Extraction and isolation of Fr.B
P. utilis leaves was collected from Dali, Yunnan, China and authenticated by one of the authors Xiaobo Li. Voucher specimen has been deposited at herbarium of School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. Preparation of Fr. B was previously described by Wu et al [9]. Dried and powdered leaves (2 kg) were re-fluxed 3 times with distilled water. The combined extraction was filtrated and then concentrated under reduced pressure to obtain a crude aqueous extract (QCJ). The sample was redissolved, and then separated by macroporous resin AB-8. After adsorption, the resin was washed with 5% ethanol without collection, followed by 40% ethanol, the elution was then evaporated under vacuum at 65 ℃ and lyophilized to obtain Fr. B.
UPLC-Q-TOF-MS analysis of Fr. B
The qualitative chemical profiles of Fr. B was analyzed by UPLC-Q-TOF-MS which was performed on a Waters ACQUITY UPLC I-Class system (Waters Corp., Milford, MA, United States) with an ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 µm, Waters Corp., United States) by gradient elution using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.4 mL/min. The gradient profile was 0–2 min (A: 90%), 2–9 min (A: 90 − 80%), 9–11 min (A: 80 − 65%), 11–14 min (A: 65 − 0%). The injection volume was 1 µl. The temperature of the column oven was set to 45℃. Mass spectrometry was carried out using a Waters VION IMS QTOF mass spectrometer (Waters Corp., Milford, MA, United States). Ionization was performed in both positive and negative electrospray ionization (ESI) mode. The MS parameters were as follows, capillary voltage, 2.5 kV; cone voltage, 40 V; source temperature, 115 ℃; desolvation temperature, 450 ℃; gas flows of cone and desolvation, 50 and 900 L/h. A MSE (Mass Spectrometry Elevated Energy) experiment in two scan functions was carried out as follows. Function 1 (low energy), m/z 50-1000, 0.2 s scan time, 0.02 s inter-scan delay, 4 eV collision energy. Function 2 (high energy), m/z 50-1000, 0.2 s scan time, 0.02 s inter-scan delay, collision energy ramp of 20–45 eV. The data were processed using UNIFI 1.8.1 software (Waters Corp., Milford, MA, United States).
Quantitative analysis of Fr. B
The quantitative analysis of Fr. B were performed on an Agilent 1200 HPLC (high-performance liquid chromatography) system, equipped with a quaternary solvent delivery system, an on-line degasser, an autosampler, a column temperature controller, and a DAD (photo-diode-array-detector) detector. Agilent Zorbax SB-C18 Column (5 µm, 4.6×250 mm) was employed during the experiment with a flow rate of 1.0 ml/min. The column temperature was maintained at 30℃, and the injection volume was 10 µl. The mobile phase was composed of water (A) and acetonitrile (B) with the following gradient elution: 0–10 min, 95% A; 10–15 min, 95%-90% A; 15–20 min, 90% A; 20–30 min, 90%-85% A; 30–40 min, 85%-80% A; 40–50 min, 80%-75% A; 50-60min, 75%-70% A; 60-70min, 70%-0% A. The wavelength was set at 360 nm.
Animals and BPH models
Male Spraue-Dawley rats (180–220 g) were procured from Shanghai Slac Laboratory Animal Co. Ltd (Shanghai, China), and housed in the Laboratory Animal Center of Shanghai Jiao Tong University (Shanghai, China). The animals were housed in groups under controlled room temperature (25 ± 2℃, 55 ± 10% relative humidity) with a 12/12 h light/dark cycle. Standard laboratory chow and water were available ad libitum. All experimental procedures were approved by the Animal Ethics Committee of Shanghai Jiao Tong University (Shanghai, China).
Following 1 week acclimation, rats were randomly assigned to 8 groups (n = 10) as following, Sham, BPH model, Finasteride, Pule'an, Low QCJ, High QCJ, Low Fr. B, High Fr. B. The scrotum of the sham animals were cut following sewing up without cutting off the both testicles. Rats in the other groups were castrated. After incision disinfection with penicillin for 1 week, Sham rats were treated with saline (s.c., 0.5 ml/kg, alternate days) and 0.5% CMC-Na (i.g. 10 ml/kg, daily). BPH group rats were received testosterone propionate (s.c., 10 mg/kg) alternate days and 0.5% CMC-Na (i.g. 10 ml/kg) daily for 4 weeks [16, 17]. Two positive groups, the rats were treated with testosterone propionate (s.c., 10 mg/kg) and received a treatment with Finasteride (i.g., 1 mg/kg) or Pule’an (i.g., 460 mg/kg). Low/High QCJ groups rats were treated with testosterone propionate (s.c., 10 mg/kg) and received a treatment with QCJ (i.g., 860 mg (dry leaf)/kg or 2580 mg (dry leaf)/kg respectively). Low/High Fr. B groups rats were treated with testosterone propionate (s.c., 10 mg/kg) and received a treatment with Fr. B (i.g., 160 mg (dry leaf)/kg or 480 mg (dry leaf)/kg respectively).
At the end of experiment, animals were sacrificed under anaesthesia after blood sample collection. The prostates were removed and ventral prostate tissues were fixed in 10% neutral buffered formalin and embedded in paraffin for both histological and immunohistochemical examinations. The remainder of each prostate was stored at -80 ℃ and used for further analyses.
Histology and Immunohistochemical localizatiion of vascular endothelial growth factor (VEGF)
Prostate tissue were fixed for one day in paraformaldehyde solution (4% in phosphate-buffered saline (PBS) 0.1 M) at room temperature, dehydrated by graded ethanol and embedded in paraffin. 10 µm thick sections collected on glass slides, deparaffinized then stained with hematoxylin and eosin (H&E) for histopathological examination using light microscopy (Olympus BX51, Japan) associated to an Imaging system (Image Pro Plus 6.0).
Slices were deparaffinized and immersed in freshly prepared 3% H2O2, blocked with goat serum for 30 min, then treated with citrate buffer. The sections were rinsed with PBS and incubated at 4 ℃ overnight with VEGF Antibody (Boster Biological Technology, Wuhan, China.), incubated with secondary antibody (goat anti-Rabbit IgG, Boster Biological Technology, Wuhan, China.) at 37 ℃ for 30 min. After washed with PBS, the sections were incubated in strept avidin-biotin complex at 37 ℃ for 30 min, and immersed in diaminobenzidine for 5 min. The haematoxylin-stained sections were dehydrated with ethanol and visualized with an optical microscope (Olympus BX51, Japan). The VEGF content was expressed as average optical density (IOD/Area).
Evaluation of dihydrotestosterone (DHT), testosterone (T), estradiol (E2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) in plasma
DHT, T and E2 levels of the rats plasma were assayed with the commercially available kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). TNF-α, IL-6 levels were assayed in plasma sample with the commercially available kits (MultiSciences Biotech Co., Ltd., Hangzhou, China). All the procedures were performed according to manufacturer’ s instructions of the kits.
Evaluation of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) in prostate tissue
Prostate samples were homogenized and assayed for MDA, SOD, GSH-Px and CAT using commercially available kits (MultiSciences Biotech Co., Ltd., Hangzhou, China). All the procedures were performed according to manufacturer’s instructions of the kits.
Statistical analysis
All data were expressed as mean ± standard deviation. The results were analyzed by one-way ANOVA followed by LSD for multiple comparisons using SPSS 21.0 software for Windows (SPSS Inc., Chicago, IL). A P value of < 0.05 was considered significant.