In vivo assessment of antidiabetic and antioxidant activities of rosemary (Rosmarinus officinalis) in alloxan-diabetic rabbits

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Journal of Ethnopharmacology 116 (2008) 64–73

In vivo assessment of antidiabetic and antioxidant activities of rosemary (Rosmarinus officinalis) in alloxan-diabetic rabbits b , Hasret Yardibi c ¨ uner Keles¸ a , Sinem G¨unes¸ Ulgen ¨ T¨ulay Bakırel a,∗ , Utku Bakırel b , Oya Ust¨ a

Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Istanbul University, Avcılar 34320, Istanbul, Turkey b Department of Internal Medicine, Faculty of Veterinary Medicine, Istanbul University, Avcılar 34320, Istanbul, Turkey c Department of Biochemistry, Faculty of Veterinary Medicine, Istanbul University, Avcılar 34320, Istanbul, Turkey Received 20 March 2007; received in revised form 14 October 2007; accepted 30 October 2007 Available online 4 November 2007

Abstract Rosemary (Rosmarinus officinalis), used in traditional Turkish folk medicine for the treatment of hyperglycaemia, is widely accepted as one of the medicinal herb with the highest antioxidant activity. Accordingly, the present study was designed to investigate the possible actions of ethanolic extract of the leaves of Rosmarinus officinalis on glucose homeostasis and antioxidant defense in rabbits. In the first set of experiments, hypoglycaemic effects of oral administration of various doses (50, 100 and 200 mg/kg) of the extract were examined in normoglycaemic and glucose-hyperglycaemic rabbits. Optimal effect was observed in both of the animal groups with a dose of 200 mg/kg of the extract and this activity was independent from the effects of insulin. In another part of experiments, acute effect of various doses of the Rosmarinus officinalis extract on blood glucose and serum insulin levels was studied in alloxan-induced diabetic rabbits. Of the three doses of extract, the highest dose (200 mg/kg) significantly lowered blood glucose level and increased serum insulin concentration in alloxan-diabetic rabbits. The last set of experiments designed to investigate the subacute effect of the Rosmarinus officinalis extract on repeated administration in alloxan-diabetic rabbits. At the doses of 100 and 200 mg/kg, antihyperglycaemic effect of extract was accompanied by a significant increase in serum insulin levels in diabetic rabbits. Furthermore, during 1 week of treatment of diabetic rabbits with a dose of 200 mg/kg of the extract showed that the extract possessed a capability to inhibit the lipid peroxidation and activate the antioxidant enzymes. It was concluded that probably, due to its potent antioxidant properties, the Rosmarinus officinalis extract exerts remarkable antidiabetogenic effect. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Rosmarinus officinalis; Diabetes; Alloxan; Rabbits; Antioxidant; Antidiabetic

1. Introduction Diabetes mellitus (DM) is a major endocrine disorder, affecting approximately 5% of the world’s population. Worldwide projections suggest that more 300 million people will have diabetes by the year 2025 and the global cost of treating diabetes and its complication could reach US $ trillion annually (King et al., 1998; Somani et al., 2006). It is characterized by abnormalities in carbohydrate, lipid, and lipoprotein metabolisms, which not only lead to hyperglycaemia but also cause many complications, such as hyperlipidemia, hyperinsulinemia, hypertension, and atherosclerosis (Sepici et al., 2004; Luo et al., 2004). Numerous studies have been demonstrated that oxidative stress, mediated

∗

Corresponding author. Tel.: +90 212 473 70 70; fax: +90 212 473 72 41. E-mail address: [email protected] (T. Bakırel).

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.10.039

mainly by hyperglycaemia-induced generation of free radicals, contributes to the development and progression of diabetes and its complications (Ceriello, 2003; Rahimi et al., 2005; Tang et al., 2006). Abnormally high levels of free radicals which cause membrane damage due to peroxidation of membrane lipids and protein glycation and the simultaneous decline of antioxidant defense mechanisms leads to cell and tissue damage (Maritim et al., 2003; Tang et al., 2006). Pancreatic ␤-cells are particularly susceptible to the deleterious effects of reactive oxygen species (ROS), because of their low expression of the antioxidant enzymes genes as compared to other tissues. Thus, the increase of ROS leads to damage of ␤-cells through the induction of apoptosis and suppression of insulin biosynthesis (El-Alfy et al., 2005; Vijayakumar et al., 2006). As a new strategy for alleviating the oxidative damage in diabetes, interest has grown in the usage of natural antioxidants. It has been postulated that many of the negative effect of oxidative stress are diminished upon

T. Bakırel et al. / Journal of Ethnopharmacology 116 (2008) 64–73

supplementation with certain dietary antioxidants such as vitamin E, C and other non-nutrient antioxidant such as flavonoids (Rahimi et al., 2005; Al-Azzawie and Alhamdani, 2006). On the other hand, many plant species are known in folk medicine of different cultures to be used for their hypoglycaemic properties and therefore used for treatment of DM (Abdel-Barry et al., 1997; Pushparaj et al., 2000). Despite this, few traditional antidiabetic plants have received proper scientific screening. The World Health Organization (WHO) has recommended that this area warrants further evaluation (WHO, 1980). Rosemary, Rosmarinus officinalis L. (Labiatae) is an evergreen perennial shrub grown in many parts of the world (Al-Sereiti et al., 1999; Porte et al., 2000). It has been reported to possess a number of therapeutic applications in folk medicines in curing or managing of a wide range of diseases such as DM, respiratory disorders, stomach problems and inflammatory diseases (EMEA, 1997; Erenmemis¸o˘glu et al., 1997; Al-Sereiti et al., 1999; K¨ult¨ur, 2007). The water decoction of rosemary leaves has been traditionally used to treat diabetic patients, especially in the western part of Turkey, without much scientific evidence of its utility. Among natural antioxidants, rosemary has been widely accepted as one of the species with the highest antioxidant activity (Peng et al., 2005). It is well known that the activity of rosemary extracts in medicine and food industry due to the presence of some important antioxidant oil and phenolic components, to prevent oxidative degradation of oil and lipid containing foods (Stefanovits-Banyai et al., 2003). Rosemary has long been recognised as having antioxidant molecules, such as rosmarinic acid, carnasol, rosmaridiphenol and these have found in ethanol-soluble fraction (Dorman et al., 1995). No detailed study has been carried out on the efficacy of Rosmarinus officinalis in the modulation of oxidative stress associated with DM in experimental animals. Hence, the present study was undertaken to investigate possible hypoglycaemic/antihyperglycaemic and antioxidant effects of ethanolic extract of Rosmarinus officinalis leaves. 2. Materials and methods 2.1. Plant material Fresh Rosmarinus officinalis L. was collected from AvcılarIstanbul, Turkey in between the month of May and July 2005. The plant was identified by Assoc. Prof. Dr. S¸u¨ kran K¨ult¨ur. A dried specimen was deposited in the Herbarium of Istanbul University, Faculty of Pharmacy (ISTE-Herbarium), with registration number 83893. The plant was dried under shade at 25 ◦ C, and the dried leaves of the plant were grounded with a blender. The powdered part was kept in nylon bags in a deep freezer until the time of use. 2.2. Preparation of plant extract The powdered leaves were extracted in soxlet with 95% ethanol at a temperature of 50 ◦ C for 12 h. The residue was removed by filtration. The alcoholic extract concentrated (yield

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6.1%) in a rotary evaporator under reduced pressure at temperature of 40–50 ◦ C and then lyophilized to get a power. 2.3. Animals Adult New Zealand rabbits of either sex, weighing 2.2– 3.1 kg, were used for experiments. All rabbits were kept in individual metal cages located in the animal laboratory of Istanbul University, Faculty of Veterinary Medicine at room temperature of 20–22 ◦ C and at 45–55% relative humidity for 12 h, each of dark and light cycle. They were fed with a standard died and water ad libitum. The rabbits were kept in those conditions for a 7-day period of adaptation prior to start of the experiment. Sixteen hours before the experiments, they were fasted overnight, but allowed free access to water. The experimental protocol has been approved by Istanbul University Veterinary Faculty Ethic Committee (Regd. No.2005/103). 2.4. Experimental procedure 2.4.1. Effect of the Rosmarinus officinalis extract on blood glucose and insulin levels of normoglycaemic rabbits Fasted rabbits were divided into five groups of seven animals per group. The first group of animals received only vehicle (Tween 80 in distilled water, 10% (v/v)) orally in a volume of 10 ml/kg and served as control. Group II received glibenclamide (Nobel Pharma, Turkey) as reference drug (5 mg/kg, p.o.) suspended in vehicle (10 ml/kg). The Rosmarinus officinalis extract, suspended in vehicle, was administered at the doses of 50, 100 and 200 mg/kg orally in a volume of 10 ml/kg to the animals of group III, IV and V, respectively. Blood samples were collected from the marginal ear vein for glucose and insulin levels estimation just prior to and at 1, 2 and 6 h after dosing. 2.4.2. Effect of the Rosmarinus officinalis extract on blood glucose and insulin levels of glucose-hyperglycaemic rabbits (oral glucose tolerance test, OGTT) Fasted rabbits were divided into five groups of seven animals each. Group I, serving as control, received only vehicle (Tween 80 in distilled water, 10% (v/v)) orally in a volume of 10 ml/kg and group II received glibenclamide as reference drug (5 mg/kg, p.o.) suspended in vehicle (10 ml/kg). A dose of 50, 100 and 200 mg/kg of the Rosmarinus officinalis extract suspended in vehicle was administered orally in a volume of 10 ml/kg to the animals of III, IV and V, respectively. The rabbits of all the groups were given glucose (2 g/kg, p.o.), 30 min after the extract and drug administration. Blood samples were collected from the marginal ear vein for glucose and insulin levels estimation just prior to glucose administration (0 h) and 1, 2 and 6 h after glucose loading. 2.4.3. Induction of diabetes Rabbits were made diabetic by a single intravenously (marginal ear vein) injection of alloxan monohydrate (Sigma Chemicals, USA) 5% (w/v) in normal saline at a dose of 120 mg/kg, body weight. Then 5 days later blood samples collected and glucose levels were determined to confirm the

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T. Bakırel et al. / Journal of Ethnopharmacology 116 (2008) 64–73

development of diabetes. The rabbits with blood glucose level >250 mg/dl were considered to be diabetic and were used in the experiment.

aldehyde (MDA) production, was measured in serum by the method of Yoshoiko et al. (1979). The results were expressed as n mol/ml.

2.4.3.1. Acute effect of the Rosmarinus officinalis extract in alloxan-induced diabetic rabbits. The diabetic rabbits were divided into five groups of seven animals each. Group I received only vehicle (Tween 80 in distilled water, 10% (v/v)) orally in a volume of 10 ml/kg and served as control. Group II received glibenclamide as reference drug (5 mg/kg, p.o.) suspended in vehicle (10 ml/kg). The Rosmarinus officinalis extract, suspended in vehicle, was administered at the doses of 50, 100 and 200 mg/kg orally in a volume of 10 ml/kg to the animals of group III, IV and V, respectively. Blood samples were collected from the marginal ear vein just prior to and at 1, 2 and 6 h after dosing. Blood glucose and insulin levels were determined.

2.5.4. Estimation of antioxidants Superoxide dismutase (SOD) activity was measured in serum by method Sun et al. (1988). One unit of SOD is defined as the amount of protein that inhibits the rate of NBT reduction by 50%. Catalase (CAT) activity was determined in serum using the modified method described by Yasmineh et al. (1995). CAT activity was expressed as kU/l.

2.4.3.2. Subacute effect of the Rosmarinus officinalis extract in alloxan-induced diabetic rabbits. The action of Rosmarinus officinalis was also tested during a longer duration of treatment. The rabbits were divided into groups containing healthy and diabetic animals. Group I (healthy rabbits, n = 7) received only vehicle orally in a volume of 10 ml/kg for 7 days and served as control. The diabetic rabbits were divided into five groups (II–VI) of seven animals each. Group II received only vehicle (Tween 80 in distilled water, 10% (v/v)) orally in a volume of 10 ml/kg for 7 days and served as diabetic control. Group III received glibenclamide as reference drug (5 mg/kg, p.o.) suspended in vehicle (10 ml/kg) for 7 days. The Rosmarinus officinalis extract, suspended in vehicle, was administered at the doses of 50, 100 and 200 mg/kg orally in a volume of 10 ml/kg for 7 days to the animals of groups IV, V and VI, respectively. Blood samples were collected from the marginal ear vein at 1st, 3rd, 5th, and 8th days after each treatment. Blood glucose, insulin, lipid peroxidation and antioxidant enzymes (superoxide dismutase and catalase) levels were measured. The body weights of the animals were also recorded at the same days. 2.5. Analytical method 2.5.1. Determination of blood glucose concentration Blood glucose concentration was determined in serum by commercially available glucose kid (Spinreact, S.A., Spain) based on Trinder’s (1969) glucose oxidase method. The glucose levels were expressed as mg/dl. 2.5.2. Determination of insulin concentration Insulin concentration was determined in serum by radioimmunoassay method using a commercially available DSL-1600 insulin kit (Diagnostic Systems Laboratories, Inc., USA). Insulin values were expressed as ␮lU/ml. 2.5.3. Determination of the lipid peroxidation (LPO) in serum The level of thiobarbituric acid reactive substances (TBARS), a commonly used marker for lipid peroxidation and malondi-

2.5.5. Statistical analysis Values are presented as means ± S.E.M. Statistical differences between the treatments and the controls were tested by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test using the “Instat” statistic computer program. A difference in the mean values of p < 0.05 or less was considered to be statistically significant. 3. Results 3.1. Effect of the Rosmarinus officinalis extract on blood glucose and insulin levels of normoglycaemic rabbits Effects of various doses of the extract obtained from Rosmarinus officinalis on blood glucose and insulin levels in normoglycaemic rabbits are shown in Table 1. The glucose and insulin levels were compared to the values obtained from animals given vehicle (control). As shown in Table 1, the extract at 50 mg/kg dose did not show any remarkable effect, while the extract at higher doses (100 and 200 mg/kg) showed significant reduction on blood glucose levels in normoglycaemic rabbits. A dose of 100 mg/kg of the extract produced a significantly fall of 14.5% in normoglycaemic rabbits in blood glucose level after 2 h of oral administration. This fall decreased at 6 h and was non-significant. Rabbits treated with 200 mg/kg of the extract showed a significant fall of 20.4% and 20.8% fall in blood glucose levels, respectively after 2 h and 6 h of oral administration. Of the three doses of the Rosmarinus officinalis extract tested, 200 mg/kg was found to be most effective on blood glucose level in normoglycaemic rabbits. Glibenclamide (5 mg/kg) induced significant reduction in blood glucose level of 16% (1 h), 28.3% (2 h) and 35.5% (6 h) when compared to the control group. In normoglycaemic rabbits, the extract of Rosmarinus officinalis at doses of 50, 100 and 200 mg/kg did not cause any significant change in insulin level at any time point. On the other hand, glibenclamide produced significant insulin-increasing effect at 2 h (43.9%) and 6 h (44.1%) in normoglycaemic rabbits. 3.2. Effect of the Rosmarinus officinalis extract on blood glucose and insulin levels of glucose-hyperglycaemic rabbits (oral glucose tolerance test, OGTT) The blood glucose and insulin levels of different doses of the Rosmarinus officinalis extract, glibenclamide and vehicle

12.20 17.58 13.63 15.97 16.17 S.E.M: mean standard error, *p < 0.05 significant from the control animals, **p < 0.01 significant from the control animals, ***p < 0.001 significant from the control animals.

11.49 16.53 13.45 14.13 14.79 124.29 80.14 119.86 107.71 98.43 4.37 2.82 *** 5.12 3.23* 1.84** ± ± ± ± ± 131.71 94.43 118.29 112.57 104.86 Control Glibenclamide Rosmarinus officinalis Rosmarinus officinalis Rosmarinus officinalis

– 5 50 100 200

118.14 120.43 116.57 111.86 106.29

± ± ± ± ±

4.88 3.22 3.30 2.34 1.80

129.14 108.43 130.00 119.57 112.86

± ± ± ± ±

2.21 5.81* 6.42 3.14 2.53

2h 1h 0h

Mean blood glucose concentration ± S.E.M. (mg/dl) Dose (mg/kg) Group

Table 1 Effect of Rosmarinus officinalis extract on blood glucose and serum insulin levels of normal fasted rabbits

6h

± ± ± ± ±

7.41 3.65*** 4.31 2.60 5.55*

13.81 13.73 14.09 14.03 14.24

± ± ± ± ±

1.11 1.60 1.26 0.88 0.84

12.63 14.19 12.35 12.76 13.25

± ± ± ± ±

0.95 0.77 1.21 0.82 1.16

2h 1h 0h

Mean serum insulin level ± S.E.M. (␮IU/ml)

± ± ± ± ±

1.02 0.66* 1.25 0.84 1.29

6h

± ± ± ± ±

0.95 1.02* 0.98 1.11 0.82

T. Bakırel et al. / Journal of Ethnopharmacology 116 (2008) 64–73

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treated rabbits after oral administration of glucose (2 g/kg) are summarized in Table 2. The blood glucose levels of the normoglycaemic rabbits reached a peak at 1 h after the oral administration of glucose and gradually decreased to the preglucose load level. In glucose-hyperglycaemic rabbits, the extract at doses of 50 mg/kg and 100 mg/kg showed a significant effect, with blood glucose levels dropping to 12.4 and 19.9%, respectively from that of control after 6 h of glucose administration. At 200 mg/kg doses of the extract a comparatively more potent reduction was observed with the blood glucose levels 30.8 and 35.6%, respectively from that of control after 2 and 6 h of glucose administration. At 6 h the magnitude of reduction was much closer to glibenclamide. It therefore appears that 200 mg/kg of the extract of Rosmarinus officinalis is the most effective dose on OGGT of normoglycaemic rabbits. Glibenclamide prevented the drastic increase of blood glucose 1 h after the glucose loading and reduced the level even below the normal values 2 and 6 h after the glucose loading. The extract of Rosmarinus officinalis could increase insulin concentrations in a time- and dose-dependent manner in glucose-loaded rabbits when compared to control, but the values were insignificant. On the other hand, glibenclamide showed more pronounced insulin-tropic activity in glucose-loaded rabbits, stimulated the blood insulin concentration after 2 h (28.6%) and 6 h (31.7%) of glucose administration. 3.3. Acute effect of the Rosmarinus officinalis extract in alloxan-induced diabetic rabbits Acute effect of various doses of the Rosmarinus officinalis extract in diabetic animals was studied using alloxan-diabetic rabbits. As shown in Table 3, the extract at 50 mg/kg and 100 mg/kg doses did not cause significant change in blood glucose and insulin levels in alloxan-diabetic rabbits at any time point. The extract at 200 mg/kg dose showed only a weak reduction (22.3%) at 2 h, while glibenclamide showed more pronounced antidiabetic activity in alloxan-diabetic rabbits, inhibited the blood glucose level dropping to 23.6% from that of control. However, a more pronounced activity was recorded at the later stage for the extract of 200 mg/kg dose. At 6 h the magnitude of reduction was much closer to glibenclamide where the blood glucose was significantly reduced 28.3% of the control. The insulin level of alloxan-diabetic rabbits increased significantly after 2 h and 6 h of administration of either the Rosmarinus officinalis extract at a dose level of 200 mg/kg or glibenclamide. The maximal percentages of increase that occurred after 6 h of the extract and glibenclamide administration were 60.3 and 75.8% in diabetic rabbits, respectively. 3.4. Subacute effect of the Rosmarinus officinalis extract in alloxan-induced diabetic rabbits In order to determine the subacute effects, three doses of the Rosmarinus officinalis extract were administered throughout 8 days consecutively. The blood glucose and insulin level of each animal was monitored on 1st, 3rd, 5th and 8th days after

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Table 2 Effect of Rosmarinus officinalis extract on blood glucose and serum insulin levels in oral glucose-loaded (OGTT) rabbits Group

Dose (mg/kg)

Mean blood glucose concentration ± S.E.M. (mg/dl) 0h

– 5 50 100 200

123.28 128.42 119.71 125.28 117.14

± ± ± ± ±

1.87 3.50 2.17 3.42 1.99

262.29 180.71 232.43 244.57 226.85

2h ± ± ± ± ±

4.57 8.84*** 13.15 9.20 11.04

202.86 116.29 190.40 173.42 140.42

6h ± ± ± ± ±

5.58 9.67*** 15.57 13.27 4.58*

141.71 87.43 124.14 113.57 91.28

0h ± ± ± ± ±

3.26 1.95*** 2.61* 6.01** 2.37***

1h

15.47 14.86 14.97 14.92 15.74

± ± ± ± ±

0.88 1.32 0.96 1.16 0.81

2h

8.76 11.58 10.67 9.44 11.17

± ± ± ± ±

0.79 0.73 1.01 0.80 1.09

6h

10.32 14.45 12.99 13.12 14.08

± ± ± ± ±

0.84 0.77* 0.75 0.92 0.95

11.87 17.39 14.23 15.30 16.61

± ± ± ± ±

0.66 1.18* 1.24 1.09 0.83

S.E.M.: mean standard error, *p < 0.05 significant from the control animals, **p < 0.01 significant from the control animals, ***p < 0.001 significant from the control animals.

Table 3 Acute effect of Rosmarinus officinalis extract on blood glucose and serum insulin levels in alloxan-induced diabetic rabbits Group

Dose (mg/kg)

Mean blood glucose concentration ± S.E.M. (mg/dl) 0h

Control Glibenclamide Rosmarinus officinalis Rosmarinus officinalis Rosmarinus officinalis

– 5 50 100 200

420.71 409.29 435.43 398.14 383.57

0h ± ± ± ± ±

11.85 6.82 9.70 19.27 14.87

432.14 423.57 450.71 418.43 397.00

Mean serum insulin level ± S.E.M. (␮IU/ml)

2h ± ± ± ± ±

10.19 14.28 20.48 8.32 17.18

411.43 314.29 408.86 360.71 319.57

6h ± ± ± ± ±

15.52 10.63** 19.20 18.99 11.14*

387.14 266.00 381.57 326.85 277.43

0h ± ± ± ± ±

S.E.M.: mean standard error, *p < 0.05 significant from the control animals, **p < 0.01 significant from the control animals.

18.21 8.70 ** 20.35 12.29 16.01**

5.98 5.90 4.77 5.93 6.21

1h ± ± ± ± ±

0.44 0.38 0.32 0.59 0.40

5.26 5.41 4.64 5.47 6.14

2h ± ± ± ± ±

0.57 0.24 0.45 0.63 0.51

6.03 8.70 6.12 7.65 8.06

6h ± ± ± ± ±

0.24 0.48** 0.39 0.56 0.33*

6.19 10.88 6.74 8.71 9.92

± ± ± ± ±

0.39 0.62** 0.76 0.65 0.47**

T. Bakırel et al. / Journal of Ethnopharmacology 116 (2008) 64–73

Control Glibenclamide Rosmarinus officinalis Rosmarinus officinalis Rosmarinus officinalis

0h

Mean serum insulin level ± S.E.M. (␮IU/ml)

± ± ± ± ± ± 14.49 6.21 9.13 7.76 9.97 11.10 115.57 396.14 323.86 342.14 317.43 279.29 122.43 414.86 311.71 364.29 343.57 302.14 112.71 452.43 383.57 400.86 379.00 363.29 S.E.M.: mean standard error. *p < 0.05 significant from the control animals. **p < 0.01 significant from the control animals. ***p < 0.001 significant from the control animals. a Compared to vehicle control. b Compared to diabetic control.

119.14 420.29 387.86 412.71 421.57 393.14 – – 5 50 100 200 Control Diabetic controla Glibenclamideb Rosmarinus officinalisb Rosmarinus officinalisb Rosmarinus officinalisb

1st day

± ± ± ± ± ±

2.29 12.96*** 19.85 24.43 12.58 14.75

3rd day

± ± ± ± ± ±

4.06 19.29*** 25.21 15.51 16.57 11.32*

5th day

± ± ± ± ± ±

4.17 14.20*** 15.42** 19.05 13.94 11.50**

8th day

± ± ± ± ± ±

3.36 18.22*** 17.09 15.36 20.20* 9.27**

13.53 5.87 6.34 5.65 5.97 6.61

± ± ± ± ± ±

0.66 0.48*** 0.54 0.72 0.51 0.36

13.76 5.65 8.85 6.58 7.30 9.47

± ± ± ± ± ±

1.04 0.57*** 0.76 0.53 0.90 0.78

13.64 5.90 10.36 7.34 9.43 10.49

± ± ± ± ± ±

0.83 0.39*** 0.58** 0.40 0.81 0.93**

8th day 5th day 3rd day 1st day

Mean serum insulin level ± S.E.M. (␮IU/ml) Dose (mg/kg) Mean blood glucose concentration ± S.E.M. (mg/dl) Group

Table 4 Subacute effect of Rosmarinus officinalis extract on blood glucose and serum insulin levels in alloxan-induced diabetic rabbits

the administration of the test samples. As shown in Table 3, the blood glucose levels of diabetic control rabbits were significantly higher than those of the control rabbits during the experiment period. The observed effect with a dose of 200 mg/kg of the Rosmarinus officinalis extract was more potent (19.7%) than that of the other doses of the extract and glibenclamide on the 3rd day. The highest reduction in blood glucose was observed on the 8th day for 100 mg/kg and 200 mg/kg dose of the extract and 200 mg/kg dose of the extract hit the highest (29.5%) and even more pronounced than glibenclamide (18.2%). The daily administration of the extract at 200 mg/kg dose and glibenclamide to alloxan-diabetic rabbits induced a significant increase in the insulin level on the 5th day when compared with diabetic control rabbits. The magnitude of the increase on which observed with administration of the extract of 200 mg/kg (77.8%) was much closer to that of glibenclamide (75.6%). According to the measurements on the day 8, glibenclamide did not show any remarkable effect, while the extract at a dose of 100 mg/kg and 200 mg/kg showed a marked increase on insulin level in alloxan-diabetic rabbits (60.5, 78.7%, respectively). During the subacute administration, rabbits treated with various doses of the Rosmarinus officinalis extract and glibenclamide were also monitored for changes in weight, but the values were insignificant (Table 4). In order to evaluate in vivo antioxidant effect of the Rosmarinus officinalis extract, MDA, SOD and CAT level in serum of each rabbit was monitored on 1st, 3rd, 5th and 8th days after the administration of the test samples. As shown in Table 5, in the serums of alloxan-diabetic rabbits, lipid peroxidation levels as evidenced by MDA determination increased significantly as compared to the control group during the experiment period. In alloxan-diabetic rabbits, the Rosmarinus officinalis extract (200 mg/kg) treatment significantly inhibited the increase in MDA both on 5th (24.8%) and 8th day (33.3%). However, glibenclamide provide a significant reduction in LPO in serum only on the 5th day (23.5%). The activities of enzymatic antioxidants (SOD and CAT) in serum of normal and experimental animals in each group are presented in Table 6. SOD and CAT activities were constantly and significantly decreased in alloxandiabetic rabbits as compared to normal rabbits. During the experiment period, alloxan-diabetic rabbits treated with the Rosmarinus officinalis extract at a dose of 50 and 100 mg/kg showed no significant difference in enzymatic antioxidants activities as compared to diabetic control rabbits. A significant elevation in serum SOD was observed in alloxan-diabetic rabbits given glibenclamide and the extract at a dose of 200 mg/kg which showed 22.1 and 24.4% increase, respectively at 5th day. A more pronounced activity (25%) was recorded at the later stage for the Rosmarinus officinalis extract at a dose of 200 mg/kg. Diabetic rabbits treated with a dose of 200 mg/kg the Rosmarinus officinalis extract had a gradual rise in serum CAT activity to reach a significant difference on 5th (32.4%) and 8th day (35.4%) as compared with diabetic control rabbits. On the other hand, the highest increase in serum CAT activity were observed on the 5th day (43.4) in alloxan-diabetic rabbits given glibenclamide and even was more pronounced than the highest dose of the Rosmarinus officinalis extract.

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0.96 0.42*** 0.93 0.51 0.75* 0.61**

T. Bakırel et al. / Journal of Ethnopharmacology 116 (2008) 64–73

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T. Bakırel et al. / Journal of Ethnopharmacology 116 (2008) 64–73

Table 5 Malondialdehyde (MDA), Superoxide dismutase (SOD) and atalase levels in serum of alloxan-induced diabetic rabbits after treated with Rosmarinus officinalis extract Group

Dose (mg/kg)

Mean serum MDA (nmol/ml), SOD (U/ml) and Catalase levels (kU/l) ± S.E.M. 1st day

3rd day

5th day

8th day

–

4.32 ± 0.22 85.39 ± 2.79 55.72 ± 1.78

4.18 ± 0.41 86.24 ± 3.42 59.63 ± 3.45

4.26 ± 0.19 88.85 ± 1.37 58.45 ± 1.82

4.29 ± 0.30 86.17 ± 3.96 59.12 ± 2.43

–

9.27 ± 0.63*** 60.31 ± 3.13*** 31.44 ± 0.95***

9.95 ± 0.58*** 55.47 ± 1.86*** 28.82 ± 1.91***

9.81 ± 0.44*** 57.28 ± 0.93*** 35.29 ± 1.43***

9.64 ± 0.91*** 62.63 ± 1.59*** 39.84 ± 1.67**

8.62 ± 0.54 57.25 ± 2.29 30.84 ± 0.77

8.14 ± 0.37 62.61 ± 1.50 35.11 ± 1.47

7.50 ± 0.28* 69.92 ± 1.84* 50.61 ± 2.61*

7.56 ± 0.41 75.37 ± 1.28 51.39 ± 3.59

8.78 ± 0.38 55.06 ± 3.05 29.09 ± 0.89

8.49 ± 0.71 59.87 ± 2.74 31.64 ± 1.51

8.18 ± 0.62 61.54 ± 3.25 36.33 ± 2.38

7.71 ± 0.57 65.46 ± 2.62 42.21 ± 4.02

Rosmarinus officinalisb MDA SOD 100 Catalase

9.02 ± 0.57 63.02 ± 4.65 33.13 ± 1.25

8.33 ± 0.49 65.76 ± 4.02 36.93 ± 2.30

7.92 ± 0.55 68.35 ± 2.92 43.52 ± 2.74

7.27 ± 0.63 74.02 ± 2.71 50.80 ± 1.72

Rosmarinus officinalisb MDA SOD 200 Catalase

8.90 ± 0.83 58.83 ± 1.93 29.50 ± 0.62

8.04 ± 0.30 64.10 ± 2.12 36.44 ± 0.88

7.38 ± 0.46* 71.27 ± 2.45* 46.72 ± 3.02*

6.43 ± 0.25* 78.31 ± 1.68* 53.94 ± 1.50*

Control MDA SOD Catalase Diabetic controla MDA SOD Catalase

Glibenclamideb MDA SOD 5 Catalase Rosmarinus officinalisb MDA SOD 50 Catalase

S.E.M.: mean standard error, *p < 0.05 significant from the control animals, **p < 0.01 significant from the control animals, ***p < 0.001 significant from the control animals. a Compared to vehicle control. b Compared to diabetic control. Table 6 Subacute effect of Rosmarinus officinalis extract on body weights in alloxan-induced diabetic rabbits Group

Dose (mg/kg)

Mean body weight ± S.E.M. (kg) (percent increase from the initial weight) 1st day

Control Diabetic controla Glibenclamideb Rosmarinus officinalis Rosmarinus officinalis Rosmarinus officinalis

– – 5 50 100 200

2.72 2.31 2.44 2.34 2.38 2.42

± ± ± ± ± ±

3rd day 0.11 0.20 0.13 0.18 0.17 0.15

2.81 2.35 2.50 2.43 2.49 2.57

± ± ± ± ± ±

5th day 0.10 (3.3%) 0.14 (1.7%) 0.11 (2.5%) 0.10 (3.8%) 0.19 (4.6%) 0.20 (6.2%)

2.86 2.37 2.58 2.47 2.60 2.68

± ± ± ± ± ±

8th day 0.13 (5.1%) 0.18 (2.6%) 0.20 (5.7%) 0.11 (5.6%) 0.14 (9.2%) 0.12 (10.7%)

2.93 2.46 2.71 2.49 2.62 2.70

± ± ± ± ± ±

0.12 (7.7%) 0.13 (6.5%) 0.18 (11.1%) 0.13 (6.4%) 0.15 (10.1%) 0.21 (11.5%)

S.E.M.: mean standard error. a Compared to vehicle control. b Compared to diabetic control.

4. Discussion The limitations of currently available pharmacological agents for control of blood glucose have stimulated research on novel antidiabetic agents with different mechanism of action (Reddy et al., 2000). The study of such medicines might offer a natural key to unlock a diabetologist’s pharmacy for the future. There are a few scientific reports relating on the antidiabetic potential of various extracts from Rosmarinus officinalis demonstrated that the infusion of the plant has hypoglycaemic effect (Erenmemis¸o˘glu et al., 1997), whereas its volatile oils have

hyperglycaemic effects (Al-Hader et al., 1994). In this study, ethanolic extract was used to examine the activity of volatile oils as well as water soluble component in the plant on treatment of diabetes. Thus, it was evaluated the activity of ethanolic extract of Rosmarinus officinalis and revealed the possible mechanism of its effect. The conclusions derived from these data revealed a defined role of the ethanolic extract of Rosmarinus officinalis in suppressing blood glucose level in normoglycaemic, glucose-hyperglycaemic and alloxan-induced diabetic rabbits. In normoglycaemic and glucose-hyperglycaemic rabbits,

T. Bakırel et al. / Journal of Ethnopharmacology 116 (2008) 64–73

hypoglycaemic action of the extract was observed to be dosedependent, with prolonged hypoglycaemia at the higher doses. As far as most effective dose is concern it has been found to be 200 mg/kg in all the groups. This dose has almost same effect as of synthetic drug glibenclamide especially during OGGT. On the other hand, the serum insulin levels remained unchanged both in the animal groups after the extract treatment. This finding indicates that the Rosmarinus officinalis extract might be producing its hypoglycaemic activity by a mechanism independent from insulin secretion, e.g. by the inhibition of endogenous glucose production (Eddouks et al., 2003) or by the inhibition of intestinal glucose absorption (Platel and Srinivasan, 1997). In a previous study, it has been suggested that 50% ethanol extract of Rosmarinus officinalis, in part, due to intestinal ␣-glucosidase (AGc) inhibitory activity of its active compound might play a role in controlling dietary glucose uptake in the small intestinal track (Koga et al., 2006). Alloxan, a beta-cytotoxin, induces “chemical diabetes” (alloxan diabetes) in a wide variety of animal species by damaging the insulin secreting cells of the pancreas. This damages a large number of ␤-cells, resulting in decrease in endogenous insulin release, which paves the ways for the decreased utilization of glucose by the tissue (Saravanan and Pari, 2005). It is well established that sulphonylureas produce hypoglycaemia by increasing the secretion of insulin from pancreas and these compounds are active in mild alloxan-induced diabetes, but they are inactive in intense alloxan diabetes (Nammi et al., 2003). Since our results showed that glibenclamide reduced blood glucose levels in hyperglycaemic animals, the state of diabetes is not severe. The acute antihyperglycaemic and insulin-tropic effects of the Rosmarinus officinalis extract (200 mg/kg) were similar to those of glibenclamide. The possible mechanism by which the plant extract mediates its antidiabetic action might be by potentiation of pancreatic secretion of insulin from existing residual ␤-cell of islets or due to enhanced transport of blood glucose to peripheral. On the other hand, subacute antihyperglycaemic and insulintropic effects of the Rosmarinus officinalis extract (100 mg/kg and 200 mg/kg) were more potent and prolonged than glibenclamide in alloxan-diabetic rabbits. Similarly, it has been reported that chronic treatment with sulphonylureas such as glibenclamide leads to a decline in their insulin-tropic activity due to a seconder failure in increasing insulin secretion such as ␤-cell exhaustion or desensitisation to glucose (Kulkarni et al., 2000; Ball et al., 2004). In this study, the progressive reduction in the blood glucose levels of alloxan-diabetic rabbits might be due to a cumulative action of the extract during the period of treatment and also associated with an increase in the blood insulin levels. The elevation in blood insulin in the extract-treated, alloxan-diabetic rabbits could be because of the insulin-tropic substances present in the extract that induce the protection of the functional ␤-cells from further deterioration or the regeneration of ␤-cells so that they remain active and produce insulin. Recently, much attention has been focused on the role of oxidative stress and it has been suggested that oxidative stress may constitute the key and common events in the pathogenesis

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of different diabetic complications (Sepici-Dincel et al., 2007). Diabetics and experimental animal models exhibit high oxidative stress due to persistent and chronic hyperglycaemia, which thereby depletes the activity of antioxidative defense system and thus promotes de novo free radicals generation (Kamalakannan and Prince, 2006). Oxygen free radicals react with all biological substances; however, the most susceptible ones are polyunsaturated fatty acids. Reactions with these cell membrane constituents lead to lipid peroxidation. (Memis¸o˘gulları and Bakan, 2004). Increased LPO impairs membrane function by decreasing membrane fluidity and changing the activity of membrane-bound enzymes and receptors (Arulselvan and Subramanian, 2007). Any compound – natural or synthetic – with antioxidant activity might totally or partly alleviate this damage (Sepici-Dincel et al., 2007). In our study MDA (as an indicator of LPO) levels in diabetes group were found to be higher than those in control group, indicating increased free radical generation. Treatment of diabetes with the highest doses of the Rosmarinus officinalis extract on subacute studies caused a decrease in MDA levels. This decrease in MDA levels may increase the activity of glutathione peroxidase (GPX) in rabbits treated with the extract and hence cause inactivation of LPO reactions (Ugochukwu et al., 2003; Afshari et al., 2007). Enzymatic antioxidant such as SOD and CAT are considered primary enzymes since they are involved in the direct elimination of ROS (Arulselvan and Subramanian, 2007). SOD is an important defense enzyme and scavenges O2 − anion form H2 O2 and hence diminishes the toxic effects due to this radical or other free radicals derived from secondary reaction (Manonmani et al., 2005). CAT is a hemoprotein, which catalyzes the reduction of hydrogen peroxides (Punitha et al., 2005) and known to be involved in detoxification of H2 O2 concentrations (Manonmani et al., 2005). Persistent hyperglycaemia leads to increased production of free radicals (Roy et al., 2005). The antioxidant enzymes such as SOD and CAT are known to be inhibited in DM as a result of non-enzymatic glycosylation and oxidation (Al-Azzawie and Alhamdani, 2006). In our study, the activities of SOD and CAT decreased in diabetic rabbits as reported earlier (Al-Azzawie and Alhamdani, 2006, Sepici-Dincel et al., 2007) which could be due to inactivation caused by alloxan-generated ROS. Long-term treatment of diabetes with the highest dose of the Rosmarinus officinalis extract had reversed the activities of these enzymatic antioxidants, which might be due to decreased oxidative stress as evidenced by decreased LPO. Several reports indicate that the compounds responsible for antioxidative activity of Rosmarinus officinalis are mainly phenolic diterpenes such as carnosoic acid, carnosol, rosmanol (Hras et al., 2000), and other phenolic acids, such as rosmarinic and caffeic acids (Carvalho Jr. et al., 2005; Perez et al., 2007). It is possible that the Rosmarinus officinalis extract due to its presence of several bioactive antioxidant principles and their synergistic properties may be caused an improving effect in antioxidant status of diabetic rabbits. In conclusion, our observations have clearly demonstrated that the Rosmarinus officinalis extract exerts remarkable hypoglycaemic and antihyperglycaemic activity due to its possible multiple effects involving both pancreatic and extra-pancreatic

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