**2. Materials and methods**

#### **2.1 Plant materials and KV extraction**

Fresh seeds of *G. kola* were purchased from the Bodija market in Ibadan, Oyo State, Nigeria, and authenticated by Professor E. Ayodele at the Department of Botany, University of Ibadan. A voucher specimen (FHI-109777) is available at the University of Ibadan, at the herbarium of the Forestry Research Institute of Nigeria (FRIN), Ibadan.

KV was extracted and isolated according to the method of [34]. The seeds were peeled, sliced, and air-dried (25–28°C). Briefly, the powdered seeds were extracted with light petroleum ether (boiling point, 40–60°C) in a soxhlet for 24 h. The defatted dried product was repacked into the soxhlet and extracted with acetone. The extract was concentrated and diluted twice its volume with water and extracted with ethylacetate (6 × 300 mL). The concentrated ethylacetate yielded a golden yellow solid termed KV.

Liquid chromatography-mass spectrometry (LC-MS) analysis of the Garcinia kola seed extract was performed on a Dionex HPLC system (Dionex Softron, Germering, Germany) equipped with a binary solvent manager and autosampler coupled to a Brucker ESI Q-TOF mass spectrometer (Bruker Daltonik GmbH, Germany) as previously described [35]. KV was separated by reversed-phase chromatography on a Thermo Fischer Scientific C18 column 5 μm, 4.6 × 150 mm (Bellefonte, USA), using gradient elution with 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) as solvent at a flow rate of 1.0 mL min<sup>−</sup><sup>1</sup> , an injection volume of 10 μL, and an oven temperature of 30°C. MS spectra were acquired in negative mode using the full scan and auto MS/MS (collision energy 25 eV) scan modes with dual spray for reference mass solution. Electrospray voltage was set to +3500 V. Dry gas flow was set to 9 L min<sup>−</sup><sup>1</sup> with a temperature of 300°C, and nebulizer gas pressure was set to 35 psi.

#### **2.2 Treatment of animals and ethical clearance**

Sixty adult male Wistar rats, weighing between 240 and 290 g, were purchased from the animal facility of the Medical Research Council, South Africa. The animals were accommodated individually in plastic cages. They were supplied with water and standard rat feed ad libitum. Animals were maintained under standard laboratory conditions at 22 ± 2°C with a 12-h light/dark cycles and humidity at 55 ± 5%. Body weights were measured from the onset of the study and monitored throughout the feeding period until sacrifice. All animals received care according to the principles of Laboratory Animal Care of the National Society of Medical Research and the National Institutes of Health Guide for the Care and Use of Laboratory Animals of the National Academy of Sciences (National Institutes of Health publication no. 80-23, revised 1978). The study was approved by the Ethical Committee of the Faculty of Health and Wellness Sciences, University of Cape Peninsula Technology, South Africa (Cape Town, South Africa) (NHREC: REC-230408-014).

#### **2.3 Experimental induction of diabetes**

The animals were fasted overnight, and diabetes was induced by a single intraperitoneal injection (50 mg/kg body weight) of freshly prepared STZ

**299**

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

solution (Sigma, USA) dissolved in 0.1 M cold citrate buffer at pH 4.5 [36]. Five days after the STZ injection, blood glucose levels were measured using a portable glucometer (Accu-Chek, Roche, Germany) in blood collected from the tail, and

The overall time period for the current study was 6 weeks. Rats were randomly

• Group 1 (N): control animals (healthy, nondiabetic animals); received dimeth-

• Group 2 (N + KV): control animals received KV dissolved in DMSO orally.

• Group 3 (D): untreated diabetic group; injected with a single dose of STZ

• Group 4 (D + KV): received KV (100 mg/kg) orally five times weekly starting 5 days post STZ injection; this served as the KV-treated diabetic group.

• Group 5 (D + INS): received subcutaneous insulin (INS) injection (2 u/kg) every other day starting 5 days post STZ injection; this served as the insulin-

At completion of the treatment periods, rats were weighed and anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). Fasting blood glucose levels were measured after 4 h of fasting (usually between 10 am and 2 pm). Blood samples were collected from the abdominal aorta into glucose tubes (containing sodium fluoride/potassium oxalate) and EDTA-containing tubes. The epididymis and testes were also excised and weighed. The tissue samples were snap

Briefly, 250 μL of phosphate buffer (50 mM NaH2PO4·2H2O, 0.5% (v/v)

TritonX-100, pH 7.5) was added to 50 mg of testicular or epididymal tissue. The homogenates were transferred into tubes and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatants were subsequently transferred to new tubes and kept at −80°C until used.

Testicular and epididymal protein levels were determined using the bicinchoninic acid (BCA) method as describe by [37]. Briefly, BCA working reagents, samples, and standards were prepared referring to the manufacturer's instructions

Malondialdehyde (MDA) levels were determined in the samples through a modern HPLC-based thiobarbituric acid (TBA) assay method. This method is highly

diabetes status was confirmed when glucose level was above 18 mmol/L.

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

divided into five groups (n = 12 per group) as follows:

**2.4 Experimental design**

ylsulfoxide (DMSO) orally.

(50 mg/kg) intraperitoneally.

treated diabetic group.

**3. Biochemical assays**

**3.2 Lipid peroxidation**

**2.5 Sample collection and preparation**

frozen in liquid nitrogen and stored at −80°C.

**3.1 Determination of protein concentration**

for the assay kit supplied by Sigma Aldrich.

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

solution (Sigma, USA) dissolved in 0.1 M cold citrate buffer at pH 4.5 [36]. Five days after the STZ injection, blood glucose levels were measured using a portable glucometer (Accu-Chek, Roche, Germany) in blood collected from the tail, and diabetes status was confirmed when glucose level was above 18 mmol/L.

## **2.4 Experimental design**

*Antioxidants*

Wistar rats.

**2. Materials and methods**

yellow solid termed KV.

9 L min<sup>−</sup><sup>1</sup>

**2.1 Plant materials and KV extraction**

B) as solvent at a flow rate of 1.0 mL min<sup>−</sup><sup>1</sup>

**2.3 Experimental induction of diabetes**

**2.2 Treatment of animals and ethical clearance**

This study was therefore designed to evaluate any potential effects of KV in boosting testicular and epididymal antioxidant status in STZ-induced diabetic

Fresh seeds of *G. kola* were purchased from the Bodija market in Ibadan, Oyo State,

Liquid chromatography-mass spectrometry (LC-MS) analysis of the Garcinia kola seed extract was performed on a Dionex HPLC system (Dionex Softron, Germering, Germany) equipped with a binary solvent manager and autosampler coupled to a Brucker ESI Q-TOF mass spectrometer (Bruker Daltonik GmbH, Germany) as previously described [35]. KV was separated by reversed-phase chromatography on a Thermo Fischer Scientific C18 column 5 μm, 4.6 × 150 mm (Bellefonte, USA), using gradient elution with 0.1% formic acid in water (solvent A) and acetonitrile (solvent

oven temperature of 30°C. MS spectra were acquired in negative mode using the full scan and auto MS/MS (collision energy 25 eV) scan modes with dual spray for reference mass solution. Electrospray voltage was set to +3500 V. Dry gas flow was set to

with a temperature of 300°C, and nebulizer gas pressure was set to 35 psi.

Sixty adult male Wistar rats, weighing between 240 and 290 g, were purchased from the animal facility of the Medical Research Council, South Africa. The animals were accommodated individually in plastic cages. They were supplied with water and standard rat feed ad libitum. Animals were maintained under standard laboratory conditions at 22 ± 2°C with a 12-h light/dark cycles and humidity at 55 ± 5%. Body weights were measured from the onset of the study and monitored throughout the feeding period until sacrifice. All animals received care according to the principles of Laboratory Animal Care of the National Society of Medical Research and the National Institutes of Health Guide for the Care and Use of Laboratory Animals of the National Academy of Sciences (National Institutes of Health publication no. 80-23, revised 1978). The study was approved by the Ethical Committee of the Faculty of Health and Wellness Sciences, University of Cape Peninsula Technology,

South Africa (Cape Town, South Africa) (NHREC: REC-230408-014).

The animals were fasted overnight, and diabetes was induced by a single intraperitoneal injection (50 mg/kg body weight) of freshly prepared STZ

, an injection volume of 10 μL, and an

Nigeria, and authenticated by Professor E. Ayodele at the Department of Botany, University of Ibadan. A voucher specimen (FHI-109777) is available at the University of Ibadan, at the herbarium of the Forestry Research Institute of Nigeria (FRIN), Ibadan. KV was extracted and isolated according to the method of [34]. The seeds were peeled, sliced, and air-dried (25–28°C). Briefly, the powdered seeds were extracted with light petroleum ether (boiling point, 40–60°C) in a soxhlet for 24 h. The defatted dried product was repacked into the soxhlet and extracted with acetone. The extract was concentrated and diluted twice its volume with water and extracted with ethylacetate (6 × 300 mL). The concentrated ethylacetate yielded a golden

**298**

The overall time period for the current study was 6 weeks. Rats were randomly divided into five groups (n = 12 per group) as follows:


#### **2.5 Sample collection and preparation**

At completion of the treatment periods, rats were weighed and anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). Fasting blood glucose levels were measured after 4 h of fasting (usually between 10 am and 2 pm). Blood samples were collected from the abdominal aorta into glucose tubes (containing sodium fluoride/potassium oxalate) and EDTA-containing tubes. The epididymis and testes were also excised and weighed. The tissue samples were snap frozen in liquid nitrogen and stored at −80°C.

Briefly, 250 μL of phosphate buffer (50 mM NaH2PO4·2H2O, 0.5% (v/v) TritonX-100, pH 7.5) was added to 50 mg of testicular or epididymal tissue. The homogenates were transferred into tubes and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatants were subsequently transferred to new tubes and kept at −80°C until used.

#### **3. Biochemical assays**

#### **3.1 Determination of protein concentration**

Testicular and epididymal protein levels were determined using the bicinchoninic acid (BCA) method as describe by [37]. Briefly, BCA working reagents, samples, and standards were prepared referring to the manufacturer's instructions for the assay kit supplied by Sigma Aldrich.

#### **3.2 Lipid peroxidation**

Malondialdehyde (MDA) levels were determined in the samples through a modern HPLC-based thiobarbituric acid (TBA) assay method. This method is highly 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.

#### **3.3 Superoxide dismutase activity**

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 μmol/mg protein.
