Preparation of BCB
The constituents of BCB are shown in Table 1. All required herbs or herbal parts were purchased from Humanherb (Daegu, Korea), a good manufacturing practice (GMP)-certified Korean herbal medicine supplier. Voucher specimens are stored at the College of Korean Medicine, Dongguk University. To prepare the BCB extract, a mixture of the dried requisite parts of Cuscutae Semen, Dioscoreae Rhizoma, Rubi Fructus, Ginseng Radix, Lycii Fructus, Angelicae Gigantis Radix, Perillae Folium, Amomi Fructus, Artemisiae Argyi Folium, Zingiberis Rhizoma Crudus, and Zizyphi Fructus (weight ratio 8:16:10:4:4:2:4:4:4:3:2, respectively) was pulverized and extracted twice with 10 volumes of water at 85-90°C for 3 h. The extract was then passed through a 50 μm filter paper, and the filtrate was concentrated by vacuum evaporation at 60°C, lyophilized to yield BCB extract (yield 6.5% based on herbal mixture weight).
BCB analysis by HPLC
Marker compounds in BCB extract were analyzed using a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a dual pump, an autosampler, a temperature regulated column oven, a diode-array spectrophotometric detector, and Chromeleon 6.8 chromatography management system software . Ellagic acid (Sigma-Aldrich, St. Louis, Mo, USA) and chlorogenic acid (Sigma-Aldrich) were used as standards. Component separations were achieved on a VDSpher C-18 column (VDSoptilab, Germany) maintained at 30°C. The column was step-gradient eluted using 0.3% trifluoroacetic acid (A) and acetonitrile (B) at a flow rate of 0.8 mL/min as follows: 10% B for 0-25 min, 60% B for 25-30 min, 100% B for 30-36 min, and 10% B for 36-40 min. Ellagic acid and chlorogenic acid were detected at 254 nm and 340 nm, respectively.
Cell culture and cell viability
Chinese hamster ovary (CHO)-K1 cells were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and cultured in DMEM (Dulbecco’s modified Eagle’s medium; WELGENE, Gyeongsan, Republic of Korea), supplemented with 10% fetal bovine serum (WELGENE) and 100 U/mL penicillin/100 μg/mL streptomycin (Thermo Fisher Scientific, Grand Island, NY, USA) at 37°C in a humidified incubator (5% CO2/95% air; Thermo Fisher Scientific, Langenselbold, Germany). Cell viabilities were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-phenyltetrazolium bromide (MTT) assay. Briefly, CHO-K1 cells were plated at a density of 5–7×103 cells per well in 96-well plates, incubated for 12 h, and treated with various concentrations of BCB (10–500 μg/mL) for 24 h. In other experiments, cells were pretreated with 50–500 μg/mL of BCB for 4 h, 1.5 mM of 4-vinylcyclohexene diepoxide (VCD; Sigma-Aldrich) was added, and cells were incubated for a further 24 h. Viable cells were stained with MTT solution (0.2 mg/mL, Sigma-Aldrich) for 3 h. Formazan crystals were completely dissolved by adding 100 μL dimethyl sulfoxide, and absorbances were measured at 540 nm using a microplate reader (Tecan, Research Triangle Park, NC, USA).
DPPH radical scavenging activity assay
The ability of BCB to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals was evaluated as previously described , and reductions in free radical levels were quantified by measuring absorbance (abs.) at 540 nm. Briefly, various concentrations of BCB (5–500 μg/mL) were mixed with 0.3 mM DPPH ethanol solution and reacted for 30 min in the dark. Absorbances were measured at 515 nm and DPPH free-radical scavenging activities were calculated as follows:
Scavenging effect (%) = [(control abs. – sample abs.)/ control abs.] × 100.
Superoxide anion free-radical scavenging activity assay
The ability of BCB to scavenge superoxide anion free radicals was determined as previously described . Briefly, BCB (5–500 μg/mL) was added to a solution containing 30 mM EDTA (pH 7.4), 30 mM hypoxanthine, and 1.42 mM nitro blue tetrazolium and pre-incubated at room temperature for 3 min. Xanthine oxidase (0.5 U/mL) was then added and allowed to react at room temperature for 20 min. Absorbances were measured at 560 nm.
Measurement of reactive oxygen species (ROS)
A DCFH-DA (2’,7’-dichlorofluorescein diacetate) assay was used to assess intracellular ROS levels. CHO-K1 cells were plated on a black 96-well plate at 1×104 cells/well and incubated with 100 μM hydrogen peroxide (H2O2) in the presence or absence of BCB (100 μg/mL). After removing medium, 10 μM DCFH-DA was added to each well, and mixtures were incubated at 37°C for 30 min. Fluorescence intensities were measured at excitation and emission wavelengths of 480 and 530 nm, respectively, using a fluorescence microplate reader (SpectraMAX Gemini, Molecular Devices, Sunnyvale, CA, USA).
Animal experimental design and treatment
Male and female C57BL/6 mice were purchased from Koatech (Pyeongtaek, Korea). Animals were bred separately by gender and given free access to drinking water and a standard diet and were maintained in a controlled environment under a 12 h light/dark cycle. All experimental procedures were performed according to the guidelines issued by the Animal Research Ethics Committee at Dongguk University Animal Center (IACUC-2016-001).
Experiment 1: Eighteen 4-week-old female mice were randomly divided into three groups: Control group, BCB 100 group, and BCB 300 group. The BCB 100 and BCB 300 groups were orally administered BCB at 100 or 300 mg/kg daily, respectively, for 30 days. At 6 pm local time on day 8 of the 30-day treatment period, all females were exposed to mating with 6-week-old males (ratio 2:1). Days on which vaginal plugs were first discovered, were designated as day 1 of pregnancy. The number of pups born to each mouse was recorded.
Experiment 2: Eighteen 4-week-old female mice were randomly divided into three groups: Control group, RU486 group, and RU486 plus BCB 300 group. Mice in the RU486 plus BCB 300 group were treated with BCB 300 mg/kg for 18-21 days. At 6 pm on day 8 during the 30-day treatment period, all females were exposed to mating with 6-week-old males (ratio 2:1). Days on which vaginal plugs were first discovered, were designated as day 1 of pregnancy. On day 4 of pregnancy, mice in the RU486 and RU486 plus BCB 300 groups were injected subcutaneously with RU486 solution (4 mg/kg, 0.08 mg/100 µL), whereas control group mice were injected with corn oil as vehicle. Seven days after RU486 injection, mice were sacrificed, uterine horns were excised, and numbers of implanted embryos in each uterine horn were counted.
Western blot analysis
For western blotting, uterus tissue was homogenized in protein lysis buffer consisting of 50 mM Tris-base (pH 7.5), 2 mM EDTA, 1% glycerol, 150 mM NaCl, 10 mM NaF, 10 mM Na-pyrophosphate, 1% NP-40 and protease inhibitors. Cell lysates (30 µg) were loaded and electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% skim milk in 0.1% TBS-T for at least 1 h, membranes were incubated with anti-phospho-IκB-α, anti-IκB-α, anti-β-actin (Cell Signaling Technology, Beverly, MA, USA), anti-MT1-MMP, anti-MMP2, anti-COX-2, or anti-iNOS (Abcam, Cambridge, MA, USA) for 16 h at 4°C. After washing with 0.05% TBS-T, membranes were treated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG for 1 h, and after washing with 0.05% TBS-T, bands were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific). Band images were obtained using the ChemiDocTM XRS+ system (Bio-Rad, Hercules, CA, USA) and band intensities were analyzed by using Image LabTM software version 2.0.1 (Bio-Rad).
Hematoxylin and eosin (H&E) staining
For histological examination, uteri were removed, fixed in 10% formalin overnight, and dehydrated in 70% ethanol. Tissues were then paraffin-embedded, sectioned, and stained with H&E and smooth muscle actin. Three randomly selected sections were chosen for histopathological analysis.
Data were analyzed using the Student’s t-test or by one-way ANOVA, and the significances of differences between means were determined using Dunnett’s test or Tukey–Kramer’s multiple comparison test. Null hypotheses of no intergroup differences were rejected for p -values of < 0.05. Results are presented as means ± SDs (in vitro) or means ± standard errors (in vivo), and the analysis was performed using SPSS (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.).