Biosci. Biotechnol. Biochem., 75 (11), 2237–2239, 2011 Note Effective Cytochrome P450 (CYP) Inhibitor Isolated from Thyme (Thymus saturoides) Purchased from a Japanese Market

Zeineb BRAHMI, Hitomi NIWA, Mio YAMASATO, Sakurako SHIGETO, Yuna KUSAKARI, y Kouichi SUGAYA, Jun-ichi ONOSE, and Naoki ABE

Department of Nutritional Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

Received April 22, 2011; Accepted July 16, 2011; Online Publication, November 7, 2011 [doi:10.1271/bbb.110328]

A highly polymethylated flavone that effectively The dry leaves (200 g) of thyme purchased from a inhibited cytochrome P450s (CYPs) 1A2 and 3A4 Japanese market (Thymus saturoides, S&B Foods, (IC50 ¼ 2:41 and 1.71 M) in vitro was isolated from Tokyo, Japan) were soaked at room temperature for thyme leaves (Thymus saturoides) purchased from a 24 h in 80% acetone (4 L). The resulting filtrate was Japanese market. Its structure was spectroscopically evaporated in vacuo to an aqueous concentrate and then identified as 40,5-dihydroxy-30,6,7,8-tetramethoxy fla- extracted three times with equal volumes of ethyl acetate vone (8-methoxycirsilineol, 1). This is the first report (EtOAc) at pH 3.0 to give an EtOAc extract (14.6 g, describing a strong inhibitor of CYP1A2 and 3A4 IC50 ¼ 6:59 0:81 mg/mL). This extract was subjected isolated from Thymus saturoides. to Sephadex LH-20 column chromatography, eluting with methanol as the solvent, to give a CYP3A4-active Key words: 8-methoxycirsilineol; cytochrome P450 fraction (980.3 mg) which had inhibitory activity of (CYP) inhibitor; Thymus saturoides; 79:9 0:8% at 7.0 mg/mL. This active fraction was polymethoxyflavone purified by repetitive preparative TLC, developed by using solvent systems of chloroform-methanol (9:1) Pharmacological and chemical investigations of and hexane-EtOAc (1:1), and an active compound aromatic and medicinal plants have provided important (1, 4.4 mg) was obtained. advances in the therapeutic approach to several types of The active compound (1) isolated from thyme was pathology. Thyme is a well-known medicinal plant used identified as a yellow amorphous powder with the worldwide as an aromatic herb to add a distinctive molecular formula of C19H18O8, based on HRESI-MS aroma and flavor to food. The leaves can be used (pos.), m=z: 375.10802 ðM þ HÞþ (calcd. as 375.10799 1–3) fresh or dried as a spice. The majority of the thyme for C19H19O8). The UV/VIS spectrum, max nm species possess various beneficial effects and have (MeOH): 349.6 (" 15,600), 280.8 (" 11,000), 254.2 antiseptic, antimicrobial, antifungal and antioxidative (" 9390), 207.4 (" 22,300), was similar to that of properties.1–6) such polymethylated flavones as (2). The IR Cytochrome P450 monooxygenases (CYPs), a large spectrum of 1 showed absorption bands, max (ATR) ubiquitous enzyme family with a heme structure, are cm1: 3422, 2922, 2851, 1600, 1571, 1458, 1307. The 1 involved in the hepatic metabolism of xenobiotic lip- H-NMR (CDCl3, 400 MHz) spectrum of 1 exhibited 17 ophilic substances. CYPs are the key enzymes catalyz- protons, H: 3.96 (3H, s 6-OCH3), 3.98 (3H, s 8-OCH3), 0 ing the oxidative metabolism of a large number of 4.01 (3H, s 3 -OCH3), 4.11 (3H, s 7-OCH3), 6.59 (1H, s exogenous compounds for detoxifying toxic, carcino- 3-H), 7.05 (1H, dd, J ¼ 8:8, 2.0 Hz 60-H), 7.41 (1H, d, genic, and most pharmaceutical agents. The extrahepatic J ¼ 2:0 Hz 20-H), 7.54 (1H, d, J ¼ 8:8 Hz 50-H), 12.54 13 expression of CYPs has recently been established, and (1H, s 5-OH), and the C-NMR (CDCl3, 100 MHz) 0 certain CYPs involved in hormone and vitamin metab- spectrum gave 19 peaks, C: 56.2 (3 -OCH3), 61.3 olism and in the metabolic activation of genotoxic (6-OCH3), 61.9 (7-OCH3), 62.2 (8-OCH3), 104.0 (C-3), substances have been reported to have important roles in 107.1 (C-4a), 108.4 (C-20), 115.3 (C-50), 120.9 (C-60), tumor formation and development.7–10) Food materials 123.4 (C-10), 133.1 (C-8), 136.7 (C-6), 145.9 (C-5), with effective inhibitors against CYPs are potential 147.0 (C-30), 149.56 (C-40), 149.65 (C-8a), 153.1 (C-7), candidates for cancer prevention strategies. 164.1 (C-2), 183.1 (C-4). The hydroxy group at H 12.54 We investigated the active compounds against CYPs was assigned as 5-OH of the flavone skeleton. The in the leaves of common thyme purchased from a active compound was identified as 40,5-dihydroxy- Japanese market to advance strategies using functional 30,6,7,8-tetramethoxyflavone (8-methoxycirsilineol, 1) foods that would either inhibit or exploit CYP enzymes based on these results, as shown in Fig. 1. 8-Methoxy- for treating cancer. We describe here the isolation, cirsilineol (1) has previously been isolated from the identification, and inhibitory activities of an active Sideritis genus11) and Thymus species,12–15) as well as compound (1) against CYPs 1A2, 2D6, and 3A4. from such other plant species as the Egyptian medicinal

y To whom correspondence should be addressed. Fax: +81-3-5477-2674; E-mail: [email protected] Abbreviations: TLC, thin layer chromatography; HRESI-MS, high resolution electron spray ionization mass spectrometry 2238 Z. BRAHMI et al.

OCH3 3' 2' R2 OCH3 4' 8 8a 1' H3CO O 5' 7 2 6' R1 = OH R2 = OH 8-methoxycirsilineol (1) H CO 6 3 R1 = OCH3 R2 = OCH3 nobiletin (2) 3 5 4a 4 R1 O

R1 R2 HO O R1 = OH R2 = OH (3) R1 = H R2 = OH (4) R1 = H R2 = H (5) OH O

R2 OH

HO O R1 = OH R2 = OH (6) R = OH R = H (7) OH 1 2 R1 = H R2 = OH (8) R1 O

Fig. 1. Chemical Structures of 8-Methoxycrisilinol (1), Nobiletin (2), and Other Flavonoids.

Table 1. Inhibitory Effects of 8-Methoxycirsilineol (1), Nobiletin erythromycin for CYP3A4.19) Safrole was purchased (2), and Other on CYP Activities from Tokyo Kasei Kogyo (Tokyo, Japan), and apige- nine, erythromycin, nobiletin and other reagents were IC50 (mM) obtained from Wako Pure Chemical Industries (Osaka, Compound CYP Japan). Cimetidine, fisetin, kaempferol, luteolin and 1A2 2D6 3A4 quercetin were from Sigma Chemicals (St. Louis, MO, 8-Methoxycirsilineol (1) 2:41 0:3 > 100 1:71 0:3 USA), and chrysin was from Kanto Chemical Industry Nobiletin (2) 0:83 0:2 > 100 20:6 5:2 Co. (Tokyo, Japan). All other reagents were of the best Luteolin (3) 26:6 4:8 > 100 57:7 16:1 grade commercially available. Apigenine (4) 4:7 0:7 > 100 30:8 7:6 8-Methoxycirsilineol 1 and nobiletin 2 showed dose- Chrysin (5) 0:3 0:1 > 100 94:7 30:9 dependent inhibitory activity toward CYPs, both com- Quercetin (6) 33:0 4:670:5 3:828:0 5:2 pounds inhibiting the CYP1A2 enzyme activity in vitro. Kaempferol (7) 2:2 0:319:7 4:618:3 5:3 Fisetin (8) 4:5 1:22:6 0:340:7 7:4 Compound 2 showed the second strongest CYP1A2 Safrolea 3:87 0:7 —— inhibitory activity toward all the tested compounds, b Cimetidine — 25:2 3:0 — following that of chrysin 5, with an IC50 value of Erythromycinc ——2:09 0:2 0:83 0:17 mM, this being much stronger than that of the Mean SD (n ¼ 3) aPositive control for CYP1A2, bPositive control for positive control, safrole (IC50 ¼ 3:87 0:7 mM). Com- CYP2D6, cPositive control for CYP3A4. pound 1 was a potent inhibitor of CYP1A2 with an IC50 value of 2:41 0:33 mM. However neither compound 1 nor 2 showed any inhibitory activity toward CYP2D6 at plant, Cleome droserifolia, and the Chinese herb, concentrations of less than 100 mM, unlike compound 1 Rabdosia rubescen.16,17) The chemical structure of 1 which had the most potent inhibitory effect of all the 0 0 was similar to that of nobiletin (2, Fig. 1), 3 ,4 ,5,6,7,8- tested compounds on CYP3A4 (IC50 ¼ 1:71 0:33 mM), hexamethoxyflavone. Compound 2 is a major compo- similar to that of the positive control, erythromycin nent of the polymethoxyflavone family of citrus fruits, (IC50 ¼ 2:09 0:2 mM) and much stronger than that of 2 and has important protective biological properties, which had an IC50 value of 20:6 5:2 mM (Table 1). including anticancer, anti-inflammatory and antiathero- These findings indicate that 8-methoxycirsilineol 1 and genic activities. There is thus strong interest in 2 as a nobiletin 2 had inhibitory effects on CYP1A2 with functional food factor (Fig. 1). Compound 2 does not nearly the same potency, but no inhibitory activity inhibit CYP3A4.18) The inhibitory effects of compounds toward CYP2D6, unlike such flavonols possessing a 1 and 2 and other flavonoids on CYP1A2, CYP2D6, and 3-OH group as quercetin 6, kaempferol 7, and fisetin 8 CYP3A4 were investigated (Table 1). These inhibitory which showed inhibitory activities. Compound 1 also effects were determined by using black 96-well micro- had the most potent inhibitory activity toward CYP3A4, titer plates (Sumitomo Bakelite, Tokyo, Japan) based on stronger than that of compound 2. Inhibitory activity is the formation of fluorescent metabolites by the CYP strongly influenced by the presence and/or position of enzymes. CYP inhibitory activity was assayed by using hydroxyl groups on the A, B, and C rings of flavonoids. the Vivid CYP Blue substrate. 7-Ethyloxymethyloxy-3- Studies on the substituent effect at C-40 of flavonoids cyanocoumarin was the substrate used with CYP1A2 have suggested that the hydroxy group had more and CYP2D6, and 7-benzyloxymethyloxy-3-cyanocou- effective CYP 3A inhibitory activity than the methoxy marin was used with CYP3A4. The positive controls or hydrogen group.20) The hydroxy groups at C-5 and were safrole for CYP1A2, cimetidine for CYP2D6 and C-40 of compound 1 might have been particularly Effective CYP Inhibitor Isolated from Purchased Thyme 2239 important for its strong inhibitory activity against 3) Morales R, ‘‘Thyme-The Genus Thymus,’’ eds. Stahl-Biskup E CYP3A4. CYP3A4 is the most abundant CYP expressed and Sa´ez F, Taylor & Francis, London and New York, pp. 20– in the human liver and the small intestine, and plays a 27 (2002). 4) Lee SJ, Umano K, Shibamoto T, and Lee KG, Food Chem., 91, dominant role in both drug metabolism and metabolic 131–137 (2005). activation of such carcinogens as aflatoxin B1 and 5) Oussalah M, Caillet S, Saucier L, and Lacroix M, Food Control, 21) polycyclic hydrocarbons. Nobiletin 2 and , 18, 414–420 (2007). both permethylated polymethoxyflavones, stimulate 6) Ito H, Urakamizaidan kenkyu Houkokusho (in Japanese), 16, many-fold the metabolism of benzo[]pyrene and 165–170 (2008). 7) Bruno RD and Njar VCO, Bioorg. Med. Chem., 15, 5047–5060 aflatoxin B1 in human liver microsomes, whereas apigenin 4, chrysin 5, quercetin 6, kaempferol 7, and (2007). 8) Kane GC and Lipsky JJ, Mayo Clin. Proc., 75, 933–942 (2000). fisetin 8 inhibit the metabolism of these compounds 9) Hasler JA, Estabrook R, Murray M, Pikuleva I, Waterman M, 22) in vitro. The substituent effects of flavones on the Capdevila J, Holla V, Helvig C, Falck JR, Farrell GR, metabolic activation of carcinogens are complicated Kaminsky LS, Spivack SD, Boitier E, and Beaune P, Mol. both in vitro and in vivo.23) Different effects of the Aspects Med., 20, 1–137 (1999). flavones against CYP isozymes may mainly depend on 10) Koga N, Matsu M, Ohta C, Haraguchi K, Matsuoka M, Kato Y, differences in their active site cavities. The crystal Ishi T, Yano M, and Ohta H, Biol. Pharm. Bull., 30, 2317–2323 structures of human CYP1A2,24) CYP3A4,25) and (2007). 26) 11) Rodriguez B, Phytochemistry, 16, 800–801 (1977). CYP2D6 isozymes have been reported. Such poly- 12) van den Broucke CO, Dommisse RA, Esmans EL, and Lemli methoxyflavones as nobiletin 2 and tangeretin have been JA, Phytochemistry, 21, 2581–2583 (1982). reported to have significant biological activities, and 13) Horwath AB, Grayer RJ, Keith-Lucas DM, and Simmonds MSJ, comparative studies on partially demethylated polyme- Biochem. Syst. Ecol., 36, 117–133 (2008). thoxyflavones are therefore important to determine the 14) Watanabe J, Shinmoto H, and Tsushida T, Biosci. Biotechnol. structure-activity relationship. Biochem., 69, 1–6 (2005). 15) Kayoko M, Hiroe K, and Nobuji N, J. Agric. Food Chem., 50, A previous study has indicated the ability of 8- 1845–1851 (2002). methoxycirsilineol 1 to inhibit the chemical mediator 16) Fushiya S, Kishi Y, Hattori K, Batkhuu J, Takano F, Singab AN, 14) release from rat basophilic leukemia RBL-2H3 cells. and Okuyama T, Planta Med., 65, 404–407 (1999). Cytotoxicity against the HL-60 cancer cell line17) and the 17) Naisheng B, Kan H, Zhu Z, Ching-Shu L, Li Z, Zheng Q, Xi S, antioxidative activity of 1 have also been reported.15,27) Min-Hsiung P, and Chi-Tang H, Food Chem., 122, 831–835 We report here for the first time that compound 1 (2010). exhibited strong CYP inhibitory effects in vitro on the 18) Takanaga H, Ohnishi A, Yamada S, Matsuo H, Horimoto S, Shoyama Y, Ohtani H, and Sawada Y, J. Pharmacol. Exp. three CYP isozymes, CYPs 1A2, 2D6, and 3A4, Ther., 293, 230–236 (2000). particularly on CYP1A2 and CYP3A4. These results 19) Huang Y-T, Onose J, Abe N, and Yoshikawa K, Biosci. suggest that thyme (Thymus saturoides) purchased from Biotechnol. Biochem., 73, 855–860 (2009). the Japanese market does not only have negative effects 20) Tsujimoto M, Horie M, Honda H, Takara K, and Nishiguchi K, on numerous food-drug interactions, but can also become Biol. Pharm. Bull., 32, 671–676 (2009). a significant food material for preventing cancer through 21) Guengerich FP, ‘‘Cytochrome P450: Structure, Mechanism and inhibition of the metabolic activation of carcinogens. Biochemistry’’ Third ed., ed. Ortiz PR, Plenum, New York, pp. 377–530 (2005). 22) Buening MK, Chang RL, Huang M-T, Fortner JG, Wood AW, Acknowledgments and Conney AH, Cancer Res., 41, 67–72 (1981). 23) Hodek P, Trefil P, and Stiborova M, Chem. Biol. Interact., 139, We thank Dr. Y. Q. Ye and Dr. H. Koshino (RIKEN) 1–21 (2002). for measuring the high-resolution mass spectra. This 24) Sansen S, Yano JK, Reynald RL, Schoch GA, Griffin KJ, Stout work was performed as a part of the Advanced Research CD, and Johnson EF, J. Biol. Chem., 282, 14348–14355 (2007). 25) Yano JK, Wester MR, Schoch GA, Griffin KJ, Stout CD, and Project of Tokyo University of Agriculture. Johnson EF, J. Biol. Chem., 279, 38091–38094 (2004). 26) Rowland P, Blaney FE, Smyth MG, Jones JJ, Leydon VR, References Oxbrow AK, Lewis CJ, Tennant MG, Modi S, Eggleston DS, Chenery RJ, and Bridges AM, J. Biol. Chem., 281, 7614–7622 1) de Lisi A, Tedone L, Montesano V, Sarli G, and Negro D, Food (2006). Chem., 125, 1284–1286 (2011). 27) Alton JJrD, Castaneda-Acosta J, Gloria CB, Kimberly LP, 2) ElHadj Ali IB, Zaouali Y, Bejaoui A, and Boussaid M, Chem. Nikolaus HF, and Gary WW, J. Nat. Prod., 63, 327–331 (2000). Biodiv., 7, 1276–1289 (2010).