Materials
WIB-801CE was provided from Whanin Pharmaceutical Corporation (Suwon, Korea). Collagen, adenosine diphosphate (ADP) and thrombin were obtained from Chrono-Log Corporation (Havertown, PA, USA). Serotonin enzyme-linked immunosorbent assay (ELISA) kit was purchased from Labor Diagnostika Nord GmbH & Corporation (Nordhorn, Germany). Pure cordycepin, aspirin, protease inhibitor cocktail, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid (AC), and other reagents were obtained from Sigma Chemical Corporation (St. Louis, MO, USA). Thromboxane B2 (TXB2) enzyme immunoassay (EIA) kit, cyclooxygenase-1 (COX-1) fluorescent activity assay kit, lactate dehydrogenase (LDH) cytotoxicity assay kit, ozagrel and prostaglandin H2 (PGH2) for TXA2 synthase (TXAS) assay were purchased from Cayman Chemical (Ann Arbor, MI, USA). Arachidonic acid (AA) release ELISA kit was purchased from Cusabio Biotech Corporation (Wuhan, Hubei, China). Anti-phosphor-cytosolic phospholipase A2 (cPLA2) (Ser505), anti-phosphor-phospholipase Cβ3 (PLCβ3) (Ser537), anti-phosphor-phospholipase Cβ3 (PLCβ3) (Ser1105), anti-phosphor-phospholipase Cγ2 (PLCγ2) (Tyr1217), anti-phosphor-p38 MAPK, anti-phosphor-extracellular signal–regulated kinase (ERK) (1/2), anti-p38 MAPK, anti-ERK (1/2) and anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugate (HRP), and lysis buffer were obtained from Cell Signaling (Beverly, MA, USA). Polyvinylidene difluoride (PVDF) membrane was from General Electric Healthcare (Piseataway, NJ, USA). Enhanced chemiluminescence solution (ECL) was from General Electric Healthcare (Chalfont St. Giles, Buckinghamshire, UK). Prothrombin time (PT) assay reagent and activated partial thromboplastin time (APTT) assay reagent were obtained from Fisher Diagnostics (Middletown, VA, USA).
Preparation of WIB-801CE
Cordyceps militaris was cultivated, and culture-solution of Cordyceps militaris-hypha was concentrated up to 50° Brix with a rotary vacuum evaporator (Eyela N3000, Rikakikai Co., Ltd., Tokyo, Japan) at 60 °C. The Brix was measured with refractometer (Atago Co., Ltd., Tokyo, Japan). The concentrate was extracted by extraction-shaker (Cosmos 660, Kyungseo Co., Ltd., Seoul, Korea) for 4 h at 40 °C one time with distilled water/95% ethanol (1:3.5, v/v), which was filtered one time using a filter paper (Advantec No.2). The filtrate was completely concentrated at 60 °C by an evaporator (Eyela N3000, Rikakikai Co., Ltd., Tokyo, Japan) under reduced pressure, and was lyophilized and stored at -20 °C until used. This was named as cordycepin-enriched WIB-801CE.
Analysis of cordycepin in WIB-801CE with HPLC
WIB-801CE was dissolved with 75% methanol, then analyzed by high performance liquid chromatography (HPLC). An Alliance 2695 liquid chromatography system (Waters Co., Milford, MA, USA), equipped with vacuum degasser, quaternary gradient pump, autosampler and photodiode array detector, was connected to Empower software. A hydrosphere C18 column (250 mm × 4.6 mm id, 5 μm, YMC Co., Ltd., Kyoto, Japan) was used at a column temperature of 30 °C. The applied-mobile phase gradient program was 0.01 M KH2PO4/methanol (95:5, v/v) at 0 min and held for 5 min; 0.01 M KH2PO4/methanol v/v) at 20 min and held for 6 min; 0.01 M KH2PO4/methanol (95:5, v/v) at 27 min and held 6 min for chromatographic balance. In this step, 99.8% of methanol was used. The flow rate was at 1.0 mL/min and sample injection volume was 10 μL. The ultra violet detection was operated at 254 nm.
Animals and administration
We investigated the ex vivo and in vivo effects of WIB-801CE using rats (Sprague-Dawley, male, 200 g) and Institute of Cancer Research (ICR) mice (male, 18 g, Daehan Biolink Co., Ltd., Chungbook, Korea). Rats for ex vivo experiment and mice for in vivo observation were divided into as follows, respectively: WIB-801CE-nontreated group (control), WIB-801CE-treated group, aspirin-treated group as positive control of in vivo, and warfarin-treated group as positive control of ex vivo.
Animals were acclimatized for a week at a temperature of 24 ± 1 °C and humidity of 55 ± 5%. Before oral administration of substances, all animals were fasted for 12 h, then were fed with standard pellets diet (Purina Inc., Korea) had free access to water. WIB-801CE [200, 400 mg/kg-body weight (BW)] and warfarin (1 mg/kg-BW) for ex vivo experiment were orally administered to the rats one per day for seven days, and WIB-801CE (200, 400 mg/kg-BW) and aspirin (100 mg/kg-BW) for in vivo observation were orally administered to the mice once a day for five days. 200 mg/kg-BW of WIB-801CE is corresponded to the minimum dose that inhibits rat platelet aggregation (data not shown). WIB-801CE, warfarin, and aspirin were dissolved with distilled water. The experiments were proved by the Ethics Committee for Animal Experiments of Whanin Pharmaceutical Corporation (Suwon, Korea/15-NE-016 for rats, 15-NE-008 for mice).
Preparation of rat platelet-rich plasma and platelet-poor plasma for ex vivo assay
After the final respective administration, all rats were fasted for 24 h, then after 2 h of WIB-801CE- and warfarin-administration were anesthetized with 20% urethane before sacrifice according to the method of Zhang et al. [17]. The blood was collected from the abdominal aorta. The blood was anti-coagulated with acid-citrate-dextrose solution (0.8% citric acid, 2.2% sodium citrate, 2.45% glucose), and was centrifuged at 250 × g for 10 min in order to obtain platelet-rich plasma (PRP). In order to remove residual red blood cells and white cells, the PRP was again centrifuged at 125 × g for 10 min. Platelet-poor plasma (PPP) was prepared by centrifuging the part of PRP at 1,300 × g for 10 min.
PRP was used to investigate ex vivo platelet aggregation, TXA2 production, serotonin release, COX-1 and TXAS activities, AA release and protein phosphorylation. PPP was used to investigate ex vivo PT and APTT. The number of platelets in PRP was adjusted with PPP to a final concentration of 5 × 108/mL. All of the above procedures were carried out at 25 °C to avoid platelet aggregation from any effect of low temperature.
Preparation of human PRP and washed platelets for in vitro assay
To investigate in vitro effects of WIB-801CE and cordycepin on fibrin clot retraction, we used human PRP and washed platelets. PRP from normal healthy human volunteers with informed consent was obtained from the Korean Red Cross Blood Center (KRBC, Changwon, Korea), and its experimental use was approved by the KRBC (Safety Supervisor Team-621-2015.02.26) and the Korea National Institute for Bioethics Policy Public Institutional Review Board (Seoul, Korea/PIRB12-072-01) with informed consent. PRP anticoagulated with acid-citrate-dextrose solution (0.8% citric acid, 2.2% sodium citrate, 2.45% glucose) was centrifuged for 10 min at 125 × g to remove a little red blood cells and white cells, which was used to investigate the effect of WIB-801CE and cordycepin on thrombin-induced fibrin clot retraction. The number of platelets in PRP was adjusted with PPP to a final concentration of 5 × 108/mL.
To observe in vitro effects of WIB-801CE and cordycepin on cytotoxicity and resting platelet aggregation, we prepared human washed platelets. The PRP was centrifuged for 10 min at 1,300 × g to obtain platelet pellets. The platelets were washed twice with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM glucose, and 1 mM Na2EDTA, pH 6.5). The washed platelets were then resuspended in suspension buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 0.49 mM MgCl2, 5.5 mM glucose, 0.25% gelatin, pH 6.9) to a final concentration of 5 × 108/mL. All of the above procedures were carried out at 25 °C to avoid platelet aggregation from any effect of low temperature. The Korea National Institute for Bioethics Policy Public Institutional Review Board (Seoul, Korea/PIRB12-072-01) approved these experiments.
In vitro cytotoxicity assay
Platelet cytotoxicity was determined by leakage of LDH from cytosol. Human washed platelets (108/mL) were incubated for 5 min at 37 °C in the presence of WIB-801CE or cordycepin, then centrifuged with 12,000 × g at room temperature for 2 min. The supernatant was measured with a synergy HT multi-model microplate reader (BioTek Instruments, Winooski, VT, USA) using LDH assay kit. LDH leakage was expressed as percentage of total LDH activity in platelets completely lysed by 0.2% triton X-100.
Measurement of ex vivo rat platelet aggregation, and in vitro human resting platelet aggregation
To evaluate antiplatelet effect of WIB-801CE under condition that generates maximally platelet aggregation, we used high dose of collagen and ADP as agonists. The concentration of collagen-induced maximal rat (Sprague-Dawley, male) platelet aggregation was 10 μg/mL [18], and 5 μM of ADP was used to aggregate rat platelets [19]. Accordingly, we used 10 μg/mL of collagen, and 5 μM of ADP to cause ex vivo rat platelet aggregation.
To measure ex vivo rat platelet aggregation, PRP (108 platelets/mL) were preincubated with or without WIB-801CE for 3 min at 37 °C, then stimulated for 5 min by collagen (10 μg/mL) and ADP (5 μM) using an aggregometer (Chrono-Log Corporation, Havertown, PA, USA) at a constant stirring speed of 1,000 rpm.
To observe in vitro effects of WIB-801CE and cordycepin on resting human platelet aggregation, the aggregation of human washed platelets (108/mL) was performed as described above in the presence of WIB-801CE or cordycepin without agonists. But, collagen (10 μg/mL) was used as positive control. Each aggregation rate was determined as an increase in light transmission. The PPP for PRP aggregation, and platelet suspension buffer (pH 6.9) for washed platelet aggregation were used as the reference (transmission 0) to regulate the base line of aggregometer. WIB-801CE and cordycepin were dissolved in distilled water.
Ex vivo measurement of TXB2
To investigate the effect on TXA2 production, the aggregation was terminated by adding ice-cold 5 mM EDTA and 0.2 mM indomethacin to inhibit subsequent conversion of AA to TXA2. The amounts of TXB2, a stable metabolite of TXA2, were determined using a TXB2 EIA kit according to the procedure described by the manufacturer.
Ex vivo Western blot for analysis of protein phosphorylation
Collagen- and ADP-activated rat PRP was centrifuged for 10 min at 1,300 × g under 4 °C to remove PPP and get platelet pellets. The platelets were suspended twice with suspension buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 0.49 mM MgCl2, 5.5 mM glucose, 0.25% gelatin, pH 7.4). The suspended platelets (250 μL) were lysed by adding an equal volume (250 μL) of lysis buffer [20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate (serine/threonine phosphatase inhibitor), 1 mM Na3VO4 (ATPase, alkaline and acid phosphatase, and protein phosphotyrosine phosphatase inhibitor), 1 μg/mL leupeptin (serine and cysteine protease inhibitor), and 1 mM phenylmethanesulfonyl fluoride (serine protease and acetylcholinesterase inhibitor), pH 7.5].
Platelet lysates were suspended in their equal volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer (62.5 mM Tris-HCl, 10% glycerol, 1% SDS, 1% β-mercaptoethanol, 0.01% bromphenol blue, pH 6.8), then were boiled to completely denature the proteins for 5 min. Aliquots containing 15 μg of protein from each sample tube were subjected to SDS-PAGE (8%, 1.5 mm gel) according to the method of Laemmli [20].
Proteins in the gel were transferred to PVDF membrane in the presence of transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol, pH 8.3). PVDF membrane was washed one time for 5 min with Tris-buffered saline with tween 20 (25 mM Tris-HCl, 140 mM NaCl, 2.7 mM KCl, 0.1% tween 20, pH 7.4), then was blocked with blocking buffer (25 mM Tris-HCl, 140 mM NaCl, 2.7 mM KCl, 0.1% tween 20, 5% skim milk, pH 7.4) for 1 h at room temperature, and subsequently was washed three times for 5 min.
The protein phosphorylation was observed using Western blotting. The dilutions for 1st antibody (anti-phosphor-cPLA2, anti-phosphor-PLCβ3, anti-phosphor-PLCγ2, anti-phosphor-p38 MAPK, anti-phosphor-ERK, anti-p38 MAPK, anti-ERK) and 2nd antibody (anti-rabbit IgG-HRP) were 1:1,000 and 1:10,000, respectively. The membranes were visualized using ECL. Blots were analyzed using the Quantity One, Ver. 4.5 (BioRad, Hercules, CA, USA).
Ex vivo determination of AA release
To investigate the effect on AA release, the aggregation was terminated adding ice-cold 5 mM EDTA and 0.2 mM indomethacin to inhibit subsequent conversion of AA to TXA2, and centrifuged with 200 × g at 4 °C for 10 min. The supernatants were used for the assay of AA release. AA release was measured with a Synergy HT multi-model microplate reader (BioTek Instruments, Winooski, VT, USA) using AA release ELISA kit.
Preparation of platelet lysates
We prepared platelet lysates to determine ex vivo COX-1 and TXAS activities. Collagen- and ADP-activated rat PRP was centrifuged for 10 min at 1,300 × g to remove PPP and get platelet pellets. The platelets were then suspended twice with suspension buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 0.49 mM MgCl2, 5.5 mM glucose, 0.25% gelatin, pH 7.4). The suspended platelets in the presence of 1% protease inhibitor cocktail were sonicated ten times in sensitivity 100% for 20 s at 4 °C with a sonicator (HD 2070, Bandelin Electronic, Bandelin, Germany) to obtain platelet lysates. Next, the platelet lysates were centrifuged at 12,000 × g for 15 min at 4 °C to remove cell debris. The supernatant was used to measure COX-1 and TXAS activity.
Ex vivo measurement of COX-1 activity
Platelet lysates containing 10 μg of protein were used. COX-1 activity was measured with a Synergy HT multi-model microplate reader (BioTek Instruments, Winooski, VT, USA) using COX-1 fluorescent activity assay kit according to the procedure described by manufacturer.
Ex vivo measurement of TXAS activity
Platelet lysates containing 20 μg of protein were used. The reaction for assay of TXAS activity was initiated by the addition of TXAS substrate PGH2 and allowed to proceed for 1 min at 37 °C. The reaction was terminated by the addition of 1 M citric acid, then was neutralized with 1 N NaOH. The concentration of TXA2 was determined as TXB2, a stable metabolite of TXA2, which was measured with a Synergy HT multi-model microplate reader (BioTek Instruments, Winooski, VT, USA) using TXB2 EIA kit.
Ex vivo determination of serotonin release
To investigate the effect on serotonin release, the aggregation was centrifuged at 4 °C for 10 min at 200 × g. The supernatants were used for the assay of serotonin release. Serotonin release was measured with a Synergy HT multi-model microplate reader (BioTek Instruments, Winooski, VT, USA) using serotonin ELISA kit.
Ex vivo measurement of PT and APTT
To investigate whether WIB-801CE shows anticoagulant characteristics, if any, has bleeding risk as side effect of anticoagulant [21], we measured PT and APTT, markers of blood coagulation. The PPP (100 μL) was preincubated in a two-channel coagulator (Behnk Elektronik GmbH & Co., KG, Norderstedt, Germany) cup (catalog number 95-662, BioMérieux, Marcyl’Etoile, France) with gentle stirring for 1 min at 37 °C. PT was determined as the time interval between the addition of PT reagent (100 μL) to the PPP and the formation of a fibrin clot. After preincubation of PPP for APTT measurement, 100 μL of APTT reagent was added to the PPP (100 μL) and incubated for 3 min at 37 °C. Following incubation, 100 μL of 25 mM CaCl2 was immediately added to the PPP containing APTT reagent. APTT was determined as the time required to form a fibrin clot.
In vivo tail bleeding time assay
We investigated whether WIB-801CE has bleeding risk, the side effect of antiplatelet substance [21]. WIB-801CE (200, 400 mg/kg-BW) and aspirin (100 mg/kg-BW), a positive control, were orally administered to mice once a day for five days. In this study, we used mice for measuring tail bleeding time according to the method of Kim and Lee [22]. After 5 min of the respective final administration, mice were anesthetized with zoletil (40 mg/kg, i.p.). The distal 0.5 cm segment of the tail was transected with operating knife, and immediately immersed in a tube containing 37 °C of saline. Tail bleeding time was determined as the time required to cause blood coagulation.
In vivo evaluation of anti-acute pulmonary thromboembolism
To confirm the endogenous antithrombotic effect, we used a mice model to generate acute pulmonary thromboembolism [23]. Mice were orally administered with WIB-801CE (200, 400 mg/kg-BW), and aspirin (100 mg/kg-BW). After 1 h of respective administration, the mixture (100 μL) of collagen (300 μg/kg-BW) plus epinephrine (30 μg/kg-BW) were injected via tail vein, and the rate of protection and mortality was observed for 15 min, which were calculated as follow: 1) Protection rate (%) = [(Number of tested mice – Number of dead mice)/Number of tested mice] × 100. 2) Mortality rate (%) = [Number of dead mice/Number of tested mice] × 100. These experiments were proved by the Ethics Committee for animal experiments of Whanin Pharmaceutical Corporation (Suwon, Korea/15-NE-009).
In vitro assay of platelet-mediated fibrin clot retraction
We investigated whether WIB-801CE or cordycepin inhibits fibrin clot retraction, an index of thrombi formation [24]. Human PRP 250 μL (108 platelets/mL) were transferred into polyethylene tube to avoid clot adherence, then were preincubated with or without WIB-801CE or cordycepin for 10 min at 37 °C, and subsequently stimulated with thrombin (0.5 U/mL) for 60 min at 37 °C. Pictures of fibrin clot were taken at 0 and 60 min using a digital camera, and its quantification was carried out by measurement of clot area using the NIH Image J Software (V1.46, National Institutes of Health, USA). Percentage of clot retraction was calculated as follows: Retraction (%) by thrombin = [1 - (final clot area/initial clot area)] × 100.
In vitro nitric oxide assay
To observe antiinflammatory effect of WIB-801CE, we used mouse leukemic macrophage RAW264.7 cells. RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and were maintained at 37 °C in 5% CO2 and 95% air in Dulbecco’s Modified Eagle’s Medium (GE Healthcare, Marlborough, USA) containing 10% fetal bovine serum, and 1% penicillin-streptomycin solution. RAW264.7 cells (5 × 104 cells) were preincubated for 30 min with or without WIB-801CE, or inducible nitric oxide synthase (iNOS) inhibitor amino guanidine (AG), and stimulated for 24 h by lipopolysaccharide (10 ng/mL). The supernatant was used for NO assay using Griess reagent. Equal volume of culture supernatant (80 μL) and Griess reagent (80 μL) were mixed. The absorbance of the mixture was measured at 540 nm using spectrophotometer (Spectramax 190, Molecular devices, LLC., Sunnyvale, CA, USA). Nitrite was used as standard of NO.
Ex vivo nitric oxide assay
To investigate the NO production, we used collagen- and ADP-stimulated PRP obtained from rats administered with the WIB-801CE (200, 400 mg/kg-BW). The PRP was centrifuged at 4 °C for 10 min at 10,000 × g to get plasma. The plasma was incubated 1 h with 400 μL methanol:diethylether (3:1 mixture v/v), and subsequently plasma proteins were precipitated by centrifuging at 4 °C for 10 min at 10,000 × g. The supernatant was used for NO assay using Griess reagent. Equal volume of supernatant (80 μL) and Griess reagent (80 μL) were mixed. After 30 min, absorbance of the mixture was measured at 540 nm using a Synergy HT multi-model microplate reader (BioTek Instruments, Winooski, VT, USA). Nitrite was used as standard of NO.
In vitro determination of antioxidant activity
To obtain antioxidant effect of WIB-801CE, we measured scavenging activity of free radical in DPPH according to the method [25, 26]. DPPH was dissolved in 99% ethanol to make 200 μM of solution. WIB-801CE and antioxidant AC were dissolved in distilled water. Equal volume of test substances and DPPH were mixed at room temperature. After 30 min, the reduction in DPPH absorbance at 517 nm was measured using spectrophotometer (Optizen 2120UV, Mecasys, Korea). The scavenging activity of DPPH radicals by substances was determined using the following equation [26]: Scavenging activity (%) = [1 – (Asample/ADPPH)] × 100. The absorbance at 517 nm by 99% ethanol, DPPH vehicle, and distilled water, vehicles of WIB-801CE and AC was 0.001 and 0.000.
Ex vivo measurement of cordycepin effect on rat platelet aggregation
This experiment was performed to investigate the effect of cordycepin on ex vivo platelet aggregation. When cordycepin (15 mg/kg per day) was administered orally to the mice for 14 days, antitumor activity was known to observe [27]. Therefore, we selected 5 and 10 mg/kg-BW per day of cordycepin in this experiment as moderate doses for administration. These doses are corresponded to about 36 and 72% of cordycepin in WIB-801CE (200 mg/kg-BW) that inhibited ex vivo rat platelet aggregation. Rats (Sprague-Dawley, male, 200 g) were acclimatized for a week at a temperature of 24 ± 1 °C and humidity of 55 ± 5%. Before oral administration of cordycepin, rats were fasted for 12 h, then were fed with standard pellets diet (Purina Inc., Korea) had free access to water. Cordycepin (5 and 10 mg/kg-BW) was orally administered to the rats one per day for seven days. Cordycepin were dissolved with distilled water. The experiments were proved by the Ethics Committee for Animal Experiments of Whanin Pharmaceutical Corporation (Suwon, Korea/15-NE-016 for rats). After the final respective administration, all rats were fasted for 24 h, then were anesthetized with 20% urethane before sacrifice. PRP preparation, platelet aggregation, measurement were performed as described before.
Protein assay
To determine COX-1, TXAS activity, and protein phosphorylation, protein concentration was measured using bicinchoninic acid assay kit (Pierce Biotechnology, USA).
Statistical analyses
The experimental results are indicated as the mean ± standard deviation accompanied by the number of observations. Data were determined by analysis of variance (ANOVA). If this analysis showed significant differences among the group means, then each group was compared by the Newman-Keuls method. Statistical analysis was carried out according to the SPSS 21.0.0.0 (SPSS, Chicago, IL, USA). p < 0.05 was considered to be statistically significant.