal locomotion, respiratory distress, uncoordinated muscle movements, weight loss and mortality were observed for 10 days. 2.5. Induction of diabetes Diabetes mellitus was induced by single intra-peritoneal injection of freshly prepared STZ (60 mg/kg bw) in 0.1 M citrate buffer (pH −4.5) in a volume of 1 ml/kg bw. Diabetes was stabilized in these STZ-treated rats over a period of 7 days. The control animals were treated with citrate buffer (pH −4.5). After 7 days the blood was collected by sinocular puncture and the plasma glucose level of each rat was determined. Rats with a fasting plasma glucose range of 280–350 mg/dl were considered diabetic and included in the study.
2.6.1. Estimation of plasma glucose and TBARS Fasting plasma glucose was estimated using glucose oxidase–peroxidase method [17]. The concentration of TBARS in the tissues was estimated by the method of Nichans and Samuelson [18]. In this method, malondialdehyde and other thiobarbituric acid reactive substances (TBARS) reacted with thiobarbituric acid in an acidic condition to generate a pink color chromophore which was read at 535 nm. 2.6.2. Oral glucose tolerance test Oral glucose tolerance test was conducted after the 60th day just prior to the animal sacrifice. After overnight fasting, plasma glucose level was estimated for 0 min in normal and experimental rats by sinocular puncture. Immediately the compounds costunolide, eremanthin (20 mg/kg bw) and insulin (3 IU/kg bw) were given to the respective experimental group rats. After 30 min glucose solution (2 g/kg bw) was administered by gavage to all the experimental rats and blood samples were taken at 30, 60, 120 and 180 min after treatment. Plasma glucose was estimated in all the experimental groups. 2.7. Non-enzymatic antioxidant 2.7.1. Estimation of reduced glutathione (GSH) Reduced glutathione in the tissues was estimated by the method of Ellman [19]. Briefly, 1.0 ml of the supernatant was treated with 0.5 ml Ellman reagent and 3.00 ml 0.2 M sodium phosphate buffer, pH 8.0, and absorbance was read at 412 nm. Reduced GSH activity is reported as nmol/g wet tissue. 2.8. Enzymatic antioxidants 2.8.1. Assay of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (EC 1.11.1.19) Superoxide dismutase in the tissues was assayed by the method of Kakkar et al. [20]. The activity of catalase in the tissues was determined by the method of Sinha [21]. The activity of GPx in the tissues was measured by the method of Rotruck et al. [22].
2.6. Experimental design and treatment schedule
2.9. Statistics
In the experiment, a total of 42 rats (18 normal; 24 STZ-induced diabetic rats) were used. The effective dose of the compounds was determined through our earlier experiments [15,16]. The rats were divided into seven groups of six each. Group 1 consisted of normal rats treated with vehicle alone (dimethysulfoxide, DMSO) 0.5%; 1 ml/kg body weight; Group 2 consisted of normal rats treated with costunolide (20 mg/kg bw); Group 3 consisted of normal rats treated with eremanthin (20 mg/kg bw); Group 4 consisted of STZ-induced diabetic rats treated with vehicle alone. Group 5 consisted of STZ-induced diabetic rats treated with costunolide (20 mg/kg bw). Group 6 consisted of STZ-induced diabetic rats treated with eremanthin (20 mg/kg bw). Group 7 consisted of STZ-induced diabetic rats treated with insulin (3 IU/kg bw). Single dose of costunolide and eremanthin was suspended in vehicle solution and was administered every day orally using intra-gastric
Statistical analysis was performed using SPSS software package Version 16.0. The values were analysed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) [23]. All the results were expressed as mean ± SD for six rats in each group. P-values < 0.05 were considered significant. 3. Results 3.1. Acute toxicity test There were no behavioural changes on the rats. Mortality was not observed during acute toxicity test. Both the compounds had not shown any behavioural changes or toxic effects up to 160 mg/kg bw.
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Table 1 Effect of costunolide and eremanthin on plasma glucose levels in normal and streptozotocin-induced diabetic male Wistar rats. Groups
Plasma glucose levels (mg/dl) 0 day
Normal Normal + costunolide (20 mg/kg bw) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
91.4 86.5 88.43 415.5 385.6 408.5 420.7
15th day ± ± ± ± ± ± ±
9.4a 14.5a 13.5a 9.4 11.5 9.3 13.5
89.6 88.4 90.11 410.41 245.6 257.65 95.67
± ± ± ± ± ± ±
30th day 11.6a 9.32a 9.7a 11.4 7.4 9.4 7.4a
90.5 85.8 89.76 422.8 184.45 196.56 90.45
± ± ± ± ± ± ±
60th day 16.7a 8.78a 11.5a 13.6 11.2a 7.7a 8.3a
86.7 90.12 85.78 427.8 137.4 140.34 87.54
± ± ± ± ± ± ±
9.23a 11.2a 17.34a 15.4 6.8a 10.23a 9.6a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats.
3.2. Estimation of plasma glucose and TBARS Changes in blood glucose levels of normal and diabetic rats treated discretely with costunolide and eremanthin are presented in Table 1. Normal untreated and normal rats treated with the compounds discretely, did not show any significant change in blood glucose levels during 60 days of treatment. However blood glucose level increased significantly in diabetic rats. The diabetic rats treated with costunolide and eremanthin at the dose of 20 mg/kg bw for 60 days showed decrease in blood glucose in a time dependent manner. 3.3. Oral glucose tolerance test The effect of costunolide and eremanthin on oral glucose tolerance test in normal and diabetic rats is given in Table 2. In STZ-treated diabetic rats, an increased plasma glucose level was observed throughout the experimental period. But the rats treated with costunolide and eremanthin showed significant (P < 0.05) decrease in plasma glucose level after 120 and 180 min. The TBARS levels of various tissues are presented in Table 3. There was a significant increase in TBARS levels in the brain, heart, liver, pancreas and kidney tissues of STZ-induced diabetic rats. Oral administration of costunolide and eremanthin for 60 days showed a significant (P < 0.05) decrease in the tissue TBARS levels, and these results were comparable with that of insulin. 3.4. Estimation of reduced glutathione (GSH) and antioxidant enzyme activities The non-enzymatic antioxidant GSH levels in liver, kidney, heart, brain and pancreas of normal, diabetic and treated groups are given in Table 4. There was a significant decrease in GSH level in brain, heart, liver, pancreas and kidneys of diabetic group when compared with normal rats. However after the administration of costunolide and eremanthin (20 mg/kg bw), there was an increase in GSH level of brain, heart, liver, pancreas and kidneys. The normal
rats treated with costunolide and eremanthin for 60 days did not exhibit any difference. The activities of glutathione peroxidase, superoxide dismutase, and catalase in the tissues of liver, kidneys, heart, brain and pancreas of normal, diabetic and treated rats are presented in Tables 5–7. The enzymatic antioxidant activities were decreased significantly in STZ-induced diabetic rats when compared with the normal untreated rats. However treatment with costunolide and eremanthin restored the enzymatic antioxidant activities in the tissues of liver, kidneys, heart, brain and pancreas almost to near normal. These results were significant (P < 0.05) and comparable with that of insulin. The normal rats treated with costunolide and eremanthin also showed normal activities of these enzymatic antioxidants. 4. Discussion Demand for natural antioxidant has been increasing due to concerns about safety of synthetic antioxidants. The present study with natural antioxidants, costunolide and eremanthin isolated from C. speciosus, is a new report for the prevention of lipid peroxidation. Oxidative stress, defined as an imbalance between oxidants and antioxidants, leads to many biochemical changes and acts as the causative factor for many diseases like diabetes, atherosclerosis, cardiovascular problems etc. [24]. Further hyperglycemia results in the generation of free radicals [25], reduced total antioxidants status (TAS) of serum [26] and oxidative damage to membranes and lipid peroxidation. The lipid peroxidation is accelerated when free radicals are formed as the result of losing a hydrogen atom from the double bond in the structure of unsaturated fatty acids. Scavenging of free radicals is one of the major antioxidation mechanisms to inhibit the chain reaction of the lipid peroxidation. Scientists have been trying to alleviate this problem by using antioxidants. Plant derived antioxidants play an important role in this. Costunolide and eremanthin isolated from the rhizome of C. speciosus were used in this study. Acute toxicity test showed that there was no marked changes in feed intake. Behavioural changes like irritation, restlessness, respi-
Table 2 Effect of costunolide and eremanthin on oral glucose tolerance in normal and streptozotocin-induced diabetic male Wistar rats after 60 days. Groups
Plasma glucose levels (mg/dl) 0 min
Normal Normal + costunolide (20 mg/kg bw) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
70.72 72.11 75.62 318.28 90.45 95.62 92.77
30 min ± ± ± ± ± ± ±
10.51a 12.71a 11.63a 14.77 9.86 10.15 12.29a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats.
108.12 105.62 110.75 355.17 138.11 146.12 102.71
60 min ± ± ± ± ± ± ±
8.72a 15.1a 13.6a 15.25 8.18a 10.11a 9.62a
135.81 128.19 138.65 420.25 188.72 196.81 120.65
120 min ± ± ± ± ± ± ±
12.82a 13.62a 14.71a 16.22 12.72 10.81 14.62a
92.33 90.16 98.21 438.32 145.46 140.71 88.19
± ± ± ± ± ± ±
180 min 11.77a 11.65a 15.81a 15.81 11.78a 9.82a 8.95a
80.75 87.6 85.82 445.55 107.63 110.72 83.85
± ± ± ± ± ± ±
10.55a 12.19a 9.52a 12.33 10.45a 12.64a 9.86a
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Table 3 Effect of costunolide and eremanthin on tissue TBARS in normal and streptozotocin-induced diabetic male Wistar rats after 60 days. Groups
Tissue TBARS (nmol/100 g tissue) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
1.59 1.53 1.56 2.85 1.62 1.70 1.64
Heart ± ± ± ± ± ± ±
0.04a 0.02a 0.04a 0.07 0.02a 0.03a 0.03a
Liver
± ± ± ± ± ± ±
0.51 0.50 0.52 0.92 0.58 0.62 0.55
.02a .03a .04a .04 .06a .05a .02a
1.36 1.27 1.33 2.8 1.42 1.52 1.8
Pancreas ± ± ± ± ± ± ±
0.06a 0.06a 0.06a 0.08 0.06a 0.04a 0.03a
± ± ± ± ± ± ±
38.85 34.40 37.18 72.00 45.79 51.05 49.19
Kidney 2.58a 2.20a 2.59a 3.63 2.97a 2.7a 2.42a
1.49 1.41 1.45 2.50 1.58 1.67 1.58
± ± ± ± ± ± ±
0.02a 0.03a 0.05a 0.02 0.05a 0.02a 0.06a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats. Table 4 Effect of costunolide and eremanthin on tissue reduced glutathione in normal and streptozotocin-induced diabetic male Wistar rats after 60 days of treatment. Groups
Reduced glutathione (nmol/g wet tissue) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
Heart
11.59 10.49 9.47 4.89 9.79 8.69 7.55
± ± ± ± ± ± ±
1.42a 1.28a .74a 1.01 1.75a 1.57a 1.22a
Liver ± ± ± ± ± ± ±
12.35 12.04 11.40 5.13 11.20 10.46 7.61
1.46a 1.64a 1.57a 1.13 1.54a 1.3a 1.18a
Pancreas
22.61 23.03 22.83 14.46 21.11 19.45 17.22
± ± ± ± ± ± ±
1.98a 2.10a 2.20a 1.33 1.86a 1.9a 2.18a
± ± ± ± ± ± ±
21.85 21.12 20.25 11.83 20.91 19.75 17.56
Kidney 1.81a 1.13a 1.46a 1.52 1.80a 2.01a 1.74a
20.68 22.51 21.40 11.00 22.26 20.68 19.34
± ± ± ± ± ± ±
1.48a 2.24a 2.39a 1.76 2.26a 1.48a 1.73a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats. Table 5 Effect of costunolide and eremanthin on tissue glutathione peroxidase in normal and streptozotocin-induced diabetic male Wistar rats after 60 days of treatment. Groups
GPx (g of GSH consumed/min/mg protein) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
Heart ± ± ± ± ± ± ±
6.93 6.71 6.55 2.19 5.34 5.16 4.11
a
0.44 0.63a 0.51a 0.29 0.36a 0.23a 0.43a
0.99 0.96 0.94 0.56 0.93 0.89 0.85
Liver
± ± ± ± ± ± ±
a
0.04 0.10a 0.04a 0.07 0.03a 0.02a 0.06a
9.15 9.22 9.14 5.00 8.00 7.79 6.59
Pancreas ± ± ± ± ± ± ±
a
0.53 0.64a 0.45a 0.25 0.33a 0.44a 0.37a
28.77 27.83 28.14 14.73 26.91 27.10 22.62
± ± ± ± ± ± ±
Kidney a
1.63 2.11a 2.23a 0.84 1.94a 2.66a 1.77a
6.51 7.13 6.92 3.17 5.96 5.14 4.26
± ± ± ± ± ± ±
0.41a 0.71a 0.62a 0.24 0.35a 0.81a 0.44a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats. Table 6 Effect of costunolide and eremanthin on superoxide dismutase in normal and streptozotocin-induced diabetic male Wistar rats after 60 days of treatment. Groups
SOD (U/mg protein) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
7.91 7.53 7.75 3.18 6.88 6.65 4.12
Heart ± ± ± ± ± ± ±
a
0.62 0.51a 0.29a 0.36 0.49a 0.55a 0.37a
9.72 8.91 8.23 4.25 8.82 8.60 7.00
Liver
± ± ± ± ± ± ±
a
0.33 0.52a 0.41a 0.72 0.61a 0.59a 0.17a
8.95 8.18 8.09 4.19 7.11 7.52 5.89
Pancreas ± ± ± ± ± ± ±
a
0.51 0.33a 0.45a 0.22 0.55a 0.43a 0.71a
4.94 4.61 4.76 2.11 4.49 4.66 3.11
± ± ± ± ± ± ±
Kidney a
0.54 0.38a 0.29a 0.11 0.30a 0.22a 0.35a
14.71 14.23 13.82 6.71 12.59 12.43 9.26
± ± ± ± ± ± ±
0.73a 0.94a 1.24a 0.11 1.17a 0.92a 0.78a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats. Table 7 Effect of costunolide and eremanthin on tissue catalase in normal and streptozotocin-induced diabetic male Wistar rats after 60 days of treatment. Groups
CAT (mol of H2 O2 consumed/min/mg protein) Brain
Normal Normal + costunolide (20 mg/kg) Normal + eremanthin (20 mg/kg bw) Diabetic control Diabetic + costunolide (20 mg/kg bw) Diabetic + eremanthin (20 mg/kg bw) Diabetic + insulin (3 IU/kg)
2.85 3.19 3.28 0.78 2.31 2.25 1.65
Heart ± ± ± ± ± ± ±
0.39a 0.24a 0.33a 0.04 0.19a 0.41a 0.21a
Each value is mean ± SD for six rats in each group. a P < 0.05 by comparison with streptozotocin-induced diabetic rats.
8.77 8.09 8.56 4.00 7.52 7.18 5.78
± ± ± ± ± ± ±
Liver 0.51a 0.68a 0.43a 0.33 0.59a 0.47a 0.55a
80.13 79.72 79.19 46.33 73.69 74.19 62.88
Pancreas ± ± ± ± ± ± ±
5.19a 5.06a 4.63a 2.18 4.11a 3.58a 3.17a
17.18 16.78 16.95 6.96 13.37 13.25 10.87
± ± ± ± ± ± ±
Kidney 0.77a 0.64a 0.81a 0.49 0.47a 0.91a 0.68a
35.19 36.11 35.82 14.98 29.41 28.72 23.17
± ± ± ± ± ± ±
1.72a 3.21a 1.48a 2.33 2.19a 1.55a 0.98a
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ratory distress, abnormal locomotion and catalapsy over a period of 10 days were not observed. This revealed the non-toxic nature of both the compounds. Daily administration of these compounds decreased the plasma glucose level significantly. Oral administration of the compounds produced significant effect when glucose challenge was given to normal and diabetic rats. Decrease in the plasma glucose was observed at 120 min after the administration of costunolide and eremanthin. Dysidine, a sesquiterpene, effectively activated the insulin signaling pathways, promoted glucose uptake and showed insulin-sensitizing activities through the inhibition of protein tyrosine phospatases [27]. The glucose lowering effect of costunolide and eremanthin which are sesquiterpenes might be due to similar action. Our results are similar to the one in which Eriobotrya japonica seeds effectively improved glucose tolerance in the KK-Ay mice [28]. Hyperglycemia results in the generation of free radicals which can exhaust antioxidant defenses thus leading to the disruption of cellular functions, oxidative damage to membranes and enhanced susceptibility to lipid peroxidation [29,30]. It has been reported that increased oxidative stress may play a role in the pathogenesis and progression of diabetic tissue damage [31–33]. STZ has been reported to generate ROS [34]. In the present study a marked increase in the level of tissue TBARS in STZ-induced diabetic rats indicated enhanced lipid peroxidation leading to tissue injury and failure of the antioxidant defense mechanism to prevent the formation of excess free radicals. The diabetic rats treated with costunolide and eremanthin for 60 days showed very low levels of tissue damage compared to untreated diabetic rats. These results showed that costunolide and eremanthin protected the various organs from tissue damage by reducing the ROS production. Studies concluded by other investigators in diabetic patients and diabetic rats reported similar findings [35,36]. The water and ethanol extracts of Helichrysum plicatum ssp. and Plicatum capitulums produced significant decrease in kidney and liver MDA levels of diabetic rats [37]. GSH, a non-enzymatic antioxidant, effectively scavenges free radicals and other ROS directly and indirectly through enzymatic reactions [38]. Depletion of tissue GSH is one of the primary factors that permit lipid peroxidation [39]. It has been proposed that antioxidants that maintain the concentration of GSH may restore the cellular defense mechanisms, block lipid peroxidation and thus protect the tissue against oxidative damage [40]. In the present investigation the GSH concentration was low in the diabetic group. This is in accordance with the results of Tagami et al. [41] who demonstrated a reduced concentration of GSH in diabetic rabbits. In the present study, oral administration with costunolide and eremanthin significantly increased the GSH levels. Increase in GSH level in turn activated the GSH dependent enzymes such as glutathione peroxidase and glutathione-S-transferase by which it could reduce the tissue damage. Apart from the non-enzymatic antioxidants, enzymatic antioxidants such as SOD and CAT play an important role in preventing cells from being exposed to oxidative damage [42]. SOD is an enzymatic antioxidant which catalyzes the conversion of superoxide radical to hydrogen peroxide (not a free radical itself, but a reactive molecule) and molecular oxygen. Other enzymatic antioxidant CAT catalyzes the reduction of hydrogen peroxides and protects the tissues against reactive hydroxyl radicals. GPx was considered biologically essential in the reduction of hydrogen peroxide. In the present study, there was a reduction in the antioxidant enzyme activities of SOD, CAT and GPx in diabetic condition. Our results are in agreement with those of Mohamed et al. [43] who reported a decrease in antioxidant enzymes such as SOD, CAT and GPx in diabetes mellitus. Daily treatment with costunolide and eremanthin to STZ-induced diabetic rats showed increased level of enzymic
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antioxidants (SOD, CAT and GPx). These results depict the protective mechanism of costunolide and eremanthin by reducing the oxidative damage and strengthening the enzymic and non-enzymic antioxidant pool in the cell. Costunolide exhibited strong nitric oxide synthase inhibitory activity in the endotoxin-activated murine macrophage [44]. It also inhibited the inducible nitric oxide synthase (iNOS) gene in a human monocyte cell line THP-1 [45] and also inhibited nitric oxide production in lipopolysaccharide (Lps) activated murine macrophages [46]. In comparison to previous reports [5,8,14,27,45,46], our study clearly demonstrated the antioxidant nature of costunolide and eremanthin by significantly decreasing the TBARS levels, by increasing the levels of GSH and enzymatic antioxidants and by reducing the levels of lipid peroxidation markers. In conclusion it can be stated that the treatment of the STZinduced diabetic rats with costunolide and eremanthin isolated from C. speciosus alleviated the oxidative stress conditions, indicating the protective nature of these compounds. Conflict of interest None. References [1] K.G. Alberti, P.Z. Zimmet, New diagnostic criteria and classification of diabetesagain? Diabet. Med. 15 (1998) 535–536. [2] J.W. Baynes, S.R. Thrope, Role of oxidative stress in diabetic complications, Diabetes 48 (1999) 1. [3] A.P. Rolo, C.M. Palmeria, Diabetes and mitochondrial function: role of hyperglycemia and oxidative stress, Toxicol. Appl. Pharm. 212 (2006) 167–178. [4] P.E. Jennings, M. McLaren, N.A. Scott, A.R. Saniabadi, J.J.F. Belch, The relationship of oxidative stress to thrombotic tendency in type 1 diabetic patients with nephropathy, Diabet. Med. 8 (1991) 860–865. [5] H.F. Al-Azzawie, M.S.S. Alhamdani, Hypoglycemic and antioxidant effect of oleuropin in alloxan-diabetic rabbits, Life Sci. 78 (2006) 1371–1377. [6] P.M. Gallan, A. Carrascosa, M. Gussinye, C. Domínguez, Biomarkers of diabetesassociated oxidative stress and antioxidant status in young diabetic patients with or without subclinical complications, Free Radic. Biol. Med. 34 (2003) 1563–1574. [7] T.J. Lyons, Oxidised low density lipoproteins: a role in the pathogenesis of atherosclerosis in diabetes? Diabet. Med. 8 (1991) 411–419. [8] F. Pourmorad, S.J. Hosseinimehr, N. Shahabimajd, Antioxidant activity, phenols, flavanoid contents of selected Iranian medicinal plants, S. Afr. J. Biotechnol. 5 (2006) 1142–1145. [9] P. Schuler, Natural antioxidants exploited commercially, in: B.J.F. Hudson (Ed.), Food Antioxidants, Elsevier, London, 1990, p. 99. [10] J. Yamahara, M. Kobayashi, K. Miki, M. Kozuka, T. Sawada, H. Fujimura, Cholagogic and antiulcer effect of Saussureae radix and its active components, Chem. Pharm. Bull. 33 (1985) 1285–1288. [11] H. Chen, C. Chou, S. Lee, J. Wang, S. Yeh, Active compounds from Saussurea lappa clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells, Antivir. Res. 27 (1995) 99–109. [12] M. Taniguchi, T. Kataoka, H. Suzuki, M. Uramoto, M. Ando, J. Magae, T. Nishimura, N. Otake, K. Nagai, Costunolide and dehydrocostus lactone as inhibitors of killing function of cytotoxic T lymphocytes, Biosci. Biotechnol. Biochem. 59 (1995) 2064–2067. [13] L. Dias Fda, C.S. Takahashi, E. Sakamoto-Hojo, W. Vichnewski, S.J. Sarti, Genotoxicity of the natural cercaricides “sucupira” oil and eremanthine in mammalian cells in vitro and in vivo, Environ. Mol. Mutagen. 26 (1995) 338–344. [14] C. Filomena, S. Giancarlo, U. Dimitar, M. Francesco, Comparative chemical composition and antioxidant activities of wild and cultivated Laurus nobilis L. leaves and Foeniculum vulgare subsp. piperitum (Ucria) coutinho seeds, Biol. Pharm. Bull. 29 (2006) 2056. [15] J. Eliza, P. Daisy, S. Ignacimuthu, V. Duraipandiyan, Normo-glycemic, Hypolipidemic effect of costunolide isolated from Costus speciosus (Koen ex. Retz.) Sm. in streptozotocin induced diabetic rats, Chem. Biol. Interact. 179 (2008) 329–334. [16] J. Eliza, P. Daisy, S. Ignacimuthu, V. Duraipandiyan, Antidiabetic and antilipidemic effect of eremanthin from Costus speciosus (Koen.) Sm., in STZ induced diabetic rats, Chem. Biol. Interact. 182 (2009) 67–72. [17] P. Trinder, Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor, Ann. Clin. Biochem. 6 (1969) 24–27. [18] W.G. Nichans, B. Samuelson, Formation of malondialdehyde from phospholipid arachidonate during microsomal lipid peroxidation, Eur. J. Biochem. 6 (1968) 126–130. [19] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77.
472
J. Eliza et al. / Chemico-Biological Interactions 188 (2010) 467–472
[20] P. Kakkar, B. Das, P.N. Viswanathan, A modified spectrophotometric assay of superoxide dismutase, Indian J. Biochem. Biophys. 21 (1984) 130–132. [21] A.K. Sinha, Colorimetric assay of catalase, Anal. Biochem. 47 (1972) 389–394. [22] J.T. Rotruck, A.L. Pope, H.E. Ganther, Selenium: biochemical role as a component of glutathione peroxidase, Science 179 (1973) 588–590. [23] B.D. Duncan, Multiple range tests for correlated and heteroscedastic means, Biometrics 13 (1957) 359–364. [24] R.C. Evans, Flavonoids and isoflavones: absorption, metabolism, and bioactivity, Free Radic. Biol. Med. 36 (2004) 827–828. [25] I. Giardino, D. Edelstein, M. Brownlee, BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intra-cellular advanced glycation endproducts in bovine endothelial cells, J. Clin. Invest. 197 (1996) 1422–1428. [26] J. Valabhji, A.J. McColl, W. Richmond, M. Schachter, M.B. Rubens, R.S. Elkeles, Total antioxidant status and coronary artery calcification in type 1 diabetes, Diabetes Care 24 (2001) 1608–1613. [27] Y. Zhang, Y. Li, Y. Guo, H. Jiang, X. Shen, Asesquiterpene quinine, dysidine from the sponge Dysidea villosa activities the insulin pathway through inhibition of PT pages, Acta Pharmacol. Sin. 30 (2009) 333–345. [28] K. Tanaka, O.S. Nishizon, N. Makino, S. Tamaru, O. Terai, I. Ikeda, Hypoglycemic activity of Eriobotrya japonica seeds in type 2 diabetic rats and mice, Biosci. Biotechnol. Biochem. 72 (2008) 686–693. [29] D. Giugliano, A. Ceriello, G. Paolisso, Oxidative stress and diabetic vascular complications, Diabetes Care 19 (1996) 257–267. [30] P.S. Van Dam, B.S. Van Asbeck, D.W. Erkelens, J.J.M. Marx, W.H. Gispen, B. Bravenboer, The role of oxidative stress in neuropathy and other diabetic complications, Diabetes Metab. Rev. 11 (1995) 181–192. [31] K. Hiramatsu, S. Arimori, Increased superoxide production by mononuclear cells of patients with hypertriglyceridemia and diabetes, Diabetes 37 (1988) 832–837. [32] S.P. Wolff, Z.Y. Jang, V.J. Hunt, Protein glycation and oxidative stress in diabetes mellitus and ageing, Free Radic. Biol. Med. 10 (1991) 339–352. [33] R. Kakkar, J. Kalra, S.V. Manth, K. Parsad, Lipid peroxidation and activity of antioxidant enzymes in diabetic rats, Mol. Cell. Biochem. 151 (1995) 113– 119. [34] F.J. Bedoya, F. Solano, M. Lucas, N-monomethyl-arginine and micotinamicle prevent streptozotocin induced double strand DNA break formation in pancreatic rat islets, Experimentia 52 (1996) 344–347.
[35] J.C. Chang, M.C. Wu, I.M. Liu, J.T. Cheng, Increase of insulin sensitivity by stevioside in fructose-rich chow-fed rats, Horm. Metab. Res. 37 (2005) 610. [36] M. Mahboob, M.F. Rahman, P. Grover, Serum lipid peroxidation and antioxidant enzyme levels in male and female diabetic patients, Singapore Med. J. 46 (2005) 322–324. [37] M. Aslan, D.D. Orhan, N. Orhan, In vivo antidiabetic and antioxidant potential of Helichrysum plicatum ssp. plicatum capitulums in streptozotocin-induceddiabetic rats, J. Ethnopharmacol. 109 (2007) 54–59. [38] W. Guoyao, Z.Y. Fang, S. Yag, R.J. Lupton, D.N. Turner, Glutathione metabolism and implication for health, J. Nutr. 134 (2004) 489–492. [39] D. Konukoglu, O. Serin, D.G. Kemerli, E. Serin, A. Hayiroglu, B. Oner, A study on the carotid artery intima-media thickness and its association with lipid peroxidation, Clin. Chim. Acta 277 (1998) 91–98. [40] S.N. Chugh, R. Kakkar, S. Kalra, A. Sharma, An evaluation of oxidative stress in diabetes mellitus during uncontrolled and controlled state and after vitamin E supplementation, J. Assoc. Physicians India 47 (1999) 380–383. [41] S. Tagami, T. Kondo, K. Yoshida, J. Hirokawa, Y. Ohtsuka, Y. Kawakami, Effect of insulin on impaired antioxidant activities in aortic endothelial cells from diabetic rabbits, Metabolism 41 (1992) 1053–1058. [42] D. Sathishsekar, S. Subramanian, Beneficial effects of Momordica charantia seeds in the treatment of STZ-induced diabetes in experimental rats, Biol. Pharm. Bull. 28 (2005) 978–983. [43] A.A.M. Mohamed, A. Mayra, M.K.R. Thanaa, A.S. Nabila, A.S. Nashami, E.G. Joseph, Association of serum sialic acid with cardiovascular metabolic risk factors in Kuwaiti children and adolescents with type 1 diabetes, Metabolism 53 (2004) 638–643. [44] H.J. Park, W.T. Jung, P. Basnet, S. Kadota, T. Namba, Syringin 4-O-beta-glucoside, a new phenylpropanoid glycoside, and costunolide, a nitric oxide synthase inhibitor, from the stem bark of Magnolia sieboldii, J. Nat. Prod. 59 (1996) 1128–1130. [45] K. Fukuda, S. Akao, Y. Ohno, K. Yamashita, H. Fujiwara, Inhibition by costunolide of phorbol ester-induced transcriptional activation of inducible nitric oxide synthase gene in a human monocyte cell line THP-1, Cancer Lett. 164 (2001) 7–13. [46] S. De Marino, N. Borbone, F. Zollo, A. Ianaro, P. Di Meglio, M. Iorizzi, New sesquiterpene lactones from Laurus nobilis leaves as inhibitors of nitric oxide production, Planta Med. 71 (2005) 706–710.