2.1 Chemicals and reagents
KET (200 mg/tablet; KETOHIM) was purchased from a local drug store. Folin-Ciocalteau’s reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), acetate buffer, 2,4,6-tripyridyl-s-triazine (TPTZ), and ferric chloride (FeCl3) were purchased from Sigma-Aldrich Co., Ltd. (Thailand). Ascorbic acid and other chemicals were of analytical grade.
2.2 Plant collection and extraction
The fresh purple CT flowers were collected from Khon Kaen Province, Thailand, in June to July 2012. Species identification was confirmed by Prof. Pranom CHANTARANOTHAI, a plant taxonomist, from Khon Kaen University (KKU), Thailand. Voucher specimens of CT were kept in the KKU Herbarium (No. Jaturon Burawat 01 [KKU]). In the extraction process, the CT flowers were washed with distilled water and air dried for two days. The dried flowers were crushed with a hammer mill crusher (AEG IP54 Lbi 07, Germany) for 30 min. Then, the crushed sample (3 kg) was extracted with 10 L of distilled water and boiled at 95–100 °C for 30 min. The soluble extract was filtered through nylon. The filtrate was dried using lyophylization. The extraction yield of aqueous extract from CT flowers was 3.97 g.
2.3 DPPH radical scavenging assay and determination of half maximum inhibition concentration (IC50)
The radical scavenging activity of the CT aqueous extract was determined using DPPH assay (Brand-Williams et al., 1995). Briefly, five concentrations of the CT flower extracts were prepared from a concentrated extract stock solution to provide a concentration plot. Each concentration of CT flower extracts (2 ml) was mixed with 2 ml DPPH in 0.004% (v/v) methanol and incubated at room temperature in dark for 30 min. The absorbance was recorded at 517 nm using an ultraviolet-visible (UV/VIS) spectrophotometer (Jasco V530, Japan). Ascorbic acid (1, 3, 5, 8, and 14 µg/ml) was used as the positive standard. All samples were measured in triplicate. The scavenging activity of the CT flower extracts or standard was calculated as the percent inhibition of DPPH radical scavenging activity using a standard formula as (absorbance of control−absorbance of sample)×100%/ absorbance of control. Data were expressed as the IC50 calculated from the point of 50% inhibition against the concentration (µg/ml) plot.
2.4 Ferric reducing antioxidant power (FRAP) assay
Reducing power capacity was determined using the FRAP assay (Benzie and Strain, 1996). Briefly, 0.1 ml of five different concentrations of CT flower extracts were mixed with 3 ml of FRAP reagent (containing 300 mmol/L acetate buffer, 10 mmol/L TPTZ, and 20 mmol/L FeCl3, in a volume ratio of 10:1:1). The CT mixture was incubated in dark for 20 min and the absorbance was recorded at 593 nm using a UV/VIS spectrophotometer. For standard preparation, six concentrations of ascorbic acid (5.76, 9.60, 17.28, 24.00, 32.00, and 48.00 µg/ml) were plotted to determine its reducing power.
2.5 Animals and treatment regime
Male Sprague-Dawley rats (180–200 g) were purchased from the National Laboratory Animal Center, Salaya, Nakhon Pathom, Thailand. The study was approved by the Animal Ethics Committee of KKU, based on the Ethics of Animal Experimentation of the National Research Council of Thailand (ref. No. 05220.127.116.11/93). Thirty-six rats were divided into six groups and each group (n=6) was treated as shown in Table 1.
2.6 Histopathological examination of the testes
On the day after termination of the CT-KET co-administration, all rats were euthanized by cervical dislocation and sacrificed to collect the male reproductive organs (i.e., testis, epididymis plus vas deferens, and seminal vesicle). These organs were subsequently removed of fats and weighed. To examine the testicular damage, testes were fixed in 10% (v/v) formalin in phosphate buffered saline (PBS) (pH 7.4), embedded in paraffin, sectioned at 4–6 μm thickness, and stained with hematoxylineosin (Iamsaard et al., 2013). All photographs were captured by a Nikon light ECLIPSE E200 microscope equipped with a DXM1200 digital camera. ImageJ was used to measure and calculate approximate average diameters of seminiferous tubules in four different axes (50 tubules per animal).
2.7 Plasma testosterone assay
At the end of the experiment, all animals were sacrificed to expose the left ventricle of the heart. Blood was collected by puncture of the left ventricular chamber using 1 ml of heparin to prevent blood clotting. The blood was centrifuged at 5000 r/min at 4 °C for 10 min to separate the plasma serum from blood cells. The plasma testosterone concentration was assayed by enzymatic immunoassay at the Radiology Unit, Srinagarind Hospital, Faculty of Medicine, KKU, Thailand.
2.8 Epididymal sperm concentration
Mature sperm were collected from the left epididymis and vas deferens. Epididymal sperm fluid was dipped and re-suspended in 1 ml PBS (37 °C, pH 7.4) and centrifuged (500g, 37 °C, 5 min) to wash and separate the mature sperm pellet from its fluid. To analyze the epididymal sperm concentration, the sperm pellets were re-suspended with 1 ml 0.3% (v/v) bovine serum albumin (BSA)-KSOM (potassium-enriched simplex optimized medium; Em-bryoMax KSOM Powdered Mouse Embryo Culture Medium; Millipore catalogue No. R-MR-020P-5D). In triplicate preparations, the sperm solutions (1:20 dilution) were used to count mature sperm using a Neubauer counting chamber and to calculate their concentration (Iamsaard et al., 2013).
2.9 Western blot analysis for testicular tyrosine protein phosphorylation
As described by Iamsaard et al. (2013), briefly, the left testis was homogenized with RIPA buffer (Cell Signaling Technology, Inc., USA) containing a cocktail of protease inhibitors. The testicular homogenate was then centrifuged at 12 000 r/mim for 10 min to collect testicular lysate. The total protein concentration of the lysate was measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., USA). Total proteins (60 μg) collected from triplicate samples were separated by 10% (0.1 g/ml) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes to detect the intensity of tyrosine phosphorylation using the 4G10 primary antibody (1:3000; Millipore Co., USA). For standard samples, BSA (AMRESCO®, USA) was used as the negative control and epidermal growth factors (EGFs) (Millipore Co., USA) as the positive control. In the detection of phosphotyrosine proteins, the enhanced chemiluminescence (ECL) substrate was applied before visualization under gel doct 4 (ImageQuant 400, GH Healthcare, USA). To quantify the levels of phosphorylation, the ImageJ program was used to analyze the relative intensity of phosphorylated protein bands among groups.
2.10 Statistical analysis
One-way analysis of variance (ANOVA) and t-test were used to examine the significance of differences among sets of data, and between pairs of data points using Sigma Stat program (Version 3.1.1). All quantitative results were expressed as mean±standard deviation (SD).