**3.1 Materials and methods**

DM2 is an endocrine disorder characterized by defects in mechanism of glucose insulin-mediated transport into the cells, resulting in persistent hyperglycemia. It is well known that glucose high levels lead to the free radical overformation, which activates lipoperoxidation—one of the main pathogenetic factors in atherosclerosis development [3, 4].

Bilberry leaf chemical composition usually consists of flavonoids, proanthocyanidins, triterpenes, and also phenolic compounds, in particular, of myricetin, which revealed an effectiveness at the DM2 initial stages of according to some authors [43, 44].

The experiment was conducted on male Wistar rats weighing 160–200 g. The experimental animals were divided into groups: (1) intact animals fed a standard vivarium chow; (2) animals fed a high-fructose diet (2 g of fructose/100 g of body weight daily) combined with daily subcutaneous dexamethasone injections in the dose of 2 mg/100 g body weight for 6 weeks; and (3) animals, which administered intragastric feeding of bilberry leaf dry extract in the dose of 9 mg/100 g of body weight for 2 weeks in addition to dexamethasone administration [45].

The glycosylated hemoglobin (HbA1C) concentration was determined in blood serum by immunoturbidimetric method. The fructosamine level was measured by the spectrophotometric method using nitro-blue tetrazolium chloride [46]. The area under the glycemic curves (AUC) during IGGTT was calculated using the computer software program MATLAB [47]. The glucose content was determined using standard kit of "Filisit-Diagnostika" (Ukraine). The basal level of immunoreactive insulin (IRI) was measured in the blood of animals by the in vitro radioimmunoassay using a standard set of reagents produced by "Immundiagnostik" (Germany) [10].

The lipoperoxidation markers were determined spectrophotometrically by the measuring of diene conjugates (DC) and TBA-reactive products (TBA-RP) content using reaction with thiobarbituric acid [48]. The antioxidant system status was evaluated via determination of reduced glutathione (GSH) concentration (spectrophotometrically by reaction with alloxan) and glutathione reductase (GR) activity [49]. Glucose homeostasis in test and control animals was evaluated at different times after the model inducing (7, 14, and 21 days) by basal glycemic indexes and intraperitoneal glucose tolerance test (IPGTT, 3 g/kg body weight) [50]. Statistical processing of the experimental data was performed using the STATISTICA software program (StatSoft Inc., USA, version 6.0). The significance of intergroup differences was estimated according to the Student's t-test [14].

#### **3.2 Results and discussion**

The dexamethasone low dose administration to laboratory animals under highfat diet caused the multiple disorder formation specific to MS and DT2. In the rats' blood serum, there was a significant increase in the glycosylated hemoglobin level, fructosamine concentration, glucose content, basal glycemia, and increased area under glycemic curves. These changes were probably developed due to a decrease in the glucose utilization by peripheral tissues caused by inhibition of the glucose transporters expression (GLUT1 and GLUT4) under the influence of dexamethasone action. Change in HbA1C level is an objective indicator of carbohydrate metabolism in diabetic patients and the effectiveness of glycemic control. An increase in

**289**

(**Tables 6** and **7**).

insulin synthesis activator [51].

*Prospects for Using the Natural Antioxidant Compounds in the Obesity Treatment*

*Variation is statistically significant in comparison with model pathology group indices (р* ≤ *0.05).*

*induced by high-fructose high-calorie diet combined with dexamethasone injection, n = 10.*

*Variation is statistically significant in comparison with model pathology group indices (р* ≤ *0.05).*

*The effect of bilberry leaf extract on carbohydrate metabolism indices under experimental diabetes mellitus* 

**Parameters Intact control Model pathology BLE + model** 

TBA-AP (nmol/g) 0.96 ± 0.27 3.56 ± 0.41\* 0.98 ± 0.39# DC (nmol/l) 22.30 ± 1.42 28.5 ± 0.94\* 26.9 ± 0.96 GSH (mmol/g) 0.25 ± 0.01 0.12 ± 0.02\* 0.19 ± 0.03 GR (nmol/min mg) of protein 18.56 ± 0.64 14.20 ± 1.06\* 17.9 ± 0.98

**Parameters Intact control Model pathology BLE + model** 

Glycosylated hemoglobin (%) 7.5 ± 0.5 9.6 ± 0.7\* 8.4 ± 0.6\*,# Fructosamine (mmol/l) 1.91 ± 0.12 3.6 ± 0.29\* 2.45 ± 0.24\*,# Basal blood glucose level (mmol/l) 4.04 ± 0.11 13.42 ± 0.38\* 12.08 ± 0.24\* AUC (glycemic) (mmol/l/min) 625.44 ± 16.56 2092.25 ± 60.48\* 1100 ± 61.56\*,# Glucose, mmol/l 4.6 ± 0.1 11.2 ± 0.2\* 8.2 ± 0.1\*,# ІRІ (pmol/l) 221.74 ± 20.79 317.97 ± 39.72\* 302 ± 36.70\*

*Variation is statistically significant in comparison with intact control group indices* 

*Variation is statistically significant in comparison with intact control group indices* 

**pathology**

**pathology**

the content of glycosylated hemoglobin is usually considered as an indirect marker for the retinopathy development, nephropathy, and other complications of diabetes. Due to the long-term hyperglycemia, albumin was subjected to nonenzymatic glycosylation, which was confirmed by the increase in fructosamine in our experiments (**Table 6**). The dry bilberry leaf extract (BLE) administration under model pathology was accompanied by a significant normalization of the studied indices

*The effect of bilberry leaf extract on the specific markers of antioxidative-prooxidative status of rat's liver in experimental diabetes mellitus induced by high-fructose high-calorie diet combined with dexamethasone* 

In particular, it was significant for the suppression of glycosylated hemoglobin (by 12.5%) and fructosamine (by 32%) concentration, hyperglycemia (by 26.8%), the area under the glycemic curves (by 47.4%), and the immunoreactive insulin level (by 5.1%) compared to untreated animals. In our opinion, it is due to the high content of different phenolic compounds in the bilberry extract, which have hypoglycemic and antioxidant effects. The mechanism of hypoglycemic effect of polyphenols is related to their impact on the process of glucose transport into the cell. The main role in hypoglycemic action of the extract of bilberry leaves belongs to myrtilline, which is a mixture of delphinidin and malvinidin esters that act as

It is proved that hyperglycemia is accompanied by free radical processes activa-

tion, which leads to complications in the main disease pathogenesis and tissue damage. Under the conditions of our experiments, the model pathology formation

*DOI: http://dx.doi.org/10.5772/intechopen.83421*

*n, number of animals in a group.\**

*n, number of animals in a group.\**

*(р* ≤ *0.05). #*

**Table 6.**

*(р* ≤ *0.05). #*

**Table 7.**

*injection, n = 10.*

#### *Prospects for Using the Natural Antioxidant Compounds in the Obesity Treatment DOI: http://dx.doi.org/10.5772/intechopen.83421*


*n, number of animals in a group.\* Variation is statistically significant in comparison with intact control group indices (р* ≤ *0.05).*

*# Variation is statistically significant in comparison with model pathology group indices (р* ≤ *0.05).*

#### **Table 6.**

*Antioxidants*

**3.1 Materials and methods**

development [3, 4].

authors [43, 44].

**3. The study of the blueberry (***Vaccinium myrtillus* **L.) extract effectiveness under experimental diabetes mellitus induced by a high-fructose diet** 

DM2 is an endocrine disorder characterized by defects in mechanism of glucose insulin-mediated transport into the cells, resulting in persistent hyperglycemia. It is well known that glucose high levels lead to the free radical overformation, which activates lipoperoxidation—one of the main pathogenetic factors in atherosclerosis

Bilberry leaf chemical composition usually consists of flavonoids, proanthocyanidins, triterpenes, and also phenolic compounds, in particular, of myricetin, which revealed an effectiveness at the DM2 initial stages of according to some

The experiment was conducted on male Wistar rats weighing 160–200 g. The experimental animals were divided into groups: (1) intact animals fed a standard vivarium chow; (2) animals fed a high-fructose diet (2 g of fructose/100 g of body weight daily) combined with daily subcutaneous dexamethasone injections in the dose of 2 mg/100 g body weight for 6 weeks; and (3) animals, which administered intragastric feeding of bilberry leaf dry extract in the dose of 9 mg/100 g of body

The glycosylated hemoglobin (HbA1C) concentration was determined in blood serum by immunoturbidimetric method. The fructosamine level was measured by the spectrophotometric method using nitro-blue tetrazolium chloride [46]. The area under the glycemic curves (AUC) during IGGTT was calculated using the computer software program MATLAB [47]. The glucose content was determined using standard kit of "Filisit-Diagnostika" (Ukraine). The basal level of immunoreactive insulin (IRI) was measured in the blood of animals by the in vitro radioimmunoassay using a standard set of reagents produced by "Immundiagnostik" (Germany) [10]. The lipoperoxidation markers were determined spectrophotometrically by the measuring of diene conjugates (DC) and TBA-reactive products (TBA-RP) content using reaction with thiobarbituric acid [48]. The antioxidant system status was evaluated via determination of reduced glutathione (GSH) concentration (spectrophotometrically by reaction with alloxan) and glutathione reductase (GR) activity [49]. Glucose homeostasis in test and control animals was evaluated at different times after the model inducing (7, 14, and 21 days) by basal glycemic indexes and intraperitoneal glucose tolerance test (IPGTT, 3 g/kg body weight) [50]. Statistical processing of the experimental data was performed using the STATISTICA software program (StatSoft Inc., USA, version 6.0). The significance of intergroup differences

The dexamethasone low dose administration to laboratory animals under highfat diet caused the multiple disorder formation specific to MS and DT2. In the rats' blood serum, there was a significant increase in the glycosylated hemoglobin level, fructosamine concentration, glucose content, basal glycemia, and increased area under glycemic curves. These changes were probably developed due to a decrease in the glucose utilization by peripheral tissues caused by inhibition of the glucose transporters expression (GLUT1 and GLUT4) under the influence of dexamethasone action. Change in HbA1C level is an objective indicator of carbohydrate metabolism in diabetic patients and the effectiveness of glycemic control. An increase in

weight for 2 weeks in addition to dexamethasone administration [45].

was estimated according to the Student's t-test [14].

**3.2 Results and discussion**

**combined with dexamethasone injections**

**288**

*The effect of bilberry leaf extract on carbohydrate metabolism indices under experimental diabetes mellitus induced by high-fructose high-calorie diet combined with dexamethasone injection, n = 10.*


*n, number of animals in a group.\* Variation is statistically significant in comparison with intact control group indices (р* ≤ *0.05).*

*# Variation is statistically significant in comparison with model pathology group indices (р* ≤ *0.05).*

#### **Table 7.**

*The effect of bilberry leaf extract on the specific markers of antioxidative-prooxidative status of rat's liver in experimental diabetes mellitus induced by high-fructose high-calorie diet combined with dexamethasone injection, n = 10.*

the content of glycosylated hemoglobin is usually considered as an indirect marker for the retinopathy development, nephropathy, and other complications of diabetes. Due to the long-term hyperglycemia, albumin was subjected to nonenzymatic glycosylation, which was confirmed by the increase in fructosamine in our experiments (**Table 6**). The dry bilberry leaf extract (BLE) administration under model pathology was accompanied by a significant normalization of the studied indices (**Tables 6** and **7**).

In particular, it was significant for the suppression of glycosylated hemoglobin (by 12.5%) and fructosamine (by 32%) concentration, hyperglycemia (by 26.8%), the area under the glycemic curves (by 47.4%), and the immunoreactive insulin level (by 5.1%) compared to untreated animals. In our opinion, it is due to the high content of different phenolic compounds in the bilberry extract, which have hypoglycemic and antioxidant effects. The mechanism of hypoglycemic effect of polyphenols is related to their impact on the process of glucose transport into the cell. The main role in hypoglycemic action of the extract of bilberry leaves belongs to myrtilline, which is a mixture of delphinidin and malvinidin esters that act as insulin synthesis activator [51].

It is proved that hyperglycemia is accompanied by free radical processes activation, which leads to complications in the main disease pathogenesis and tissue damage. Under the conditions of our experiments, the model pathology formation was accompanied by the oxidative stress development, which was confirmed by significant increase in the content of TBA-AP and DC (basic lipoperoxidation products) in the liver—by 270.8 and 27.8%, respectively, compared with healthy rats. At the same time, antioxidant defense was reduced as evidenced by the decrease in the content of GSH and GR in the liver by 52 and 23.5%, respectively, compared to intact control animals. The above changes confirmed the oxidative stress development under pathology. The BLE administration under the model pathology led to the antioxidative-prooxidative balance normalization, which was evidenced by the significant suppression of lipoperoxidation and antioxidant status improvement, which was reflected in the relevant indicator dynamics. We suggested that these changes were the result of remarkable antioxidant and antiradical properties of the investigated extract components.

Thus, the effectiveness of the BLE under the experimental DT2 primary was due to the expressive antioxidant properties of the biologically active substances that are part of its composition.
