**4. Results**

*Antioxidants*

**3.3 Superoxide dismutase activity**

μmol/mg protein.

**3.4 Catalase activity**

specific because it quantifies the genuine MDA-(TBA)2 adduct formed [38]. The quantitative analysis of MDA was performed using a modified method of Cuny et al. [39] on a Spectra SYSTEM™ HPLC (Agilent Technology, 1200 series, Germany). Briefly, 50 μL of sample was mixed with 375 μL orthophosphoric acid 0.44 M, 125 μL thiobarbituric acid, and 225 μL distilled water. This mixture was heated at 100°C for 60 min and cooled on ice. Thereafter, 775 μL of alkaline methanol was added, and the sample was subsequently vortexed and centrifuged at 3500 rpm for 3 min at 4°C. The supernatant (1 mL) was collected; 500 μL of n-hexane was added and centrifuged at 14,000 rpm for 2 min. The supernatant (500 μL) was collected in chromatographic tubes and injected into the HPLC system. The readings were performed after 10 min, and sample concentration MDA levels were expressed in μmol/g of tissue.

Superoxide dismutase (SOD) activity was determined by a modified method from Ellerby and Bredesen [40]. Briefly, samples were run in duplicate, in a 96-well plate; 15 μL of 6-HD was added to 6 μL of supernatant. An amount of 170 μL of diethylenetriaminepentaacetic acid (DETAPAC) solution (0.1 mM) in SOD assay buffer and readings were taken immediately at 490 nm for 4 min at 1 min intervals. The activity of SOD was calculated from a linear calibration curve and expressed as

The catalase (CAT) activity was assessed according to Aebi [41]. The CAT induced decomposition of hydrogen peroxide (H2O2) into water and oxygen. The rate of disintegration is proportional to the concentration of CAT activity. The CAT activity was determined by measuring the change in absorbance of H2O2 and sample mixture. Briefly, the CAT assay was performed in duplicate; 150 μL H2O2 was added to 20 μL of sample. Readings were determined by using a spectrophotometer (Thermo Electron Corporation, Multiskan Spectrum, USA) at 240 nm wavelength.

The activity of glutathione peroxidase (GPx) is derived from the oxidation of reduced β-NADPH in a conjugated glutathione reductase (GR) system using H2O2 (12 mM) as a substrate. Glutathione peroxidase reacts with H2O2 oxidizing reduced glutathione (GSH) to oxidized glutathione (GSSG). In brief, the GPx assay was performed in duplicate in a 96-well UV Costar plate. Each well contained 215 μL assay buffer (AB: 50 mM potassium phosphate, 1 mM EDTA, pH 7.0), 5 μL GSH (30.7 mg/mL in water), 5 μL GR (0.1 U/mL in AB), and 20 μL of sample, and 5 μL NAD(P)H was added to the mixture. Two readings were recorded [38]. The first was the background of oxidation at 340 nm for 3 min in 30 s intervals for samples (A1) and blank (A1b). The second reading was performed after adding 50 μL H2O2. This reading monitored the decrease of H2O2 due to NAD(P)H oxidation at 340 nm for 2 min. The GPX activity was expressed in

Data are expressed as mean ± standard deviation (mean ± SEM). One-way analysis of variance (ANOVA) was used to test for significance between the groups.

The CAT activity was expressed as μmol/mg of protein.

**3.5 Glutathione peroxidase activity**

**300**

μmol/mg of protein.

**3.6 Statistical analysis**

## **4.1 Plasma glucose levels in diabetic and nondiabetic groups before initiation of treatments**

**Figure 3** shows the non-fasted plasma glucose levels in both nondiabetic (N) and diabetic (STZ) groups before the start of KV and insulin (IN) treatments. The average fasting glucose level was significantly higher in the D group than the N group (28.19 ± 2.25 mmol/L versus 9.93 ± 0.51 mmol/L, p < 0.05).

#### **4.2 Evaluation of body weights after subjecting the rats to various treatments**

The induction of diabetes with STZ resulted in a significant loss of body weight (**Figure 4**). KV administration to normal rats did not affect body weight compared to untreated nondiabetic rats. On the other hand, the body weights of KV-treated diabetic rats significantly increased compared to diabetic control. Injection of the standard antidiabetic drug, insulin, also improved body weight gains in diabetic rats compared to the untreated diabetic rats.

### **4.3 Evaluation of testicular and epididymal weights of rats subjected to various treatments**

**Figure 5** presents the testicular weights of rats treated with STZ, KV, and/or IN. Diabetes induction decreased testicular weight in rats, whereas KV treatment of diabetic rats reversed this alteration. This is evident by a significant increase in testicular weight in KV-treated diabetic rats in comparison to untreated diabetic rats. However, insulin treatment did affect testicular weight in diabetic rats when compared to untreated diabetic rats.

**Figure 6** presents epididymal weights of rats subjected to different treatments. Diabetic rats had a lower epididymal weight compared to the nondiabetic rats (0.431 g ± 0.062 g versus 0.529 ± 0.058 g, p < 0.05). Treatment of diabetic rats with KV significantly increased epididymal weight in comparison to

#### **Figure 3.**

*Glucose levels in diabetic and nondiabetic rats before KV and IN treatments. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group.*

#### **Figure 4.**

*Effect of KV and insulin on body weight of rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated with insulin (standard drug).*

#### **Figure 5.**

*Effect of KV and insulin treatment on testicular weight of rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated with insulin (standard drug).*

untreated diabetic rats. KV treatment did not affect the epididymal weight of normal rats as no apparent difference was observed between N + KV group and N group (0.529 ± 0.058 g versus 0.475 ± 0.09 g, p > 0.05). On the other hand, the epididymal weight of insulin-treated diabetic rats was not significantly different compared to the untreated diabetic rats (0.454 ± 0.050 g versus 0.431 ± 0.062 g, p > 0.05).

#### **4.4 Assessment of lipid peroxidation of testicular and epididymal tissues of rats subjected to various treatments**

MDA levels in the testis are presented in **Figure 7** for both nondiabetic and diabetic groups treated with or without KV or insulin. The testicular MDA level was significantly higher in the D group compared to the N group (0.014 ± 0.001 μmol/g versus 0.010 ± 0.002 μmol/g, p < 0.05). The MDA level in the testes of nondiabetic rats treated with KV (N + KV) was significantly lower than the N group. The testes of diabetic rats treated with KV (D + KV) showed significantly lower testicular MDA level than the diabetic control group. Also, insulin treatment significantly lowered MDA levels in diabetic rats compared to the diabetic control group. It is noteworthy that a significant reduction in MDA

**303**

**Figure 8.**

*with insulin (standard drug).*

*Potential Antioxidative Effects of Kolaviron on Reproductive Function in Streptozotocin-Induced…*

*Effect of KV and insulin treatment on epididymal weight of rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated* 

*Effect of KV and insulin treatment on testicular tissue LPO in rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated* 

*Effect of KV and insulin treatment on epididymal LPO in rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated* 

*DOI: http://dx.doi.org/10.5772/intechopen.84822*

**Figure 6.**

**Figure 7.**

*with insulin (standard drug).*

*with insulin (standard drug).*

*Potential Antioxidative Effects of Kolaviron on Reproductive Function in Streptozotocin-Induced… DOI: http://dx.doi.org/10.5772/intechopen.84822*

#### **Figure 6.**

*Antioxidants*

**Figure 4.**

*(standard drug).*

**302**

p > 0.05).

**Figure 5.**

*with insulin (standard drug).*

**subjected to various treatments**

untreated diabetic rats. KV treatment did not affect the epididymal weight of normal rats as no apparent difference was observed between N + KV group and N group (0.529 ± 0.058 g versus 0.475 ± 0.09 g, p > 0.05). On the other hand, the epididymal weight of insulin-treated diabetic rats was not significantly different compared to the untreated diabetic rats (0.454 ± 0.050 g versus 0.431 ± 0.062 g,

*Effect of KV and insulin treatment on testicular weight of rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated* 

*Effect of KV and insulin on body weight of rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated with insulin* 

**4.4 Assessment of lipid peroxidation of testicular and epididymal tissues of rats** 

MDA levels in the testis are presented in **Figure 7** for both nondiabetic and diabetic groups treated with or without KV or insulin. The testicular MDA level was significantly higher in the D group compared to the N group (0.014 ± 0.001 μmol/g versus 0.010 ± 0.002 μmol/g, p < 0.05). The MDA level in the testes of nondiabetic rats treated with KV (N + KV) was significantly lower than the N group. The testes of diabetic rats treated with KV (D + KV) showed significantly lower testicular MDA level than the diabetic control group. Also, insulin treatment significantly lowered MDA levels in diabetic rats compared to the diabetic control group. It is noteworthy that a significant reduction in MDA

*Effect of KV and insulin treatment on epididymal weight of rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated with insulin (standard drug).*

#### **Figure 7.**

*Effect of KV and insulin treatment on testicular tissue LPO in rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated with insulin (standard drug).*

#### **Figure 8.**

*Effect of KV and insulin treatment on epididymal LPO in rats. Data are presented as mean ± SEM. (\*) indicates significant difference with p < 0.05. N, nondiabetic control group; D, diabetic group; N + KV, nondiabetic group treated with KV; D + KV, diabetic group treated with KV; D + IN, diabetic group treated with insulin (standard drug).*

levels was observed in the testes of KV-treated diabetic rats when compared to the D + IN group (0.007 ± 0.001 μmol/g versus 0.012 ± 0.001 μmol/g, p < 0.05).

**Figure 8** shows the MDA level in epididymis of both nondiabetic and diabetic groups treated with KV and/or insulin. The MDA level was significantly higher in epididymal tissue of the D group compared to the N group in epididymal tissue (0.009 ± 0.004 μmol/g versus 0.006 ± 0.002 μmol/g, p < 0.05). No significant differences in MDA levels were observed in the epididymal tissue of nondiabetic rats supplemented with KV (N + KV) compared to the untreated (N) rats. Furthermore, a significantly lower epididymal MDA level was observed in the D + KV group when compared to the D group. KV treatment normalized epididymal MDA level in diabetic rats, and this effect is comparable to that of insulin, the standard antidiabetic drug.
