Ephedrine was purchased from Dainippon Pharma Co., Ltd. (Tokyo, Japan). Methylephedrine and pseudoephedrine were purchased from Alps Pharmaceutical Ind. Co. Ltd. (Gifu, Japan). Norephedrine was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). 6-Methoxykynurenic acid was purchased from Chemicia Scientific, LLC (San Diego, CA, USA). trans-Cinnamic acid was purchased from Wako Co. (Tokyo, Japan). 6-Hydroxykynurenic acid was synthesized from 6-methoxykynurenic acid . Syringin, kaempferol 3-O-rhamnoside 7-O-glucoside, and isovitexin 2″-O-rhamnoside were isolated from Ephedra Herb . The methanol and water used for liquid chromatography coupled with photodiode array detection (LC-PDA) analysis were LC grade.
Recombinant human HGF (purity ≥98 % by SDS-PAGE and HPLC analysis) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA). SU11274 (purity ≥98 % by HPLC analysis) was purchased from Sigma-Aldrich (St Louis, MO, USA). The antibodies (Abs) used were as follows: anti-p-Met (Tyr1234/1235) monoclonal Ab (mAb) (CST#3077), anti-Met mAb (CST#8198), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPD) mAb (CST#2118), and horseradish peroxidase (HRP)-labeled anti-rabbit IgG Ab (CST#7074); all were obtained from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).
Preparation of EFE and Ephedra Herb extract
Preparation of EFE and Ephedra Herb extract was carried out as described by Oshima et al. . Ephedra Herb (200 g, E. sinica, Japanese pharmacopoeia grade) was added to water (2000 ml), extracted at 95 °C for 1 h, and filtered, after which the residue was washed with water (200 ml). The extract was centrifuged at 1800g for 10 min, after which half of the supernatant was concentrated under reduced pressure to obtain Ephedra Herb extract (14.1 g), while the other half was passed directly through DIAION™ SK-1B ion-exchange resin (100 ml) which was treated with 1 M HCl (30 ml) and water (100 ml) prior to use, then washed with water (100 ml). The unadsorbed fraction (1100 ml) was adjusted to pH 5 using 5 % NaHCO3 aq. (60 ml), and the solution was then evaporated under reduced pressure to obtain EFE (11.8 g).
LC-PDA analysis of Ephedra Herb extract and EFE
One milliliter of methanol was added to 50 mg samples of Ephedra Herb extract and EFE, which were exposed to ultrasonic waves for 30 min and centrifuged. The supernatants were filtered through 0.45-µm membrane filters, after which 20 µl of each sample was subjected to LC-PDA analysis.
LC-PDA analysis was performed using an LC-10A HPLC system consisting of an SCL-10Avp system controller, an LC-10ATvp pump, a DGU-12A degasser, an SIL-10A auto injector, a CTO-10Avp column oven, and an SPD-M10Avp photodiode array detector equipped with a semi-micro cell (Shimadzu Inc., Tokyo, Japan). An Inertsil ODS-3 column (4.6 × 150 mm, 5 µm) from GL Sciences (Tokyo, Japan) was used for the separation. The column was maintained at 40 °C in the column oven. The mobile phase consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in methanol (B). The flow rate was 1.0 ml/min. The mobile phase gradient was adjusted as follows: 5 % B (0–10 min), 5–75 % B (10–70 min), 75–100 % B (70–80 min), 100 % B (80–90 min).
Cell lines and culture
MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Sigma-Aldrich, St. Louis, MA, USA) containing 10 % fetal calf serum (FCS) (Invitrogen Corp., Carlsbad, CA, USA) at 37 °C in an atmosphere containing 5 % CO2.
Trans-well migration assay
MDA-MB-231 cells (5 × 104 cells/100 µl) were suspended in 100 μl DMEM containing EFE (10, 20, or 40 μg/ml), Ephedra Herb extract (40 μg/ml), or SU11274 (5 μM). The cells were poured into the upper well of a trans-well permeable support system (Corning Inc., Acton, MA, USA). DMEM (600 µl) containing 50 ng/ml HGF was added to the lower well of the trans-well system, which was incubated for 20 h at 37 °C. Finally, the number of cells that had migrated from the upper layer to the lower well was counted.
MDA-MB-231 cells (5 × 104 cells/100 μl) were suspended in 100 μl of 10 % FCS-DMEM containing 40 μg/ml EFE, 40 μg/ml Ephedra Herb extract, or 5 μM SU11274. After 20 h, 10 μl of Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added to each sample, after which the resulting mixture was incubated at 37 °C for 2 h. Formazan absorbance (450 nm) was quantified using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Detection of phosphorylated c-Met (p-Met), c-Met, and GAPDH
MDA-MB-231 cells (2 × 106 cells/4 ml) were incubated in 4 ml of 10 % FCS-DMEM for 48 h, washed three times with DMEM, and incubated in 4 ml DMEM for 24 h. After the cells were washed three times with DMEM, they were incubated for 15 min at 37 °C in 4 ml DMEM or DMEM containing 50 ng/ml of HGF with or without 0.5, 1, or 10 μg/ml EFE, 10 μg/ml Ephedra Herb extract, or 5 μM SU11274. After the cells were washed three times with cold PBS without Ca2+ and Mg2+ (PBS(-)) they were treated with 1 ml Complete Lysis-M with phosphatase inhibitor (Roche Diagnostics Co., Indianapolis, IN, USA) for 5 min on an ice bath. The lysates were collected and centrifuged, after which the supernatants were incubated with 5× sodium dodecyl sulfate (SDS) loading buffer for 5 min at 95 °C. The lysates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and electro-transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked at room temperature for 1 h with 5 % non-fat dry milk in Tris-buffered saline (10 mM Tris–HCl, pH 7.5, 100 mM NaCl) containing 0.1 % Tween 20 (TBS-T). After the membrane was washed with TBS-T, it was incubated with the anti-p-Met (Tyr1234/1235) mAb (CST#3077), anti-Met mAb (CST #8198), or anti-GAPDH Ab (SC-25778) overnight at 4 °C and washed with TBS-T. Horseradish peroxidase-labeled anti-rabbit IgG Ab (CST#7074) was applied for 1 h at room temperature, after which the membranes were washed with TBS-T. The Abs were detected with an enhanced chemiluminescent (ECL) reaction (GE Healthcare Japan, Tokyo, Japan) and imaged using an ImageQuant Las 4000 mini system (GE Healthcare Japan).
Measurement of c-Met tyrosine-kinase activity and determination of IC50 values
Met kinase activity was measured using the ADP-Glo kinase assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, 10 μl of a reaction mixture containing 2 μg/ml of recombinant Met kinase domain, 0.2 μg/ml poly(E4Y1), and 10 μM ATP was incubated with Ephedra Herb extract or EFE at room temperature for 60 min. The kinase reactions were terminated by the addition of 10 μl ADP-Glo reagent, after which the resulting mixture was incubated for 40 min at room temperature. Next, 20 μl of Kinase Detection Reagent was added, after which the mixture was incubated for 30 min at room temperature. Luminescence was measured with an EnSpire multi-plate reader (Perkin Elmer, Foster City, CA, USA). The experiments were repeated three times. Each IC50 was calculated using a four-parameter logistic model (Prism 5.0, GraphPad Software, San Diego, CA, USA).
ICR male mice (5 weeks of age, 8 mice per group) were orally administered water, 350 mg/kg EFE, or 700 mg/kg Ephedra Herb extract for 3 days. On the third day, paw-licking was induced in the mice by intraplantar injection of 20 μl of 2.5 % formalin 6 h after extract/water administration. After the injection, the mice were individually placed into a glass cage, in which the amount of time that the animal spent licking the injected paw was measured as an indicator of pain. Paw-licking was recorded for 30 min in two phases, the first phase (0–5 min) and second phase (15–30 min).
The protocol for animal experiments was approved by the Ethics Review Committee for Animal Experimentation of the National Institute of Health Sciences.
Evaluation of anti-influenza activity
Madin–Darby canine kidney (MDCK) cells (3 × 104 cells/100 μl) were incubated in 100 μl of 10 % FCS-minimal essential medium (MEM) in a 96-well plate for 24 h and washed with MEM. Next, the cells were incubated for 72 h at 37 °C in 100 μl of MEM or MEM containing a twofold serial dilution of 10 μM oseltamivir, 50 μg/ml EFE, or 50 μg/ml Ephedra Herb extract with or without 100 TCID50 of influenza virus A/WSN/33(H1N1). Living cells were then stained with crystal violet, after which the absorbance (560 nm) of each cell sample was quantified using a microplate reader. These experiments were performed externally by AVSS Corporation. Each IC50 was calculated using a four-parameter logistic model (Prism 5.0, GraphPad Software).
Repeated oral dose toxicity assessment
Special pathogen-free ICR mice (Crl:CD1) (5 weeks old) were obtained from Charles River Laboratories (Boston, MA, USA). The mice were kept in a laboratory animal facility with temperature and relative humidity maintained at 20–26 °C and 30–70 %, respectively, a 12 h-light–dark cycle, and 8–10 air charges per hour. The mice were housed in polycarbonate cages and offered CE-2 pellet feed (Nippon Formula Feed Mfg. Co., Ltd., Ehime, Japan) and groundwater that was disinfected with 0.5 % chlorine and filtered through a 5-μm filter. The mice were acclimated for 7 days before the start of the study. The mice were grouped into three groups: water, EFE, or Ephedra Herb extract. Each group included five male mice and five female mice.
The dosages of EFE and Ephedra Herb extract were converted to 50-fold of the human maximum dose of Ephedra Herb extract, equivalent to 6 g of cut crude drug. The doses of EFE and Ephedra Herb extract were 632 mg/kg/day and 755 mg/kg/day, respectively. The mice were orally administered water, EFE, or Ephedra Herb extract once per day for 14 days. Clinical signs and mortality were assessed several times per day. Body weight, food consumption, and water consumption were measured twice per week throughout the experiment. After 14 days, all mice were anesthetized by isoflurane inhalation, after which blood samples were collected from the abdominal aorta.
Daiich Negishi Clinical Laboratory, Inc. assessed the following hematological parameters: red blood cell count (RBC), hematocrit (Ht), hemoglobin concentration (Hb), platelet count (PLT), white blood cell count (WBC), and WBC differential count (stab neutrophils, segmented neutrophils, lymphocytes, and monocytes). Serum biochemical parameters were measured by the Nagahama Institute for Biochemical Science at Oriental Yeast Co., Ltd. The selected serum biochemical parameters were albumin (ALB), aspartate aminotransferase (ALT), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), leucine aminopeptidase (LAP), gamma-glutamyl transpeptidase (γ-GT), and total bilirubin (T-BIL).
After the collection of the blood samples, the organs were harvested from each mouse and weighed. Colon weight was measured after washing out the colon contents with saline solution.
All data are expressed as mean ± standard deviation (SD). Data were analyzed by ANOVA. Significant differences between the control and treatment groups were determined by Student’s t test, Dunnett’s test, and Tukey’s test using GraphPad Prism 5J software (MDF Co., Ltd., Tokyo, Japan). p < 0.05 was considered statistically significant.