An Anti-Estrogenic Lignan Glycoside, Tracheloside, from Seeds of Carthamus Tinctorius

Biosci. Biotechnol. Biochem., 70 (11), 2783–2785, 2006 Note An Anti-Estrogenic Lignan Glycoside, Tracheloside, from Seeds of Carthamus tinctorius

y Hye Hyun YOO,1;2 Jeong Hill PARK,1 and Sung Won KWON1;

1College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea 2Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea

Received May 25, 2006; Accepted July 11, 2006; Online Publication, November 7, 2006 [doi:10.1271/bbb.60290]

The lignan glycoside, tracheloside, was isolated from S. Han of Seoul National University, Korea. A voucher seeds of Carthamus tinctorius (Compositae) as an anti- specimen of this plant (NP-03-011) has been deposited estrogenic principle against cultured Ishikawa cells by at the Herbarium of the College of Pharmacy, Seoul employing a bioassay-linked HPLC-ELSD method. National University, Korea. Milled seeds of C. tinctorus Tracheloside significantly decreased the activity of (2.0 kg) were extracted with MeOH. After filtering and alkaline phosphatase (AP), an estrogen-inducible mark- evaporating the solvent, the resulting extract was er enzyme, with an IC50 value of 0.31 g/ml, a level of suspended in water, and then partitioned between EtOAc inhibition comparable to that of tamoxifen (IC50 = and n-BuOH to afford dried EtOAc-soluble (8 g) and 0.43 g/ml). n-BuOH-soluble residues (6.5 g). Each of the residues was tested against Ishikawa cells, and the EtOAc- Key words: Carthamus tinctorius; tracheloside; anti-es- soluble fraction was found to be active (Inhibition at trogenic activity; Ishikawa cell; bioassay- 4 mg/ml = 92%). Fifty microliters of the EtOAc-soluble linked HPLC-ELSD residue (20 mg/ml MeOH) was injected into an ODS column (Kanto Mightysil, RP-18, 5 mm, 4:6 250 mm), Interest in estrogen analogs as antagonists has and the column was eluted with 1% acetic acid in increased tremendously because of the clinical applica- CH3CN/1% acetic acid in H2O (30/70, 0 min ! 100/0, tion of anti-estrogens for treating breast cancer and their 15 min ! 100/0, 30 min) at a rate of 1 ml/min. The possible chemopreventive effects on women who are at eluent from the column was introduced through a micro- high risk for developing breast cancer. Indeed, tamox- splitter valve into ELSD and a fraction collector in the ifen, a non-steroidal anti-estrogen, is extensively utilized ratio of 1:9, respectively. The eluent collected in a 96- in the prevention and treatment of breast cancer in well plate was dried and used for determining the anti- women. However, the effectiveness of tamoxifen in estrogenic activity. Figure 1 displays the HPLC-ELSD treating breast cancer has been limited, because of the chromatogram (A) and the bioactivity profile (B) of possible increased risk of endometrial carcinoma.1) the EtOAc-soluble fraction of C. tinctorius seeds. The Therefore, the search for novel anti-estrogens without activity profile indicates a retention time for the side effects, especially those from natural origins, has component exhibiting strong AP inhibitory activity of been extensive. As part of our current project to discover around 11 min, and the HPLC-ELSD chromatogram novel plant-derived anti-estrogens as chemopreventive shows a peak corresponding to the active component. agents, we utilized Ishikawa cells. Ishikawa cells are On this basis, the active component, tracheloside, was estrogen responsive human endometrial adenocarcinoma isolated by using prep-TLC. cells, and the alkaline phosphatase (AP) activity in these The anti-estrogenic activity was evaluated by using cells is markedly stimulated by estrogens.2) We report Ishikawa cells as described previously.3) Briefly, here on the isolation of the active component from Ishikawa cells provided by R. B. Hochberg (Yale C. tinctorius seeds and the biological potential of University, New Haven, CT, USA) were incubated its anti-estrogenic activity by using a bioassay-linked overnight with an estrogen-free DMEM/F12 medium in HPLC-ELSD method. 96-well plates (5 104/well). To determine the anti- Seeds of Carthamus tinctorus L. (Compositae) were estrogenic activity, the cells were incubated with 1 nM purchased from the Kyung-Dong Market, Seoul, Korea estradiol (E2) and a test compound in a total volume of in March 2001 and were identified by Emeritus Prof. D. 200 ml of medium/well at 37 C for 4 days. The enzyme

y To whom correspondence should be addressed. Tel: +82-2-880-7844; Fax: +82-2-886-7844; E-mail: [email protected] 2784 H. H. YOO et al.

Fig. 1. HPLC-ELSD Chromatogram (A) and Anti-Estrogenic Activity Profile (B) of the EtOAc-Soluble Fraction from Carthamus tinctorius Seeds.

activity was measured by reading at 405 nm the O liberation of p-nitrophenol with a microplate reader at OH CH3O 15-s intervals to give a total of 16–20 readings. The maximum slope of the lines generated from the kinetic O data was calculated by using a computer program. The GlcO percentage induction for determining the estrogenic H activity was calculated as [(slope for sample slope for cells)/(slope for estrogen slope for cells)] 100. To determine the anti-estrogenic activity, the percentage OCH induction was determined as [(slope for sample slope 3 for cells)/(slope for DMSO slope for cells)] 100. OCH3 The data represent the average of triplicate determina- tions. Tracheloside alone did not significantly induce Fig. 2. Chemical Structure of Tracheloside. AP activity in the absence of estradiol (E2). However, when the cells were treated with 1 nM E2, the AP activity was increased by approximately 10-fold, and the phytoestrogens may have beneficial effects on breast induction was markedly inhibited by tracheloside in a and prostate cancer which are both influenced by 6) dose-dependent manner (Fig. 3). The IC50 value for hormones. Tracheloside, a lignan glycoside, is known tracheloside was 0.31 mg/ml, comparable to that of to occur abundantly in the Trachelospermum species. tamoxifen (IC50 = 0.43 mg/ml). Tracheloside had no Lignan glycosides are generally known to be phytoes- effect on the cell viability at any of the concentrations trogens or cytotoxic agents.7) Tracheloside is structurally tested, as judged by parallel studies of cytotoxicity with similar to the well-known lignan phytoestrogen, arctiin, a sulforhodamine B assay (data not shown).4) in which side-chain C-2 of a five-membered ring is A variety of plant chemicals that bind to the estrogen changed from a hydrogen to a hydroxyl group (Fig. 2), receptor have been identified and may exert estrogenic- and thus tracheloside can also be considered to function like action by various mechanisms.5) Representative of as a phytoestrogen. It is noteworthy that arctiin is these phytoestrogens are the isoflavonoids and diphe- transformed to estrogenic and anti-estrogenic substances nolic compounds contained in the bean subfamily of by human intestinal bacteria,8) suggesting that trachelo- Leguminosae. However, the potency of most of these side might also be metabolized by human intestinal phytoestrogens is low compared to such endogenous or bacteria in the same manner. There is therefore the synthetic steroid estrogens as 17-estradiol or ethinyl- possibility that an intake of tracheloside via the diet or estradiol, and these phytoestrogens therefore sometimes a herbal medicine such as safflower seeds might show exhibit antagonistic actions against estrogens. Epide- estrogenic action in the body, contrary to the effect of its miological data have suggested that the consumption of constituent, tracheloside. In addition, tracheloside and An Anti-Estrogenic Lignan Glycoside 2785

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Fig. 3. Effect of Tracheloside on the Induction of AP in Ishikawa Cells. The cells were treated with various concentrations of tracheloside with ( ) or without ( )E2(1nM) for 96 hr in an estrogen-free medium, and the AP level was measured by using p-nitrophenyl phosphate as the substrate. Each value is presented as the mean S.E. arctiin are known to be respectively transformed into the Effects of steroid hormones and antisteroids on alkaline aglycones, trachelogenin and arctigenin by gastric juice phosphatase activity in human endometrial cancer cells and intestinal bacteria in rats.9) Further studies on the in (Ishikawa line). Cancer Res., 46, 2771–2774 (1986). vivo metabolism of tracheloside will be needed to clarify 3) Pisha, E., and Pezzuto, J. M., Cell-based assay for the the estrogen-related effect of tracheloside in vivo. determination of estrogenic and anti-estrogenic activ- ities. Methods Cell Sci., 19, 37–43 (1997). There have been few reports to date concerning the 4) Rubinstein, L. V., Shoemaker, R. H., Paull, K. D., anti-estrogenic properties of lignans, and some have Simon, R. M., Tosini, S., Skehan, P., Scudiero, D. A., 10) been to be reported inactive in anti-estrogenic tests. Monks, A., and Boyd, M. R., Comparison of in vitro Therefore, this result for the anti-estrogenic activity of anticancer-drug-screening data generated with a tetrazo- tracheloside in Ishikawa cells will be helpful to our lium assay versus a protein assay against a diverse panel understanding of the hormone-like action of lignans and of human tumor cell lines. J. Natl. Cancer Inst., 82, will provide informative data concerning the effect of 1113–1118 (1990). safflower seeds as a herbal medicine, although further 5) Davis, S. R., Dalais, F. S., Simpson, E. R., and Murkies, studies using in vivo models are needed. The LC-ELSD A. L., Phytoestrogens in health and disease. Recent Prog. method used in this investigation in conjunction with the Horm. Res., 54, 185–210 (1999). Ishikawa cell system permitted the anti-estrogenic 6) Adlercreutz, H., Mousavi, Y., Clark, J., Hockerstedt, K., Hamalainen, E., Wahala, K., Makela, T., and Hase, T., potential of tracheloside from seeds of C. tinctorius to Dietary phytoestrogens and cancer: in vitro and in vivo be evaluated by a rapid and simple procedure. studies. J. Steroid Biochem. Mol. Biol., 41, 331–337 (1992). Acknowledgments 7) Adlercreutz, C. H., Goldin, B. R., Gorbach, S. L., Hockerstedt, K. A., Watanabe, S., Hamalainen, E. K., We thank Dr. S. K. Lee of Ewha Womans University Markkanen, M. H., Makela, T. H., Wahala, K. T., and for helpful discussions. This work was supported by Adlercreutz, T., Soybean phytoestrogen intake and grant no. R01-2006-000-10593-0 from the Basic Re- cancer risk. J. Nutr., 125, 757S–770S (1995). search Program of the Korea Science & Engineering 8) Xie, L. H., Ahn, E. M., Akao, T., Abdel-Hafez, A. A., Foundation and Seoul National University (370C- Nakamura, N., and Hattori, M., Transformation of arctiin 20050126). to estrogenic and antiestrogenic substances by human intestinal bacteria. Chem. Pharm. Bull., 51, 378–384 (2003). References 9) Nose, M., Fujimoto, T., Takeda, T., Nishibe, S., and Ogihara, Y., Structural transformation of lignan com- 1) Simard, J., Sanchez, R., Poirier, D., Gauthier, S., Singh, pounds in rat gastrointestinal tract. Planta Med., 58, S. M., Merand, Y., Belanger, A., Labrie, C., and Labrie, 520–523 (1992). F., Blockade of the stimulatory effect of estrogens, OH- 10) Chang, L. C., Gills, J. J., Bhat, K. P. L., Luyengi, L., tamoxifen, OH-toremifene, droloxifene, and raloxifene Farnsworth, N. R., Pezzuto, J. M., and Kinghorn, D., on alkaline phosphatase activity by the antiestrogen EM- Activity-guided isolation of constituents of Cerbera 800 in human endometrial adenocarcinoma Ishikawa manghas with antiproliferative and antiestrogenic activ- cells. Cancer Res., 57, 3494–3497 (1997). ities. Bioorg. Med. Chem. Lett., 10, 2431–2434 (2000). 2) Holinka, C. F., Hata, H., Kuramoto, H., and Gurpide, E.,