**3.4 17β hydroxy steroid dehydorgenase enzyme activity**

17β HSD (EC 1.1.1.62) activity was measured by following the protocol of Jarabek et al. [24]. In brief, 10% homogenate of the ovarian tissues were prepared in normal Phosphate Buffered Saline (PBS; pH 7.4) and 250 μl of the supernatant was mixed with 250 μl of 440 μM sodium pyrophosphate buffer (pH 10.2), 10 μl ethanol containing 0.3 μM estradiol (Sigma, St. Louis, USA) and 240 μl of 25 mg% BSA. Enzyme activity was measured after addition of 50 μl of 0.5 μM NAD to the mixture in a spectrophotometer at 340 nm against a blank (without NAD). One unit of enzyme activity was the amount causing a change in absorbance of 0.001/min at 340 nm.

#### **3.5 Evaluation of SOD activity in ovary**

Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed following the method of Das et al. [25]. Just after sacrifice, 10% homogenates of all ovarian tissues from group 1 and set-III mice were prepared in 150 mM phosphate buffered saline (PBS, pH 7.4) and centrifuged for 30 min at 12,000 g at 4°C. The supernatant was again centrifuged for 60 min at 12,000 g at 4°C and then processed for enzymatic activity based on a modified spectrophotometric method

*Cannabis Effect on Female Reproductive Health DOI: http://dx.doi.org/10.5772/intechopen.91620*

using nitrite formation by superoxide radicals. A 0.5 ml of homogenate was added to 1.4 ml of reaction mixture comprised of 50 mM phosphate buffer (pH 7.4), 20 mM L-methionine, 1% (v/v) Triton X- 100, 10 mM hydroxylamine hydrochloride, 50 mM ethylene diamine tetraacetic acid (EDTA) followed by a brief preincubation at 37°C for 5 min. Next, 0.8 ml of riboflavin was added to all samples along with a control containing buffer instead of sample and then exposed to two 20 W fluorescent lamps fitted parallel to each other in an aluminum foil coated wooden box. After 10 min of exposure, 1 ml of Greiss reagent was added and absorbance of the color formed was measured at 543 nm. One unit of enzyme activity is defined as the amount of SOD inhibiting 50% of nitrite formation under assay conditions.

#### **3.6 Estimation of catalase activity in ovary**

Catalase (CAT; EC 1.11.1.6) activity was measured following the procedure of Sinha [26]. This method is based on the fact that dichromate in acetic acid is reduced to chromic acetate when heated in the presence of H2O2 with the formation of perchromic acid as an unstable intermediate. The chromic acetate thus produced is measured calorimetrically. The catalase preparation is allowed to split H2O2 for different periods of time. The reaction is stopped at a particular time by the addition of dichromate/acetic acid mixture and the remaining H2O2 is determined by measuring chromic acetate calorimetrically after heating the reaction mixture. There is production of green color at the end of the process. Immediately after sacrifice, 20% homogenate of ovarian tissues from groups 1 to 3 were prepared in PBS (10 mM; pH = 7.0) and then centrifuged at 12,000 g for 20 min at 4°C. Supernatant was taken for enzyme estimation. About 5 ml of PBS was added to 4 ml of H2O2 (200 mM) and then 1 ml of enzyme extract was added. After 1 min 1 ml of this solution was taken in a tube and 2 ml of K2Cr2O7 (5%) solution was added. Then, it was boiled for 10 min and absorbance was measured at 570 nm. The activity of CAT was expressed as amount of H2O2 degraded per minute.

## **3.7 Estimation of lipid peroxidation (LPO) assay by thiobarbituric acid reactive substances (TBARS) level estimation in ovary**

After sacrifice of the mice of all the groups, the ovarian tissues were dissected out on a sterile watch glass placed in ice box, cleaned from adherent tissues and processed immediately for estimation of lipid peroxidation. Ovarian tissues of groups 1–3 experimental mice were weighed and homogenized in a tenfold excess of 20 mM Tris-HCl buffer (pH 7.4) and the 10% homogenates were centrifuged for 15 min at 3000 g at 4°C. The supernatant was subjected to thiobarbituric acid (TBA) assay by mixing with 8.1% sodium dodecyl sulfate (SDS), 20% acetic acid, 0.8% TBA and then digested it for 1 h at 95°C. The reaction mixture was immediately cooled in running water, vigorously shaken with 2.5 ml of n-butanol and pyridine reagent (15:1) and centrifuged for 10 min at 1500 g (Ohkawa et al. ) [27]. The absorbance of the upper phase was measured at 534 nm. Total thiobarbituric acid reactive substances (TBARS) were expressed as malondialdehyde (MDA; nmol/g tissue weight) taking 1,1,1,1-tetraethoxy propane (TEP) as standard. The standard curve was calibrated using 10 nM TEP.

#### **3.8 Glutathione peroxidase (GPx) estimation in ovary**

Glutathione peroxidase (GPx; EC 1.11.1.9) activity was assayed as described by Mantha et al. [28]. The reaction mixture (1 ml) contained 50 μl sample, 398 μl of

50 mM phosphate buffer (pH 7.0), 2 μl of 1 mM EDTA, 10 μl of 1 mM sodium azide, 500 μl of 0.5 mM NADPH, 40 μl of 0.2 mM GSH, and 1 U glutathione reductase. The reaction mixture was allowed to equilibrate for 1 min at room temperature. After this, the reaction was initiated by addition of 100 mMH2O2. The absorbance measured kinetically at 340 nm for 3 min. The GPx activity was expressed as nmol of oxidized NADPH oxidized to NADP+ per min per mg of protein using an extinction coefficient (6.22 mM<sup>1</sup> cm<sup>1</sup> ) for NADPH.
