JOURNAL OF MEDICINAL FOOD J Med Food 18 (1) 2014, 1–10 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.3116

FULL COMMUNICATION

Antiplatelet Effects of Rhus verniciflua Stokes Heartwood and Its Active Constituents—Fisetin, Butein, and Sulfuretin—in Rats Jun-Hyeong Lee,1 Mikyung Kim,1 Kyung-Hwa Chang,1 Cheol Yi Hong,2 Chun-Soo Na,2 Mi-Sook Dong,3 Dongho Lee,3 and Moo-Yeol Lee1 1

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College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do, Korea. Lifetree Biotechnology Institute, Lifetree Bitotech Co. Ltd., Suwon, Gyeonggi-do, Korea. 3 School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.

ABSTRACT Rhus verniciflua stokes (RVS) is known to promote blood circulation by preventing blood stasis, although the active ingredients and the underlying mechanism are unclear. Platelets are the primary cells that regulate circulation and contribute to the development of diverse cardiovascular diseases by aggregation and thrombosis. The study assessed the antiplatelet activity of RVS and sought to identify the active constituents. Pretreatment of washed platelets with RVS heartwood extract blunted the aggregatory response of platelets to collagen. In the subfractions, fisetin, butein, and sulfuretin were identified as effective inhibitors of platelet aggregation by collagen, thrombin, and adenosine-50 -diphosphate. Antiplatelet activities of all three compounds were concentration dependent, and fisetin had longer in vitro duration of action compared with butein or sulfuretin. Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase activation by collagen was prevented by fisetin, whereas butein and sulfuretin failed to inhibit ERK and p38 activation was not affected by any of the compounds. Rats orally administered 100 mg/(kg$day - 1) fisetin for 7 days were resistant to arterial thrombosis, although total extract of RVS heartwood exhibited little effect at a dose of 1000 mg/(kg$day - 1). RVS heartwood may have cardiovascular protective activity by inhibiting platelet aggregation. The active constituents are fisetin, butein, and sulfuretin, and fisetin is orally effective against thrombosis.

KEY WORDS:  antiplatelet  butein  fisetin  Rhus verniciflua stokes  sulfuretin

activities, considerable attention has been focused on practical medicinal applications of RVS. Although RVS has diverse beneficial effects, daily use requires particular caution because of its toxicity, which is mainly attributed to urushiol, an oily organic compound. Urushiol is allergenic and causes irritation, inflammation, blisters, and dermatitis in sensitive individuals. Therefore, its use as a pharmaceutical material has been limited.15 Detoxification of RVS extract has been tried in an attempt to overcome the toxicity problem. Strategies have included removal of urushiol or lowering the toxicity by polymerizing urushiol, but these approaches require technical validation.10,15 Urushiol is mainly present in the bark and only a minor amount is detectable in duramen.7 Therefore, use of bark-free heartwood could be a promising way to avoid toxicity, although the biological activities of bark and heartwood may not be the same. Platelets are cells that have a primary role in hemostasis under normal physiological conditions through aggregation. However, dysregulation of platelet activity, which is mainly caused by alteration in the vasculature microenvironment, leads to thrombosis. Abnormal activation or hyperaggregation of platelets is involved in atherosclerosis and inflammation, which contributes to the development of acute coronary syndrome, stroke, and the ischemic complications of peripheral

INTRODUCTION

R

hus verniciflua stokes (RVS) is a species in the family Anacardiaceae that grows in East Asian countries, including Korea, China, and Japan. RVS is popularly known as the lacquer tree because it is cultivated and tapped for the toxic sap, which is used as a highly durable lacquer to make Korean lacquerware. It is also known as poison ivy, reflecting its toxicity. In Western countries, little attention has been paid to practical use of RVS. However, in addition to application as a lacquer, RVS has been used as an ingredient of traditional herbal medicine in Korea.1–3 Reported diverse biological functions of RVS include antioxidation,4 anti-inflammation,2,5 antitumorigenic,6 antimutagenic,7 antimicrobial,8,9 and neuroprotection.1,10,11 Along with these biological activities, RVS extract has been linked with cardiovascular protection by improving blood circulation, although the active ingredients and mechanism of action are unclear.3,12–14 Based on these Manuscript received 4 December 2013. Revision accepted 26 August 2014. Address correspondence to: Moo-Yeol Lee, PhD, College of Pharmacy, Dongguk University, 32 Dongguk-ro, Ilsandong-gu, Goyang, Gyeonggi-do 410-820, Republic of Korea, E-mail:[email protected]

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vascular disease. Platelets are critical regulators of blood circulation and thus serve as a primary target for treating atherothrombotic disorders. Antiplatelet therapies play a key role in treating various cardiovascular diseases, such as myocardial infarction, stroke, and atherosclerosis. In this study, we assessed the antiplatelet activity of RVS heartwood extract based on the traditional literature and empirical data, hypothesizing that RVS might contain active substances improving blood circulation.3,14 Employing fractionation methods, subfractions as well as total extract of bark-free heartwood were examined in an attempt to identify the active constituents. Additionally, their mechanisms of action and in vivo relevancy were investigated. MATERIALS AND METHODS Reagents Collagen and adenosine-50 -diphosphate (ADP) were purchased from Chrono-log Corporation (Havertown, PA, USA). Hematologic reagents for prothrombin time (PT) and activated partial thromboplastin time (aPTT) measurements were from Fisher Diagnostics (Middletown, VA, USA). All antibodies, including anti-extracellular signal-regulated kinase (ERK)1/2, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-p38, anti-phospho-p38 (Thr180/Tyr182), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, and anti-mouse IgG, were obtained from Cell Signaling Technology (Beverly, MA, USA). Other chemicals and sources were as follows: thrombin and ferric chloride (FeCl3) (Sigma-Aldrich, St. Louis, MO, USA), U0126 and SB203580 (Merck KGaA, Darmstadt, Germany), pentobarbital sodium (Hanlim Pharm, Seoul, Korea), bicinchoninic acid (BCA) protein assay kits (Pierce Biotechnology, Rockford, IL, USA), and Immobilon Western detection reagents (Millipore, Billerica, MA, USA). All other chemicals used were of the highest purity available and purchased from standard suppliers. Preparation of RVS heartwood extract RVS was harvested in Gangwon-do, Republic of Korea. The bark was removed and the heartwood was air-dried. Wood was fragmented into 11 · 1 · 0.2 cm3 pieces using a wood chip-maker. The wooden chips were extracted for 3 h in hot water (90–100C) and impurities were removed by filtration. The filtered extracts were concentrated to over 15% solid content using a vacuum evaporator and the concentrate was freeze-dried in a lyophilizer. The yield was *4%. The final extract was a dark brown powder and was kept in refrigerator until use. Fustin as the reference material was analyzed in the extract by conventional high-performance liquid chromatography (HPLC) using a Varian 920-LC (Agilent Technologies, Santa Clara, CA, USA) and YMC-Pack ODSA column (4 lm, 150 · 4.6 mm; YMC, Kyoto, Japan) to confirm the consistency of quality. Fustin was an abundant flavonoid in RVS and measured 2.4–3.6% (w/w) in RVS extract. For the experiments, powder was dissolved in distilled water and filtered again through a 0.2-lm membrane filter (Millipore).

Fractionation of RVS heartwood extract The extracted dark brown powder was suspended in H2O and sequentially partitioned with hexane and ethyl acetate (EtOAc) (Fig. 1). The EtOAc extract was subjected to silica gel column chromatography using a chloroform (CHCl3) methanol gradient system (100:1–1:1) as the mobile phase to yield 10 fractions (F1–F10). Fraction F5 was chromatographed on silica gel with a CHCl3 - methanol gradient system (40:1–3:1) to afford 10 subfractions (F5-1–F5-10). F5-7 was precipitated using EtOAc and the residue was further purified by preparative HPLC (YMC Pack ODS-A, 5 lm, 250 · 20 mm i.d., 30–50% methanol in H2O, flow rate 8.0 mL/ min) to afford butein. F6 was chromatographed on silica gel with a hexane-EtOAc gradient system (5:1–0:1) to afford 12 subfractions (F6-1–F6-12). Fraction F6-8 was purified by preparative HPLC (YMC Pack ODS-A, 5 lm, 250 · 20 mm i.d., 30–50% methanol in H2O, flow rate 8.0 mL/min) to afford sulfuretin. F9 was precipitated using CHCl3 to yield fisetin. All compounds were identified on the basis of 1H nuclear magnetic resonance (NMR) spectral data obtained with a VNMRS 500 MHz spectrometer (Agilent Technologies) referring to reference data.16,17 Animals All animal experiments were conducted in accordance with protocols approved by the ethics committee of Animal Service Center at Dongguk University. Male Sprague-Dawley rats (5–6 weeks of age, 130–150 g body weight) were purchased from Daehan Biolink (Eumseong, Korea) and were acclimated for 1 week before experiments. The laboratory animal facility was maintained at a constant temperature and humidity with a 12 h light/dark cycle. Commercial rodent chow (RodFeed; Daehan Biolink) and water were provided ad libitum. Preparation of platelet-rich plasma and washed platelets Platelet-rich plasma (PRP) and washed platelets (WPs) were prepared as described previously.18 Briefly, blood was collected from the abdominal aorta of rats anesthetized with ether using either sodium citrate (3.2% trisodium citrate; for PRP) or acid-citrate-dextrose (ACD; 85 mM trisodium citrate, 66.6 mM citric acid, and 111 mM glucose; for WPs) as anticoagulants (sodium citrate:blood = 1:9, ACD:blood = 1:6). After centrifugation at 250 g for 15 min, PRP was obtained from the supernatant. Platelets were spun down by further centrifugation of PRP at 500 g for 10 min, and were washed once with washing buffer solution (140 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 0.5 mM Na2HPO4, 10 mM NaHCO3, 1 mM CaCl2, 0.55 mM glucose, 22 mM trisodium citrate, and 0.3% bovine serum albumin [pH 6.5]) by suspension and centrifugation. WPs were prepared by resuspending the platelet pellets in suspension buffer solution (134 mM NaCl, 2.9 mM KCl, 1 mM MgCl2, 0.34 mM Na2HPO4, 12 mM NaHCO3, 10 mM HEPES, 1 mM CaCl2, 5 mM glucose, and 0.3% bovine serum albumin [pH 7.4]). The cell density was adjusted to 2 · 108 cells/mL with platelet-poor plasma or suspension buffer solution for PRP or WPs, respectively.

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In vitro platelet aggregation study Platelet aggregation experiments were performed using a four-channel aggregometer (490-X; Chrono-log Corporation) as described previously.18 WPs or PRP was treated with testing materials or their vehicles (distilled water for RVS extract and dimethyl sulfoxide for purified fisetin, butein, and sulfuretin) for the indicated times and platelet aggregation was induced by 2.5 lg/mL collagen, 0.12– 0.14 U/mL thrombin, or 16 lM ADP, which is the minimal concentration inducing submaximal aggregation. Plasma coagulation tests Plasma coagulability was assessed by measuring PT and aPTT as previously described.19 Blood plasma was incubated with testing materials for 10 or 30 min. PT or aPTT was measured with a Coagulator2 coagulation analyzer (Behnk Elektronik, Norderstedt, Germany) using thromboplastin-D or CaCl2 and APTT-XL reagents (Fisher Diagnostics), respectively, according to the manufacturer’s instructions. Assessment of ERK and p38 activation in platelets Activation of ERK1/2 and p38 was assessed by conventional western blot analysis with activation-dependent, phospho-specific antibodies and suitable HRP-conjugated secondary antibodies. To avoid protein loss during membrane stripping, phospho-protein and total protein were detected separately with the same samples in different gels. After treatment of WPs with the testing materials, platelet sediment was obtained by centrifugation at 12,000 g for 2 min. Platelets were lysed using a solution comprised of 50 lM HEPES, 50 lM NaCl, 50 lM sucrose, 1% Triton X-100, protease inhibitor cocktail, and phosphatase inhibitor cocktail. Following centrifugation, platelet lysate was obtained from supernatants and protein contents were quantified with a BCA protein assay kit (Pierce Biotechnology). Platelet lysates were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a polyvinylidene difluoride membrane, immunoreactive proteins were detected with primary antibodies for ERK, phospho-ERK, p38, phospho-p38, and b-actin in combination with HRP-conjugated secondary antibodies and Immobilon Western detection reagents (Millipore) as described previously.20 Chemiluminescence images were obtained and analyzed with a ChemiDoc XRS + system and Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).

artery was exposed and dissected free of nerves and connective tissue. An ultrasonic flowprobe MA0.7PSB (Transonic Systems, Ithaca, NY, USA) was placed around the arterial segment and was connected to a TS420 perivascular flowmeter module (Transonic Systems) to monitor blood flow. To induce thrombosis, filter paper (1 · 1 mm2, Whatman No. 1) was saturated with 50% FeCl3 and applied to the external surface of the femoral artery segment proximal to the flow probe 2 h after RVS extract or fisetin administration. The time needed for occlusion to occur was measured for up to 90 min, and occlusion time was assigned a value of 90 min for vessels that did not occlude within 90 min. As a positive control, clopidogrel was injected intraperitoneally at a dose of 30 mg/kg. Statistical analyses The mean and standard error of the mean were calculated for all experimental groups. The data were subjected to oneway analysis of variance (ANOVA) followed by Dunnett’s test to determine significant differences from the control. For the comparison of IC50 in Table 1, Duncan’s multiple-range test was employed. The half-maximal inhibitory concentration (IC50) was calculated by linear regression analysis. Statistical analyses were performed using SigmaStat Version 3.5 (Systat Software, San Jose, CA, USA). In all cases, a P-value < .05 was used to determine significance. RESULTS Antiplatelet effect of RVS heartwood extract and its subfractions RVS heartwood extract and its subfractions were tested for their ability to inhibit aggregation. Treatment with total extract resulted in a significant decrease in aggregation by collagen, indicating the antiplatelet activity of RVS heartwood (data not shown). RVS heartwood extract was further fractionated and tested to isolate and identify the active constituents (Fig. 1). A similar result was obtained using EtOAc partitioning (Figs. 1 and 2), but hexane partitioning failed to inhibit platelet aggregation (data not shown). The material obtained by EtOAc partitioning inhibited platelet aggregation induced by all agonists tested, including collagen, thrombin, and ADP. The ranges of effective concentration were broad—from 0.1 to 1000 mg/mL—in aggregation induced by 2.5 lg/mL collagen Table 1. IC50 Values for Fisetin, Butein, and Sulfuretin on Platelet Aggregation Induced by Collagen, Thrombin, and Adenosine-50 -Diphosphate

In vivo arterial thrombosis study In vivo antiplatelet effects were evaluated in an arterial thrombosis animal model as described previously with slight modifications.21 Briefly, rats were orally administered RVS extract, fisetin, or an equal volume of vehicle (water) for 7 days. Ninety minutes after the final administration, anesthesia was induced with an intraperitoneal injection of 50 mg/kg sodium pentobarbital. Approximately 10 mm of the right femoral

Fisetin Butein Sulfuretin

Collagen

Thrombin

ADP

46 – 3 19 – 1 44 – 11

226 – 14* 331 – 7* 238 – 29*

48 – 5 34 – 5 41 – 9

Values are mean – standard error (n = 3–4) and unit is lM. *Significantly different from IC50 values obtained from collagen or ADP experiment (P < .05). ADP, adenosine-50 -diphosphate.

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FIG. 1. Overall scheme for extraction and purification of antiplatelet constituents from RVS heartwood. The bold font indicates the fraction possessing antiaggregation activity. C.C., column chromatography; EtOAc, ethyl acetate; H, hexane; MeOH, methanol; Ppt, precipitation; RVS, Rhus verniciflua stokes.

or 16 lM ADP (Fig. 2, left panel, and black circles and white squares of the right panel). In contrast, the EtOAc-partitioned material was effective in a relatively narrow range, 1.5–2.5 mg/ mL against 0.12–0.14 U/mL thrombin-mediated aggregation (Fig. 2, right panel, white circles). The concentration–response curve was steeper compared with the cases of collagen and ADP-induced aggregation. To identify effective constituents, the EtOAc partition was further fractionated; F5, F6, and F9 were effective at 1 mg/mL concentration (Fig. 1). In the subsequent experiments, subfractions F5-7 and F6-8 exhibited antiplatelet activity (Fig. 1). From F9, F5-7, and F6-8, three single compounds were purified, and all exhibited a potent antiaggregation effect. NMR spectral data identified F9, F5-7, and F6-8 as fisetin, butein, and sulfuretin, respectively (Supplementary Figs S1–S3; Supplementary Data are available online at www.liebertpub.com/jmf).16,17 Their structures are shown in Figure 3; the presence of these compounds in RVS has been reported previously.22

FIG. 2. Antiplatelet effect of the EtOAc partition. WPs were treated with the indicated concentrations of EtOAc partition for 5 min. Aggregation was induced by the application of 2.5 lg/mL collagen, 0.12– 0.14 U/mL thrombin, or 16 lM ADP. Representative tracings are presented among three repeated collagen experiments in the left panel. Arrowhead indicates the application of collagen. Aggregation inhibitory effect against various platelet agonists—thrombin, collagen, and ADP—is plotted in the right panel. Values are mean – standard error (n = 3). ADP, adenosine-50 -diphosphate; WPs, washed platelets.

Ineffectiveness of RVS heartwood against plasma coagulation To test whether RVS heartwood extract affected plasma coagulation, PT and aPTT were assessed with plasma treated with RVS heartwood extract or its subfractions for 10 or 30 min. PT and aPTT are the representative indicators measuring the activities of contact activation pathway and tissue factor pathway, respectively.23 All extracts and subfractions failed to prolong PT and aPTT, indicating that RVS heartwood does not have any effect on plasma clotting (data not shown). Aggregation inhibitory effect of fisetin, butein, and sulfuretin Antiaggregation effects were tested with purified fisetin, butein, and sulfuretin in WPs. Following the treatment of platelets with each compound for 5 min, aggregation was evoked by collagen, thrombin, or ADP. Fisetin pretreatment resulted in a significant decrease in aggregation elicited by collagen, which was concentration dependent in the range of 10–100 lM (Fig. 4, left panel, and black circles of the right panel). Similar inhibitory effect was observed in aggregation induced by thrombin or ADP (Fig. 4, right panel). However, higher concentrations of 100–350 lM were required to inhibit thrombin-induced aggregation (Fig. 4, right panel, open circles). Comparable results were obtained with butein and sulfuretin (Fig. 5). Both compounds prevented platelet aggregation induced by all agonists in a concentration-dependent manner. The calculated IC50 values are presented in Table 1. Fisetin, butein, and sulfuretin exhibited relatively higher IC50 values in thrombininduced aggregation, compared with the aggregation by collagen or ADP, suggesting that they are more effective against aggregation by collagen and ADP than thrombin. Antiaggregation effect of all these compounds was also observed in PRP as well as WPs (Supplementary Fig. S4).

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FIG. 4. Inhibitory effect of fisetin on platelet aggregation induced by collagen, thrombin, or ADP. WPs were treated with the indicated concentrations of fisetin for 5 min and aggregation was elicited by collagen, thrombin, or ADP. Representative tracings among three independent experiments are presented in the left panel. The arrowhead indicates the application of collagen. Antiaggregation effects against thrombin, collagen, or ADP are plotted in the right panel. Values are mean – standard error (n = 4).

MAPK inhibition, the effect of fisetin, butein, and sulfuretin on ERK1/2 and p38 activations was examined. After incubating with 100 lM fisetin, 25 lM butein, or 100 lM sulfuretin for 5 min, WPs were treated with 2.5 lg/mL collagen for 5 min. As previously reported,24 collagen stimulated ERK2 (p42 MAPK, lower bands in doublets) and p38, evidenced by the increases in phospho-ERK2 and phosphop38, respectively (Fig. 7A). Fisetin significantly suppressed collagen-stimulated ERK2 phosphorylation, but did not prevent p38 activation. Both butein and sulfuretin exhibited little, if any, effect on the activation of both ERK2 and p38. Lower concentrations of fisetin were tested, since 100 lM of fisetin almost completely prevented ERK2 activation. Inhibition of ERK2 was well correlated with the concentration tested in a range of 25–100 lM (Fig. 7B), which was also in agreement with the antiaggregation activity of fisetin (Fig. 4). To obtain the internal and loading controls, total ERK or p38 MAPK and b-actin levels were detected on duplicate blots. U0126 and SB203580 were used as positive controls for ERK kinase and p38 MAPK inhibition, respectively. FIG. 3.

Chemical structures of fisetin, butein, and sulfuretin.

Duration of antiplatelet activity In addition to concentration dependency, pretreatment time dependency was examined with regard to antiplatelet activity. The tested compounds were used to treat platelets for 2, 5, 10, or 30 min and aggregation was elicited by collagen. Fisetin blunted the aggregatory response of platelet consistently up to 30 min at 100 lM (Fig. 6). Both 25 lM butein and 100 lM sulfuretin efficiently suppressed the aggregation of platelets treated for 2 and 5 min. Unexpectedly, such an aggregation inhibitory effect was significantly attenuated as pretreatment time increased (Fig. 6), indicating the short duration of action and the loss of efficacy during longer exposure to butein and sulfuretin. Effect on ERK and p38 signaling in platelet aggregation To test whether antiplatelet activity was related to ERK1/ 2 [p44/42 mitogen-activated protein kinase (MAPK)] or p38

In vivo antithrombotic effect of RVS heartwood extract and fisetin The antiaggregation effect of RVS heartwood extract and fisetin observed in the in vitro experiments was tested in an animal model to confirm in vivo relevancy. RVS extract or fisetin was orally administered to rats for 7 days and arterial thrombosis was induced 2 h after the last administration by applying FeCl3 to the femoral artery. Administration of 100 mg/(kg$day - 1) fisetin significantly prolonged the time needed for arterial occlusion to form from 15.8 – 2.4 to 44.7 – 11.3 min. This result suggested the capability of fisetin to prevent thrombus formation caused by vascular injury (Fig. 8). In rats treated with 1000 mg/(kg$day - 1) extract, the arterial occlusion time was 12.9 – 1.0 min, which was not different from that of control, indicating a minimal effect of extract under this experimental condition. Clopidogrel, an antiplatelet drug, was used as a positive control and was confirmed to inhibit thrombosis significantly at 30 mg/kg.

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FIG. 5. Antiaggregation effect of butein and sulfuretin. After a 5-min treatment with (A) butein or (B) sulfuretin, aggregation was induced by platelet agonists collagen, thrombin, and ADP. Aggregation inhibition against thrombin, collagen, or ADP is presented in each panel. Values are mean – standard error (n = 3).

DISCUSSION This study investigated the antiplatelet effect of RVS heartwood based on the traditional use and empirical data describing a cardiovascular protective effect of RVS. To avoid a safety issue, bark-free heartwood was examined to avoid the

FIG. 6. Time-dependent effect of fisetin, butein, or sulfuretin on platelet aggregation. WPs were treated with 100 lM fisetin, 25 lM butein, or 100 lM sulfuretin for 2, 5, 10, or 30 min, and aggregation was induced by the application of collagen. Values are mean – standard error (n = 3). *Significantly different from 2-min pretreatment groups (P < .05).

toxicity problem of urushiol, which is present mainly in bark. Total extract of RVS heartwood blunted the aggregatory response of platelets to agonists. Activity-guided fractionation was employed in the search of active components. Fisetin, butein, and sulfuretin were found to possess antiaggregatory activity. Fisetin had a longer duration of action than butein or sulfuretin, and inhibition of ERK activation appeared to be involved in the antiaggregation effect of fisetin, although the mechanism of action was not fully elucidated. In vivo animal experiments confirmed that fisetin was orally effective, which was evidenced by a reduction of arterial thrombosis in animals receiving fisetin. These results are in agreement with traditionally described efficacy of RVS and provide a theoretical basis for the practical use of RVS to protect the cardiovascular system, although clinical validation remains to be done.3,14 There is a tremendous need for antiplatelet drugs, and several classes of medicine are clinically available. Antiplatelet drugs are used for preventive purposes in most cases, and accordingly need long-term use. Therefore, natural products originating from plants are desirable as a source for such pharmaceuticals if the absence of toxicity or side effects has been guaranteed empirically. In this context, bark-free heartwood of RVS is an attractive source of antiplatelet medication. Bark-extract RVS has been tested for its antiplatelet activity.13 The EtOAc extract contained active compounds, which proved to be isomaltol and pentagalloyl glucose. IC50 values for isomaltol and pentagalloyl glucose were 3.1 mM

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FIG. 7. Effect of fisetin, butein, and sulfuretin on ERK and p38 activation by collagen. (A) After incubating with 100 lM fisetin, 25 lM butein, or 100 lM sulfuretin for 5 min, WPs were treated with 2.5 lg/mL collagen for 5 min. Platelet lysates were obtained and subjected to western blot analysis. Activation of ERK and p38 was assessed with activationdependent, phospho-specific antibodies and suitable horseradish-peroxidase-conjugated secondary antibodies. Representative images from three independent experiments were presented. (B) Concentration-dependent effect of fisetin was tested for ERK inhibition. Representative images (left panel) and relative band densities of phospho-ERK against total ERK (right panel) are presented. Values are mean – standard error (n = 3). *Significantly different from control, collagen-only group (P < .05). ERK, extracellular signal-regulated kinase.

FIG. 8. Effect of RVS extract and fisetin on arterial thrombosis. RVS extract or fisetin was administered to rats for 7 days at a dose of 1.0 g/(kg$day - 1) or 100 mg/(kg$day - 1), respectively. Vascular occlusion was induced after 2 h of final administration by applying FeCl3 to the femoral artery. Blood flow was monitored by ultrasonic flowmetry. The positive control was treated with 30 mg/kg clopidogrel by intraperitoneal injection. Values are mean – standard error (n = 7, except n = 4 for clopidogrel-treated group). *Significantly different from control (P < .05).

and 160 lM in collagen-induced aggregation, respectively, and comparable values were obtained with ADP or arachidonic acid. Overall, these values are higher, and thus efficacy appears to be lower, compared with the fisetin, butein, or sulfuretin in our study (Table 1). Isomaltol and pentagalloyl glucose were not detected in RVS heartwood in our whole and extensive fractionation analysis (unpublished data). In contrast, RVS bark contains fisetin, butein, and sulfuretin, but Jeon et al. failed to detect antiplatelet activity with the fractions containing them. This may be attributed to the trace amounts of these compounds in fractions or the concentration of the fractions that they tested. There was not-so-dramatic a difference in the quantity of butein and sulfuretin, but the amount of fisetin was quite different between bark and heartwood. The amount of fisetin was more than 1.6% in the EtOAc partition of heartwood (Fig. 1), while comprising only 0.2% of the EtOAc fraction of bark.13 It must be noted that the extraction methods were not exactly the same, limiting direct comparison between the studies. In summary, active constituents are distinctly different between heartwood and bark, although both heartwood and bark possess antiplatelet activity. Heartwood appears to have higher efficacy than bark, considering the amount and in vitro efficacy of active compounds. Fisetin and sulfuretin are flavonoids and butein is a chalconoid that is structurally close to flavonoids (Fig. 3). They are well known for their antioxidant and anti-inflammatory activities, although their clinical relevancy is elusive.25–28 Butein and sulfuretin have never been tested for an effect on

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platelets. This is the first study that reports their antiplatelet activity. One of the remaining questions is the loss of activity. Antiplatelet effects of butein and sulfuretin in WPs gradually decreased with time (Fig. 6). Loss of biological activity is generally related to a metabolic change of parent chemicals. WPs are a simple experimental system composed of only platelets, which has nothing to do with systemic metabolism. Platelets were once thought not to develop metabolic systems such as cytochrome P450 well. However, it was recently reported that platelets possess a metabolic system for flavonoids and that such metabolism may influence the activity of flavonoids, raising the possibility of metabolic inactivation of butein and sulfuretin in platelets.29 There may be other mechanisms underlying a short duration of action, such as a desensitization of cell signaling affected by butein or sulfuretin. Duration of action is a critical factor determining efficacy, and a short duration of action may be disadvantageous. Further study will be needed to clarify the reason for inactivation and to assess in vivo efficacy. Together with other flavonoids with 3,7,40 -trihydroxyflavone structure, such as quercetin and kaempferol, fisetin has been tested for its aggregation inhibitory effect in vitro.30 Fisetin was effective in limiting aggregation by agonists, including arachidonic acid and ADP. The reported effective concentration (several tens of lM) is comparable to this study, even though results were from rabbit platelets (Fig. 4). Our study tested the in vivo effect by employing an arterial thrombosis animal model. Mice fed with fisetin for 1 week were resistant to thrombosis, whereas total extract of RVS heartwood exhibited a minimal effect (Fig. 8). One of the difficulties in this experiment was the determination of doses in the absence of pharmacokinetic data. Hence, a relatively large amount, 100 mg/(kg$day - 1) or 1000 mg/(kg$day - 1), was administered to mice for fisetin and total extract, respectively. Total extract may have more complicate activities in in vivo and unlimited number of physiological factors affect in vivo effects. To observe a positive result with total extract, the treatment schedule needs fine-tuning. The in vivo effect of RVS heartwood deserves further study. Molecular targets of fisetin, butein, and sulfuretin were not identified clearly in this study. These constituents affect diverse signaling pathways related with inflammation, cellular growth, and apoptosis.31–33 However, most of the signals are not relevant to platelets, because platelets lack a nucleus and transcriptional activity, and they are not proliferative. Through the thorough literature search, MAPKs were hypothesized as relevant and potential targets for antiaggregation activity. MAPKs are a family of serine-threonine kinase activated by many extracellular stimuli, including growth factors and hormones. They are versatile signaling molecules mediating diverse cellular functions.24,34 MAPKs are mainly involved in growth and inflammation, but are also related with platelet aggregation. MAPKs are comprised of three families, including ERK1/2, p38, and c-Jun N-terminal kinase, and platelets express all three types of MAPKs. Among them, ERK2 and p38 positively contribute to aggregation.24,34–36 Although all the downstream signaling pathways of these MAPKs were not identified, protein kinase C and phospholipase A2 are known to be ef-

fectors of MAPKs in platelets. As a possible mechanism, the effect on ERK2 and p38 was examined; fisetin suppressed ERK2 activation by collagen (Fig. 7). Indeed, similar ERK inhibition by fisetin has been reported in osteoclasts and synovial cells.37,38 The overall effect of fisetin on MAPKs is controversial because both activation and inhibition have been observed in diverse cells.37–41 Therefore, it appears to be dependent on the cell type.42 In platelets, fisetin inhibits ERK2 or its upstream molecules, which may be targets of fisetin. Oral administration of 100 mg/(kg$day - 1) fisetin for a week was effective against thrombosis, while RVS extract exhibited a marginal efficacy. Currently, it is not clear why RVS extract showed negligible antithrombotic activity. According to this study, fisetin is *0.7% of RVS extract (Fig. 1). Therefore, the content of an active constituent, fisetin, may not be enough to have efficacy. Numerically, 100 mg/kg fisetin is equal to 14.2 g/kg RVS, which appears to be an impractical dose. However, there are a number of important points that should be considered. (1) This study was focused on the qualitative rather than quantitative observations. Accordingly, a relatively high dose was tested to assure potential efficacy, and lower dose efficacy was not examined; however, fisetin is effective in lower doses. The dose of RVS required for its efficacy will be much less than the amounts used in this study. (2) Pharmacokinetic information for RVS and fisetin is not available, especially for long-term or repeated administration. If longer and morerepeated administration results in cumulative effects, then the efficacy may be higher than single or short exposure. (3) In addition to fisetin, other constituents, such as butein and sulfuretin, may be effective. Then, the effective dose of RVS extract may be much lower than the numerical calculation based on only fisetin. (4) Different from drug, RVS is frequently consumed intentionally as food. Thus, larger amount than drug can be administered for longer periods. Taken together, the function of RVS may be stronger than simple calculation and further study will be needed to validate the clinical relevancy of RVS uptake. One of the notable characteristics of fisetin is fluorescence behavior. When fisetin is present with protein such as albumin, its interaction with protein generates strong fluorescence signals over wide ranges of wavelength.43,44 This property practically limits a wide range of assays utilizing fluorescence signals, such as fluorescence-activated cell sorting and calcium measurement using fluorescent indicators. To investigate the mechanism underlying antiaggregation effects, calcium signals or secretion will need to be assessed in platelets. However, such studies are not feasible due to an interference of fluorescence signal by fisetin. Experiments based on fluorescence should be performed with caution when testing fisetin.

ACKNOWLEDGMENTS This research was supported by Industrialization Support Program for Bio-technology of Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

ANTIPLATELET EFFECT OF RHUS VERNICIFLUA STOKES

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