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Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, Wasabi
Mechanisms of Ageing and Development 116 (2000) 125 – 134 www.elsevier.com/locate/mechagedev
Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, Wasabi Yasujiro Morimitsu a,*, Kazuhiro Hayashi a, Yoko Nakagawa a, Hiroyuki Fujii a, Fumihiko Horio b, Koji Uchida a, Toshihiko Osawa a a Laboratory of Food and Biodynamics, Nagoya Uni6ersity Graduate School of Bioagricultural Sciences, Nagoya 464 -8601, Japan b Di6ision of Biomodeling, Nagoya Uni6ersity Graduate School of Bioagricultural Sciences, Nagoya 464 -8601, Japan
Received 3 December 1999; accepted 24 March 2000
Abstract 6-Methylsulfinylhexyl isothiocyanate (MS-ITC) was isolated from wasabi (Wasabia japonica, Japanese domestic horseradish) as a potential inhibitor of human platelet aggregation in vitro through our extensive screening of vegetables and fruits. In the course of an another screening for the induction of glutathione S-transferase (GST) activity in RL34 cells, MS-ITC was inadvertently isolated from wasabi as a potential inducer of GST. MS-ITC administered to rats or mice also showed both activities in vivo. As a result from elucidation of the platelet aggregation inhibition and the GST induction mechanisms of MS-ITC, the isothiocyanate moiety of MS-ITC plays an important role for antiplatelet and anticancer activities because of its high reactivity with sulfhydryl (RSH) groups in biomolecules (GSH, cysteine residue in a certain protein, etc.). © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Isothiocyanate; Wasabi; Platelet aggregation; Phase II enzyme; Glutathione S-transferase; Cancer chemoprevention
* Corresponding author. Present address: Department of Nutrition and Food Science, Laboratory of Food Chemistry, Ochanomizu University, Otsuka 2-1-1, Bunkyo-ku, Tokyo 112-8610, Japan. Tel./fax: + 81-3-59785757. E-mail address: [email protected] (Y. Morimitsu). 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 1 4 - 7
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1. Introduction Glucosinolates are found in cell vacuoles of various pants in the family Cruciferae such as horseradish, mustard, broccoli, and wasabi (Wasabia japonica, syn. Eutrema wasabi ). When plant cells are damaged, glucosinolates are hydrolyzed by myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) which is also produced in the same family, and produce isothiocyanates (Fig. 1). Most of these Crucifer isothiocyanates (cf. allyl isothiocyanate) are well known for having antimicrobial, fungicidal, and pesticidal activities (Soledade et al., 1998; Tajima et al., 1998). A major wasabi flavor compound, 6-methylthiohexyl isothiocyanate (MS-ITC) (n= 6) termed ‘green note’ flavor, has been shown the antimicrobial and platelet aggregation inhibition activities (Morimitsu et al., personal communication; Kumagai et al. 1994). On the other hand, epidemiological studies have demonstrated that consumption of cruciferous vegetables is associated with a lower incidence of cancers (Lee et al., 1989; Olsen et al., 1991; Mehta et al., 1995). Induction of phase II enzymes such as the glutathione S-transferase (GST) and/or quinone reductase (QR) have been demonstrated in broccoli (Aspry and Bjeldanes, 1983; Fahey et al., 1997), cabbage (Whitty and Bjeldanes, 1987), and Brussel sprouts (Nijhoff et al., 1993; Wallig et al., 1998). Many natural isothiocyanates derived from cruciferous vegetables and some fruits have been shown to induction of phase II enzymes in cultured cells and rodents (Leonard et al., 1981; Yang, et al., 1994; Barcelo et al., 1996). We have studied on physiological, pharmaceutical or therapeutic effects of the Allium genus, onion and garlic, especially for their antithrombotic activity. Our continuous attempt was succeeded in isolation of new sulfur-containing compounds, termed AC series, from the methanol extract of onion, which inhibited human platelet aggregation (Kawakishi and Morimitsu, 1988; Morimitsu and
Fig. 1. The enzymatic formation of isothiocyanates from Brassica sp.
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Kawakishi, 1990). Our screening for potential inhibitors of human platelet aggregation has been extending for daily vegetables and fruits, chiefly the cruciferous vegetables, because the family Cruciferae, except for Allium, is well-known vegetable for containing organosulfur compounds. Finally, the ethyl acetate (EtOAc) extract of wasabi was found to show a potent inhibitory activity for human platelet aggregation in vitro. Incidentally, an another screening for the induction of GST activity in RL34 cells resulted in finding the EtOAc extract of wasabi. In this study, we describe the results of two individual screenings, isolation and identification of MS-ITC from wasabi. Furthermore, a proposed mechanism for both the platelet aggregation inhibition and the GST induction by MS-ITC was discussed.
2. Materials and methods
2.1. Measurement of antiaggregation acti6ity Antiaggregation activity was measured by the reported method (Morimitsu and Kawakishi, 1990) using a dual-channel aggregometer (NKK, Hematracer I). Platelet aggregation was induced by various agonists at minimum concentrations as follows (final conc.): collagen (1 mg/ml), AA (0.5 mM), ADP (10 mM), PAF (5 mM), A23187 (13.2 mM), U46619 (5 mM), STA2 (5 mM), PMA (25 mM), OAG (200 mM) and thrombin (0.5 U/ml).
2.2. Inhibitory assay for PG biosynthesis The preparation of rabbit renal microsomes and the inhibitory assay were carried out using the reported procedure (Sankawa et al., 1982) with a slight modification. The reaction mixture (0.2 ml) contained 0.1 M sodium phosphate buffer (pH 7.5), 10mM tryptophan, 0.25 mM hemoglobin, 4.0 mM reduced glutathione, 20 mM [1-14C] arachidonic acid (1.35 mCi), rabbit renal microsomes (100 mg protein) and a test sample (DMSO solution). The reaction (37°C, 20 min) was started by the addition of microsomes and terminated by the addition of 1 N HCl (50 ml). The reaction mixture was then extracted with ether. After the ether was removed, the residue was dissolved in acetone and applied to a TLC plate. Following the TLC plate was developed with a solvent mixture [CHCl3/MeOH/AcOH = 90/5/5 (v/v)], radioactivities of PGE2, PGF2a and arachidonic acid were measured by an imaging plate reader (Bio-Image Analyser, FUJIX BA-100).
2.3. Measurement of the GST induction acti6ity in RL34 cells The GST induction Activity in RL34 cells was measured by using the conditions reported previously (Fukuda et al., 1997; Uchida et al., 1999).
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Fig. 2. The chemical structures of platelet aggregation inhibitors, which were also identified as potential inducers of GST in RL34, cell.
3. Results
3.1. The result of screening for platelet aggregation inhibitors and the isolation of MS-ITC from wasabi Our screening for searching platelet aggregation inhibitors was undergone on many extracts of various vegetables and fruits. Each extract (less than 100 mg) was added to human platelets (200 ml, 1×108 platelets/ml), and two inducers, arachidonic acid (AA) and ADP, were typically used. In the course of our screening, the EtOAc extracts of cruciferous vegetables were found to show potent inhibitory activities. Especially, the EtOAc extracts of wasabi, radish, turnip, and cabbage exhibited highly inhibitory activities. The EtOAc extract of wasabi showed the potent inhibitory activity even though the adding amount of extract was diminished. After continuous fractionations of the EtOAc extract (1.7 g) of wasabi (1.3 kg) by conventional methods (silica gel column chromatography and reversed-phase HPLC), three potent inhibitors of human platelet aggregation were ultimately isolated and identified as known 6-methylsulfinylalkyl isothiocyanates by spectroscopic analyses (Etoh et al., 1990). Their chemical structures were shown in Fig. 2 and their yields were 14 mg, 70 mg (MS-ITC; major), and 11 mg in order of central chain length respectively. The IC50 value of MS-ITC for human platelet aggregation was 21.9 mM, which is stronger than a positive control, aspirin (IC50 value: 458 mM, Table 1).
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Table 1 IC50 values of platelet aggregation inhibitors from wasabi IC50 (mM)
5-Methylsulfinylpentyl isothiocyanate 6-Methylsulfinylhexyl isothiocyanate (MS-ITC) 7-Methylsulfinylheptyl isothiocyanate Aspirin Indomethacin
ADP
AA
168 148 231 – –
20.0 21.9 12.4 458 12.2
3.2. The studies on structure– acti6ity relationship and a possible mechanism for platelet aggregation inhibition of MS-ITC The structure – activity relationship of 6-methylsulfinylalkyl isothiocyanates for human platelet aggregation was examined (Fig. 3). It could be summarized as follows, the oxidized form of sulfur atom was important and the central chain length 6 or 8 was suitable for their activity. Moreover, the isothiocyanate moiety of each MS-ITC derivative was necessary for inhibition of platelet aggregation. After incubation with MS-ITC (5 or 10 min), the treated platelets were washed two times with phosphate buffered saline, and MS-ITC potently inhibited platelet aggregation induced by thrombin (Fig. 4A). Then, MS-ITC could not show any
Fig. 3. The inhibitory effect of 6-methylsulfinylalkyl isothiocyanate derivatives for human platelet aggregation. Each sample was added 25 mM to 200 ml platelets’ solution. Platelet aggregation was induced by ADP (left) or AA (right).
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Fig. 4. The pre-incubation effect of MS-ITC for inhibition of the washed platelet aggregation (left), and the proposed mechanisms of platelet aggregation inhibition (right) by MS-ITC. (A) a. Control; b. no pre-incubation then washed two times; c. after incubation with MS-ITC (100 mM, 5 min), the pre-incubated platelets were washed two times with phosphate buffered saline (pH 7.2). (B) PLA2, using human platelets as a source of this enzyme; PG synthase, using rabbit renal microsomes as a source of this enzyme; TX synthase, using the assay kit from Cayman Chemical.
effects on arachidonic acid cascade (phspholipase A2, PG synthase, and TX synthase) in human platelets using (detail data not shown, Fig. 4B). As above results from studies on the inhibition mechanism of MS-ITC, it could not suppress the enzyme reactions in the platelet arachidonic acid cascade, but was possible to interact with sulfhydryl (SH) group on platelets (including certain receptors of agonists). 95% of MS-ITC underwent conjugation with GSH non-enzymatically to form dithiocarbamate within 60 min at 37°C. We also confirmed that MS-ITC also reacted with thiol groups of BSA and certain proteins on platelets (data not shown). Additional proofs: MS-ITC and its GSH-conjugate were detected in SD rats’ serums and platelets after administration by gavage. And inhibitory effect of MS-ITC on platelet aggregation of SD rats in vivo was confirmed by feeding with MS-ITC (0.05, 0.15 and 0.25%) for 2 weeks.
3.3. The result of screening for the GST induction acti6ity in RL34 cells On the other hand, our another screening for induction of GST activity in RL34 cells was also undergone on many extracts of various vegetables and fruits at the concentration of 25 and 2.5 mg/ml. Similarly the EtOAc extracts of cruciferous vegetables were found to show potent induction activities, especially wasabi and cresson. Finally, MS-ITC and the same 6-methylsulfinylalkyl isothiocyanates were inadvertently isolated from wasabi as a potential inducer of GST (Fig. 5).
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3.4. The studies on structure– acti6ity relationship and a possible mechanism for the GST induction by MS-ITC The structure – activity relationship of 6-methylsulfinylalkyl isothiocyanates for the GST induction was also examined (data not shown). It could be summarized as follows, only the isothiocyanate moiety of each MS-ITC derivative was necessary for their potent GST induction activity. As a result from western blot analysis of GST expression induced by MS-ITC in RL34 cells, increasing of protein levels of both GST-Ya and GST-Yp were detected (data not shown). And as a result from time dependent GSH level by MS-ITC, the GSH level in RL34 cells was initially decreased, then increased gradually to 1.8 times higher than its basal level after 12
Fig. 5. The HPLC profile of wasabi extracts and the GST activities of each fraction. Solid line indicated the HPLC profile. MS-ITC was finally isolated from Fr. 3.
Fig. 6. The time-course of the GSH levels in RL34 cells after adding MS-ITC. MS-ITC was added to RL34 at the concentration of 25 mM. GSH levels were measured by the conventional method.
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h (Fig. 6). As a result from these studies on the GST induction mechanism of MS-ITC, it was possible to interact with sulfhydryl group in RL34 cells (especially, GSH), because of the isothiocyanate’s highly reactivity with nucleophiles. After adding MS-ITC to RL34 cells, slight oxidative stress was immediately occurred, then this stimuli results in signal transductions and gene expressions related to phase II enzymes. Additional proofs: The effect of MS-ITC on levels of phase II enzymes of female ICR mice treated with MS-ITC by gavage in daily dose of 15 mmol for 5 days was examined. The five organs (lung, stomach, small intestine, liver and kidney) of each mouse after treated with MS-ITC were excised, frozen, and stored at − 80°C until analyzed. As a result from determination of GST and QR activities, MS-ITC was found to be a potential inducer of GST and QR in stomach, small intestine and significantly in liver. Especially, MS-ITC enhanced the GST activity in liver, owing to increasing protein levels of Class a GST isozyme (GST-Ya). Furthermore the GST induction activity of MS-ITC in liver was stronger than that of sulforaphane (ca. 1.9 times), a well-known phase II inducer isolated from broccoli (Prochaska and Talalay, 1988).
4. Discussion MS-ITC was isolated from wasabi as a potential inhibitor of human platelet aggregation and a potential inducer of GST in vitro through our extensive screening of vegetables and fruits. MS-ITC also showed the both biological activities in vivo. These biological activities were mainly caused by the isothiocyanate’s highly reactivity with nucleophiles such as GSH, sulfhydryl residues of proteins and so on. Concerning about platelet aggregation inhibition by MS-ITC, the conjugation reaction between isothiocyanate group and thiol groups of certain proteins on platelets resulted in the formation of dithiocarbamates on the surface of platelets so immediately. This protein modification reaction may play an important role for inhibition of binding of agonists to their receptors on platelets. Equally the non-enzymatically conjugation reaction between isothiocyanate group and GSH in RL34 cells at initial step after the addition of MS-ITC resulted in slight GSH depletion. The Michael addition acceptors were reported to be able to induce phase II enzyme activities in vitro (Talalay, 1992). Isothiocyanates belongs in this category. Furthermore, we isolated benzyl isothiocyanate (BITC) from papaya as a potential inducer of GST, and BITC was also decreased the GSH level, then was induced oxidative stress in RL34 cells measured by the flow cytometer (Nakamura et al., 2000). We assume that this slight oxidative stress may lead to increase the protein levels of GST, after induction of signal transductions and gene expressions in cells. Therefore, MS-ITC can be concluded to be a useful phytochemical, possessing antiplatelet and anticancer properties, in Japanese traditional food, wasabi.
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Acknowledgements The authors thank Mr. Inoue et al. (Kinjirushi Wasabi Co., Ltd.) for providing us the Wasabi research samples. This research project was mainly supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
References Aspry, K.E., Bjeldanes, L.F., 1983. Effects of dietary broccoli and butylated hydroxyanisole on liver-mediated metabolism of benzo[a]pyrene. Food Chem. Toxicol. 21, 133 – 142. Barcelo, S., Gardiner, J.M., Gescher, A., Chipman, J.K., 1996. Cyp 2E1-mediated mechanism of anti-genotoxicity of the broccoli constituents sulforaphane. Carcinogenesis 17, 277 – 282. Etoh, H., Nishimura, A., Takasawa, R., Yagi, A., Saito, K., Sakata, K., Kishima, I., Ina, K., 1990. Methylsulfinylalkyl isothiocyanates in wasabi, Wasabia japonica MATSUM. Agric. Biol. Chem. 54, 1587–1591. Fahey, J.W., Zhang, Y., Talalay, P., 1997. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that against chemical carcinogens. Proc. Natl. Acad. Sci. USA 94, 10367 – 10372. Fukuda, A., Nakamura, Y., Ohigashi, H., Osawa, T., Uchida, K., 1997. Cellular response to the redox active lipid peroxidation products: induction of glutathione S-transferase by 4-hydroxy-2-nonenal. Biochem. Biophys. Res. Commun. 236, 505 – 509. Kawakishi, S., Morimitsu, Y., 1988. New inhibitor of platelet aggregation in onion oil. Lancet 1, 330. Kumagai, H., Kashima, N., Seki, T., Sakurai, H., Ishii, K., Ariga, T., 1994. Analysis of volatile compounds in essential oil of upland wasabi and their effects on platelet aggregation. Biosci. Biotech. Biochem. 58, 2131–2135. Lee, H.P., Gourley, L., Duffy, S.W., Esteve, J., Lee, J., Day, N.E., 1989. Colorectal cancer and diet in an Asian population — a case-control study among Singapore Chinese. Int. J. Cancer 434, 1007–1016. Leonard, T.B., Popp, J.A., Graichen, M.E., Dent, J.G., 1981. Naphtylisothiocyanate induced alterations in hepatic drug metabolizing enzymes and liver morphology: implications concerning anticarcinogenesis. Carcinogenesis 2, 473–482. Mehta, B.G., Liu, J., Constantinou, A., Thomas, C.F., Hawthorne, M., You, M., Gerhauser, C., Pezzuto, J.M., Moon, R.C., Moriarty, R.M., 1995. Cancer chemopreventive activity of brassinin, a phytoalexin from cabbage. Carcinogenesis 16, 399 – 404. Morimitsu, Y., Kawakishi, S., 1990. Inhibitors of platelet aggregation from onion. Phytochemistry 29, 3435–3439. Nakamura, Y., Ohigashi, H., Masuda, S., Murakami, A., Morimitsu, Y., Naito, Y., Kawamoto, Y., Osawa, T., Imagawa, M., Uchida, K., 2000. Redox regulation of glutathione S-transferase P1 induction by benzyl isothiocyanate: correlation of enzyme induction with the formation of 2%,7%dichrolofluoresein diacetate-detectable reactive oxygen intermediates. Cancer Res. 60, 219 – 225. Nijhoff, W.A., Groen, G.M., Peters, W.H.M., 1993. Induction of rat hepatic and intestinal glutathione S-transferase and glutathione by dietary naturally occurring anticarcinogens. Int. J. Oncol. 3, 1131–1139. Olsen, G.W., Mandel, J.S., Gibson, R.W., Wattenberg, L.W., Schuman, L.M., 1991. Nutrients and pancreatic cancer: a population-based case-control study. Cancer Causes Contr. 2, 291 – 297. Prochaska, H.J., Talalay, P., 1988. Regulatory mechanisms of monofunctional and bifunctional anticarcinogenic enzyme inducers in murine liver. Cancer Res. 48, 4776 – 4782. Sankawa, M., Pedras, C., Ebizuka, Y., Noguchi, H., Kinoshita, T., Iitaka, Y., 1982. Depside as potent inhibitor of prostaglandin biosynthesis: a new active site nodel for fatty acid cyclooxygenase. Prostaglandins 24, 21–34.
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Soledade, M., Pedras, C., Sorensen, J.L., 1998. Phyoalexin accumulation and antifungal compounds from the Crucifer wasabi. Phytochemistry 49, 1959 – 1965. Tajima, H., Kimoto, H., Taketo, Y., Taketo, A., 1998. Effects of synthetic hydroxy isothiocyanates on microbial systems. Biosci. Biotechnol. Biochem. 62, 491 – 495. Talalay, P., 1992. The role of enzyme induction in protection against carcinogenesis. In: Watttenberg, L., Lipkin, M., Boone, C.W., Kelloff, G.J. (Eds.), Cancer Chemoprevention. CRC Press, Boca Raton, FL, pp. 469–478. Uchida, K., Shiraishi, M., Naito, Y., Torii, Y., Nakamura, Y., Osawa, T., 1999. Activation of stress signaling pathways by the end-product of lipid peroxidation. 4-Hydroxy-2-nonenal is a potential inducer of intercellular peroxide production. J. Biol. Chem. 274, 2234 – 2242. Wallig, M.A., Kingston, S., Staack, R., Jeffery, E.H., 1998. Induction of rat pancreatic glutathione S-transferase and quinone reductase activities by a mixture of glucosinolate breakdown derivatives found in Brussels sprouts. Food Chem. Toxicol. 36, 365 – 373. Whitty, J.P., Bjeldanes, L.F., 1987. The effects of dietary cabbage on xenobiotics-metabolizing enzymes and the binding of aflatoxin B1 to hepatic DNA in rats. Food Chem. Tozicol. 25, 581 – 587. Yang, C.S., Smith, T.J., Hong, J.Y., 1994. Cytochrome P450 enzymes as targets for the chemoprevention against chemical carcinogenesis and toxicity: opportunities and limitations. Cancer Res. 54, 1982–1986.
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Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, Wasabi Yasujiro Morimitsu a,*, Kazuhiro Hayashi a, Yoko Nakagawa a, Hiroyuki Fujii a, Fumihiko Horio b, Koji Uchida a, Toshihiko Osawa a a Laboratory of Food and Biodynamics, Nagoya Uni6ersity Graduate School of Bioagricultural Sciences, Nagoya 464 -8601, Japan b Di6ision of Biomodeling, Nagoya Uni6ersity Graduate School of Bioagricultural Sciences, Nagoya 464 -8601, Japan
Received 3 December 1999; accepted 24 March 2000
Abstract 6-Methylsulfinylhexyl isothiocyanate (MS-ITC) was isolated from wasabi (Wasabia japonica, Japanese domestic horseradish) as a potential inhibitor of human platelet aggregation in vitro through our extensive screening of vegetables and fruits. In the course of an another screening for the induction of glutathione S-transferase (GST) activity in RL34 cells, MS-ITC was inadvertently isolated from wasabi as a potential inducer of GST. MS-ITC administered to rats or mice also showed both activities in vivo. As a result from elucidation of the platelet aggregation inhibition and the GST induction mechanisms of MS-ITC, the isothiocyanate moiety of MS-ITC plays an important role for antiplatelet and anticancer activities because of its high reactivity with sulfhydryl (RSH) groups in biomolecules (GSH, cysteine residue in a certain protein, etc.). © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Isothiocyanate; Wasabi; Platelet aggregation; Phase II enzyme; Glutathione S-transferase; Cancer chemoprevention
* Corresponding author. Present address: Department of Nutrition and Food Science, Laboratory of Food Chemistry, Ochanomizu University, Otsuka 2-1-1, Bunkyo-ku, Tokyo 112-8610, Japan. Tel./fax: + 81-3-59785757. E-mail address: [email protected] (Y. Morimitsu). 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 1 4 - 7
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1. Introduction Glucosinolates are found in cell vacuoles of various pants in the family Cruciferae such as horseradish, mustard, broccoli, and wasabi (Wasabia japonica, syn. Eutrema wasabi ). When plant cells are damaged, glucosinolates are hydrolyzed by myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) which is also produced in the same family, and produce isothiocyanates (Fig. 1). Most of these Crucifer isothiocyanates (cf. allyl isothiocyanate) are well known for having antimicrobial, fungicidal, and pesticidal activities (Soledade et al., 1998; Tajima et al., 1998). A major wasabi flavor compound, 6-methylthiohexyl isothiocyanate (MS-ITC) (n= 6) termed ‘green note’ flavor, has been shown the antimicrobial and platelet aggregation inhibition activities (Morimitsu et al., personal communication; Kumagai et al. 1994). On the other hand, epidemiological studies have demonstrated that consumption of cruciferous vegetables is associated with a lower incidence of cancers (Lee et al., 1989; Olsen et al., 1991; Mehta et al., 1995). Induction of phase II enzymes such as the glutathione S-transferase (GST) and/or quinone reductase (QR) have been demonstrated in broccoli (Aspry and Bjeldanes, 1983; Fahey et al., 1997), cabbage (Whitty and Bjeldanes, 1987), and Brussel sprouts (Nijhoff et al., 1993; Wallig et al., 1998). Many natural isothiocyanates derived from cruciferous vegetables and some fruits have been shown to induction of phase II enzymes in cultured cells and rodents (Leonard et al., 1981; Yang, et al., 1994; Barcelo et al., 1996). We have studied on physiological, pharmaceutical or therapeutic effects of the Allium genus, onion and garlic, especially for their antithrombotic activity. Our continuous attempt was succeeded in isolation of new sulfur-containing compounds, termed AC series, from the methanol extract of onion, which inhibited human platelet aggregation (Kawakishi and Morimitsu, 1988; Morimitsu and
Fig. 1. The enzymatic formation of isothiocyanates from Brassica sp.
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127
Kawakishi, 1990). Our screening for potential inhibitors of human platelet aggregation has been extending for daily vegetables and fruits, chiefly the cruciferous vegetables, because the family Cruciferae, except for Allium, is well-known vegetable for containing organosulfur compounds. Finally, the ethyl acetate (EtOAc) extract of wasabi was found to show a potent inhibitory activity for human platelet aggregation in vitro. Incidentally, an another screening for the induction of GST activity in RL34 cells resulted in finding the EtOAc extract of wasabi. In this study, we describe the results of two individual screenings, isolation and identification of MS-ITC from wasabi. Furthermore, a proposed mechanism for both the platelet aggregation inhibition and the GST induction by MS-ITC was discussed.
2. Materials and methods
2.1. Measurement of antiaggregation acti6ity Antiaggregation activity was measured by the reported method (Morimitsu and Kawakishi, 1990) using a dual-channel aggregometer (NKK, Hematracer I). Platelet aggregation was induced by various agonists at minimum concentrations as follows (final conc.): collagen (1 mg/ml), AA (0.5 mM), ADP (10 mM), PAF (5 mM), A23187 (13.2 mM), U46619 (5 mM), STA2 (5 mM), PMA (25 mM), OAG (200 mM) and thrombin (0.5 U/ml).
2.2. Inhibitory assay for PG biosynthesis The preparation of rabbit renal microsomes and the inhibitory assay were carried out using the reported procedure (Sankawa et al., 1982) with a slight modification. The reaction mixture (0.2 ml) contained 0.1 M sodium phosphate buffer (pH 7.5), 10mM tryptophan, 0.25 mM hemoglobin, 4.0 mM reduced glutathione, 20 mM [1-14C] arachidonic acid (1.35 mCi), rabbit renal microsomes (100 mg protein) and a test sample (DMSO solution). The reaction (37°C, 20 min) was started by the addition of microsomes and terminated by the addition of 1 N HCl (50 ml). The reaction mixture was then extracted with ether. After the ether was removed, the residue was dissolved in acetone and applied to a TLC plate. Following the TLC plate was developed with a solvent mixture [CHCl3/MeOH/AcOH = 90/5/5 (v/v)], radioactivities of PGE2, PGF2a and arachidonic acid were measured by an imaging plate reader (Bio-Image Analyser, FUJIX BA-100).
2.3. Measurement of the GST induction acti6ity in RL34 cells The GST induction Activity in RL34 cells was measured by using the conditions reported previously (Fukuda et al., 1997; Uchida et al., 1999).
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Fig. 2. The chemical structures of platelet aggregation inhibitors, which were also identified as potential inducers of GST in RL34, cell.
3. Results
3.1. The result of screening for platelet aggregation inhibitors and the isolation of MS-ITC from wasabi Our screening for searching platelet aggregation inhibitors was undergone on many extracts of various vegetables and fruits. Each extract (less than 100 mg) was added to human platelets (200 ml, 1×108 platelets/ml), and two inducers, arachidonic acid (AA) and ADP, were typically used. In the course of our screening, the EtOAc extracts of cruciferous vegetables were found to show potent inhibitory activities. Especially, the EtOAc extracts of wasabi, radish, turnip, and cabbage exhibited highly inhibitory activities. The EtOAc extract of wasabi showed the potent inhibitory activity even though the adding amount of extract was diminished. After continuous fractionations of the EtOAc extract (1.7 g) of wasabi (1.3 kg) by conventional methods (silica gel column chromatography and reversed-phase HPLC), three potent inhibitors of human platelet aggregation were ultimately isolated and identified as known 6-methylsulfinylalkyl isothiocyanates by spectroscopic analyses (Etoh et al., 1990). Their chemical structures were shown in Fig. 2 and their yields were 14 mg, 70 mg (MS-ITC; major), and 11 mg in order of central chain length respectively. The IC50 value of MS-ITC for human platelet aggregation was 21.9 mM, which is stronger than a positive control, aspirin (IC50 value: 458 mM, Table 1).
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Table 1 IC50 values of platelet aggregation inhibitors from wasabi IC50 (mM)
5-Methylsulfinylpentyl isothiocyanate 6-Methylsulfinylhexyl isothiocyanate (MS-ITC) 7-Methylsulfinylheptyl isothiocyanate Aspirin Indomethacin
ADP
AA
168 148 231 – –
20.0 21.9 12.4 458 12.2
3.2. The studies on structure– acti6ity relationship and a possible mechanism for platelet aggregation inhibition of MS-ITC The structure – activity relationship of 6-methylsulfinylalkyl isothiocyanates for human platelet aggregation was examined (Fig. 3). It could be summarized as follows, the oxidized form of sulfur atom was important and the central chain length 6 or 8 was suitable for their activity. Moreover, the isothiocyanate moiety of each MS-ITC derivative was necessary for inhibition of platelet aggregation. After incubation with MS-ITC (5 or 10 min), the treated platelets were washed two times with phosphate buffered saline, and MS-ITC potently inhibited platelet aggregation induced by thrombin (Fig. 4A). Then, MS-ITC could not show any
Fig. 3. The inhibitory effect of 6-methylsulfinylalkyl isothiocyanate derivatives for human platelet aggregation. Each sample was added 25 mM to 200 ml platelets’ solution. Platelet aggregation was induced by ADP (left) or AA (right).
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Fig. 4. The pre-incubation effect of MS-ITC for inhibition of the washed platelet aggregation (left), and the proposed mechanisms of platelet aggregation inhibition (right) by MS-ITC. (A) a. Control; b. no pre-incubation then washed two times; c. after incubation with MS-ITC (100 mM, 5 min), the pre-incubated platelets were washed two times with phosphate buffered saline (pH 7.2). (B) PLA2, using human platelets as a source of this enzyme; PG synthase, using rabbit renal microsomes as a source of this enzyme; TX synthase, using the assay kit from Cayman Chemical.
effects on arachidonic acid cascade (phspholipase A2, PG synthase, and TX synthase) in human platelets using (detail data not shown, Fig. 4B). As above results from studies on the inhibition mechanism of MS-ITC, it could not suppress the enzyme reactions in the platelet arachidonic acid cascade, but was possible to interact with sulfhydryl (SH) group on platelets (including certain receptors of agonists). 95% of MS-ITC underwent conjugation with GSH non-enzymatically to form dithiocarbamate within 60 min at 37°C. We also confirmed that MS-ITC also reacted with thiol groups of BSA and certain proteins on platelets (data not shown). Additional proofs: MS-ITC and its GSH-conjugate were detected in SD rats’ serums and platelets after administration by gavage. And inhibitory effect of MS-ITC on platelet aggregation of SD rats in vivo was confirmed by feeding with MS-ITC (0.05, 0.15 and 0.25%) for 2 weeks.
3.3. The result of screening for the GST induction acti6ity in RL34 cells On the other hand, our another screening for induction of GST activity in RL34 cells was also undergone on many extracts of various vegetables and fruits at the concentration of 25 and 2.5 mg/ml. Similarly the EtOAc extracts of cruciferous vegetables were found to show potent induction activities, especially wasabi and cresson. Finally, MS-ITC and the same 6-methylsulfinylalkyl isothiocyanates were inadvertently isolated from wasabi as a potential inducer of GST (Fig. 5).
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3.4. The studies on structure– acti6ity relationship and a possible mechanism for the GST induction by MS-ITC The structure – activity relationship of 6-methylsulfinylalkyl isothiocyanates for the GST induction was also examined (data not shown). It could be summarized as follows, only the isothiocyanate moiety of each MS-ITC derivative was necessary for their potent GST induction activity. As a result from western blot analysis of GST expression induced by MS-ITC in RL34 cells, increasing of protein levels of both GST-Ya and GST-Yp were detected (data not shown). And as a result from time dependent GSH level by MS-ITC, the GSH level in RL34 cells was initially decreased, then increased gradually to 1.8 times higher than its basal level after 12
Fig. 5. The HPLC profile of wasabi extracts and the GST activities of each fraction. Solid line indicated the HPLC profile. MS-ITC was finally isolated from Fr. 3.
Fig. 6. The time-course of the GSH levels in RL34 cells after adding MS-ITC. MS-ITC was added to RL34 at the concentration of 25 mM. GSH levels were measured by the conventional method.
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h (Fig. 6). As a result from these studies on the GST induction mechanism of MS-ITC, it was possible to interact with sulfhydryl group in RL34 cells (especially, GSH), because of the isothiocyanate’s highly reactivity with nucleophiles. After adding MS-ITC to RL34 cells, slight oxidative stress was immediately occurred, then this stimuli results in signal transductions and gene expressions related to phase II enzymes. Additional proofs: The effect of MS-ITC on levels of phase II enzymes of female ICR mice treated with MS-ITC by gavage in daily dose of 15 mmol for 5 days was examined. The five organs (lung, stomach, small intestine, liver and kidney) of each mouse after treated with MS-ITC were excised, frozen, and stored at − 80°C until analyzed. As a result from determination of GST and QR activities, MS-ITC was found to be a potential inducer of GST and QR in stomach, small intestine and significantly in liver. Especially, MS-ITC enhanced the GST activity in liver, owing to increasing protein levels of Class a GST isozyme (GST-Ya). Furthermore the GST induction activity of MS-ITC in liver was stronger than that of sulforaphane (ca. 1.9 times), a well-known phase II inducer isolated from broccoli (Prochaska and Talalay, 1988).
4. Discussion MS-ITC was isolated from wasabi as a potential inhibitor of human platelet aggregation and a potential inducer of GST in vitro through our extensive screening of vegetables and fruits. MS-ITC also showed the both biological activities in vivo. These biological activities were mainly caused by the isothiocyanate’s highly reactivity with nucleophiles such as GSH, sulfhydryl residues of proteins and so on. Concerning about platelet aggregation inhibition by MS-ITC, the conjugation reaction between isothiocyanate group and thiol groups of certain proteins on platelets resulted in the formation of dithiocarbamates on the surface of platelets so immediately. This protein modification reaction may play an important role for inhibition of binding of agonists to their receptors on platelets. Equally the non-enzymatically conjugation reaction between isothiocyanate group and GSH in RL34 cells at initial step after the addition of MS-ITC resulted in slight GSH depletion. The Michael addition acceptors were reported to be able to induce phase II enzyme activities in vitro (Talalay, 1992). Isothiocyanates belongs in this category. Furthermore, we isolated benzyl isothiocyanate (BITC) from papaya as a potential inducer of GST, and BITC was also decreased the GSH level, then was induced oxidative stress in RL34 cells measured by the flow cytometer (Nakamura et al., 2000). We assume that this slight oxidative stress may lead to increase the protein levels of GST, after induction of signal transductions and gene expressions in cells. Therefore, MS-ITC can be concluded to be a useful phytochemical, possessing antiplatelet and anticancer properties, in Japanese traditional food, wasabi.
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Acknowledgements The authors thank Mr. Inoue et al. (Kinjirushi Wasabi Co., Ltd.) for providing us the Wasabi research samples. This research project was mainly supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
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