Journal of Chromatography B, 941 (2013) 1–9

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Metabolic profile of irisolidone in rats obtained by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry Guozhe Zhang a , Jiahong Sun b , Yoshihiro Kano a , Dan Yuan a,∗ a b

Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China Department of Pharmacology, West Virginia University, PO Box 6001, Morgantown, WV 26506-0002, USA

a r t i c l e

i n f o

Article history: Received 15 July 2013 Accepted 22 September 2013 Available online 7 October 2013 Keywords: Irisolidone Metabolic profile UHPLC/Q-TOF MS Metabolite Rat

a b s t r a c t Irisolidone, a major isoflavone found in Pueraria lobata flowers, exhibits a wide spectrum of bioactivities, while its metabolic pathway in vivo has not been investigated. In this study, an ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the in vivo metabolism of irisolidone in rats. Plasma, bile, urine, and feces were collected from rats after a single 100 mg/kg oral dose of irisolidone. Protein precipitation, solid phase extraction (SPE) and ultrasonic extraction were used to prepare samples of plasma, bile/urine, and feces, respectively. A total of 46 metabolites were detected and tentatively identified based on the mass spectral fragmentation patterns, elution order or confirmed using available reference standards. The metabolic pathways of irisolidone in rats included decarbonylation, reduction, demethylation, demethoxylation, dehydroxylation, hydroxylation, sulfation, and glucuronidation. The relative content of each metabolite was also determined to help understand the major metabolic pathways of irisolidone in rats. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Flavonoids, as plant secondary metabolites, include many structurally similar classes such as flavones, isoflavones, flavans, anthocyanins, proanthcyanidins, flavanones, chalcones, and aurones. Flavones and isoflavones are of ecotoxicological importance since they are present in the heartwood of tree species used for wood pulp [1,2], and are to be found in a variety of fruits and vegetables. Plants of the Leguminosae family (e.g. soy, lupin) contain isoflavones that are important components in the diets of humans and animals. Flos Puerariae, as a traditional Chinese medicine, has been used in China since ancient times to help with recovery from alcohol intoxication. In China and Japan, phytochemicals extracted from Flos Puerariae have recently become popular herbal medicines for treating alcohol intoxication and liver injury. The content of kakkalide (irisolidone-7-O-␤-d-xylosylglucoside, KA) in the

Abbreviations: BiA, biochanin A; ESI, electrospray ionization; CLog P, calculated 1-octanol/water partition coefficient; Ir, irisolidone; KA, kakkalide; SPE, solid phase extraction; Te, tectorigenin; UHPLC/Q-TOF MS, ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry. ∗ Corresponding author. Tel.: +86 024 23986502; fax: +86 024 23986502. E-mail addresses: yuandan [email protected], [email protected] (D. Yuan). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.09.033

P. lobata flower and in its aqueous extract accounts for more than 2 and 10%, respectively. It has been found that human fecal bacteria can transform KA into irisolidone (Ir, Fig. 1) in anaerobic medium [3]. Whether given orally or intraperitoneally, Ir exhibits more potent bioactivity than KA [4–6]. In addition, some isoflavone aglycone metabolites, such as tectorigenin, glycitein, and genistein, also exhibited more potent bioactivity than their glycoside precursors [4]. These results indicate that flavone glycoside is in essence a prodrug, while aglycone may be the real active component in vivo. Irisolidone, as the aglycone of kakkalide, exhibits a wide spectrum of bioactivities such as anti-inflammatory [7], antioxidative [8], antiviral [9], anti-tumor [10], and estrogenic effects [6] and protecting against ethanol-induced damage and hepatic injury [3]. Compared with tectorigenin (Te) and genistein, Ir has the most potent inhibitory effect on the growth of Helicobacter pylori (HP) [11], suggesting that the C-4 -OCH3 group may be the active group involved. Although it has been reported that Ir is produced from the metabolism of KA both in vitro [3,6] and in vivo [12], the metabolism of Ir has not yet been studied in detail either in vitro or in vivo. Accordingly, there is a need to study the detailed metabolism of Ir. In this paper, an UHPLC/Q-TOF MS method was employed to characterize the metabolites of Ir in rat biological samples, including plasma, bile, urine, and feces, in order to provide evidence of the metabolic pathways of isoflavones in vivo.

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G. Zhang et al. / J. Chromatogr. B 941 (2013) 1–9

10 min. Then, 5 ␮L samples of the supernatants were injected into the UHPLC/Q-TOF MS system for analysis.

Fig. 1. Structure, nomenclature and diagnostic fragmentations of irisolidone.

2. Experimental 2.1. Chemicals and reagents Irisolidone (purity >98%) and other authentic standards (purity >95%) were separated in our laboratory. Their structures were determined using ultraviolet (UV), infrared, 1 H and 13 C NMR, and mass spectrometry (MS) methods. The formic acid and acetonitrile used were of HPLC grade (Fisher Scientific) and ultra-pure water (18.2 M) was prepared with a Milli-Q water purification system (Millipore, France). 2.2. Animal experiments Male Sprague-Dawley rats (220–250 g) purchased from the Animal Center of Shenyang Pharmaceutical University (Shenyang, China) were housed in an animal room with a standardized temperature (25–28 ◦ C), humidity (50–60%) and a 12 h light/dark cycle, with free access to a soy-free diet and tap water for one week. Rats were fasted for 12 h before the experiments, and were allowed free access to water and sugar over the period of sample collection. Ir was dispersed in 0.5% carboxymethylcellulose solution at 10.0 mg/mL, and then sonicated for 5 min to obtain a homogeneous suspension. Whole blood samples were collected from the suborbital vein and placed in heparinized polythene tubes at 0, 1, 2, 4, 8, 12, 24, 36 and 48 h after oral administration of Ir at a dose of 100 mg/kg BW, then immediately centrifuged at 3500 rpm for 10 min at 4 ◦ C to obtain plasma. Urine and feces were collected for 0–48 h after administration. Urine samples were made acidic by adding 1% acetic acid and then immediately stored at −20 ◦ C; feces samples were also stored at −20 ◦ C after drying. For the study of bile, rats were anaesthetized by intraperitoneal administration of urethane, and then a plastic cannula was surgically inserted into the bile ducts. After collecting blank bile for one hour, bile was collected for 0–36 h after the oral dose. All the biological samples were stored at −20 ◦ C. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University. 2.3. Sample preparation 2.3.1. Plasma samples Two hundred microliter of mixed plasma was diluted with 600 ␮L acetonitrile containing 1% acetic acid, and vortex-mixed for 2 min. After centrifuging for 10 min at 10,000 rpm to precipitate proteins, the supernatants were transferred to other tubes and evaporated to dryness under a stream of nitrogen gas at room temperature. The residue obtained was reconstituted with 100 ␮L methanol–water (80:20, v/v) and centrifuged at 13,000 rpm for

2.3.2. Urine samples Solid phase extraction (SPE) was used for extracting the metabolites from urine with Bond Elut C18 cartridges (3 mL, 500 mg). Each cartridge was conditioned with 3 mL methanol followed by 2 mL distilled water. Spiked urine samples were centrifuged at 3500 rpm for 10 min, and then 100 ␮L of these supernatants were loaded on to the cartridges. The SPE cartridges were washed with 2 mL 1% acetic acid in water to elute the matrix and then with 2 mL methanol to elute the metabolites at a rate of 30 drops/min. The methanol layer was evaporated to dryness under a stream of nitrogen gas at room temperature. The residue of urine was reconstituted with 600 ␮L methanol–water (80:20, v/v), and then centrifuged at 13,000 rpm for 10 min, and 2 ␮L samples of the supernatants were injected into the UHPLC/Q-TOF MS system for analysis. 2.3.3. Bile samples The method for preparing the bile samples was the same as that for preparing urine samples, except that 100 ␮L bile extract was reconstituted with 300 ␮L methanol–water (80:20, v/v). 2.3.4. Feces samples Feces samples weighing 1 g were extracted with 20 mL methanol–water (80:20, v/v). Then, after ultrasonic extraction for 20 min and centrifugation at 3500 rpm for 10 min, the supernatants were passed through a 0.22 ␮m membrane filter. Finally, 2 ␮L samples of the filtrates were injected into the UHPLC/Q-TOF MS system for analysis. 2.4. UHPLC conditions Separations were performed on an Acquity UPLC system (Waters) with an Acquity UPLC column (HSS C18 100 mm × 2.1 mm, 1.8 ␮m) at 40 ◦ C and at a flow rate of 0.45 mL/min. Biological samples were maintained at 4 ◦ C in the auto sampler. A VanGuard (Waters) pre-column (HSS C18 , 5 mm × 2.1 mm, 1.8 ␮m) was used with a mobile phase consisting of (A) 0.2% formic acid in water (v/v) and (B) 0.2% formic acid in acetonitrile (v/v). The gradient elution was as follows: 0–0.5 min, a linear gradient from 5 to 15% B; 0.5–4 min, 15 to 25% B; 4.1–5 min, 35 to 42% B; 5–7 min, 42 to 70% B; 7–7.1 min, 70 to 100% B; 7.1–9 min, 100% B, a linear gradient back to 5% B. The injection volume was 5 ␮L for plasma and 2 ␮L for other fluids. 2.5. Q-TOF/MSE parameters Analyses were performed using a Micromass-Q-TOF Premier mass spectrometer (Waters) coupled with an electrospray ionization (ESI) source operated in positive ion mode. There are sensitivity and resolution mode available for acquiring mass data. For ions originating from a given source and accelerated by a fixed potential, the mass resolving power of a TOF MS will increase as the flight path is lengthened. However, a longer flight path will sure reduce the total signal as fewer ions strike the detector. Therefore, the sensitivity mode is more sensitive, but the resolution mode offers higher mass resolution. In the present study, the sensitivity mode was used for there are some trace amount of metabolites in biological samples. The MS tune parameters were as follows: the cone and desolvation gas flow were 50 L/h and 700 L/h, respectively; the temperature of the source and desolvation were set at 130 ◦ C and 350 ◦ C, respectively; the capillary and the cone voltage were set at 3.0 kV and 40 eV, respectively; the micro-channel plates (MCPs) were operated at 1750 V and the Q-TOF mass spectrometer was operated in MSE mode with a low collision energy set at 6 eV in the

G. Zhang et al. / J. Chromatogr. B 941 (2013) 1–9

first function and a collision energy ramp from 20 to 40 eV in the second function. Centroid mode data were collected over the range of m/z 100–1000 in both functions, and the scan time was 0.2 s with an interscan delay of 0.02 s. For the dynamic range enhancement (DRE) lock mass, a 2 ng/mL solution of leucine-enkephalin generating an [M+H]+ ion (m/z 556.2771) was infused through the Lock Spray probe at 10 ␮L/min. 3. Results and discussion 3.1. Mass spectrometric analysis of Ir It is important to study the detailed fragmentation pathways of Ir because the mass fragmentation patterns of metabolites might be similar to those of the parent drug. By comparing the probable metabolite product ion spectrum with that of Ir, the structure of the metabolite could be confirmed. Furthermore, some product ions can help us to predict the structure of metabolites. This process is assisted by using Mass Fragment software. The nomenclature and diagnostic fragmentations of Ir are shown in Fig. 1. The i,j A+ and the i,j B+ labels designate primary product ions containing intact A and B rings, respectively, in which the superscripts indicate the C-ring bonds that have been broken. The C-ring bonds are numbered with a small font, the carbon atoms in the A, B, and C rings are labeled with a larger font, and the primary fragmentations are indicated. There are eleven principal fragment ion species in the product ion mass spectrum of the [M+H]+ ion of Ir. The empirical formula, observed and calculated mass/charge ratios, double bond equivalents, and mass errors are given in Table 1. The errors between the observed masses and calculated ones ranged from 0 to −1.5 mDa, indicating good mass accuracy. March et al. have studied the product ion mass spectrums of the aglycone ion from genistein-7-O-glucoside both in negative and positive ion mode [13]. Compared with genistein, the structure of Ir has a methoxyl group instead of a hydroxyl group at 4 -C-site, and an additional methoxyl group at the 6-C-site and, so, the fragment ions of Ir can be predicted according to those of genistein. The proposed fragment ion structures are given in Fig. 2. Because of space limitations, only one or two fragment ions with the same m/z are shown. 3.2. Classes of metabolites identified using MetaboLynxTM Use of the UHPLC/Q-TOF MS technique offers a higher peak capacity, greater resolution, increased sensitivity, and richer data on the exact mass of the molecular ion and fragment ions. It could be found that neat MS data of metabolites were extracted from high abundance endogenous in the Extracted Ion Chromatogram (EIC) mode. A total of 46 Ir metabolites were detected in rat plasma, bile, urine, and feces (Fig. 3). The Retention time, Calculated Mass, Observed mass (Molecular/main fragment Ions), Mass Error, Metabolite Description, Formula, and Matrix of the 46 metabolites are given in Table 2. The major metabolic pathways of Ir in rats are shown in Fig. 4. At least one structure of metabolites with the same m/z was presented. The structures of the metabolites were characterized by mass spectral fragmentation patterns, the elution order or confirmed by comparison of the chromatographic retention times and mass spectra with available reference standards. The results obtained are shown in detail below for each group. 3.2.1. Parent compound or its isomers (M0, M0-1, M0-2, and M0-3) The metabolites M0, M0-1, M0-2, and M0-3 exhibited the same protonated molecular ion at 315.09 (C17 H14 O6 , retention times 6.61, 4.86, 5.14 and 6.49 min) and were identified as the parent

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drug or its isomers. M0 and M0-3 were identified as Ir and 5,7dihydroxy-8,4 -dimethoxyisoflavone, respectively, by comparing the retention times and accurate mass spectra with those of authentic standards. M0-1 and M0-2 were the isomers of Ir. 3.2.2. Hydroxylated metabolites (M10 and M10-1) M10 and M10-1 exhibited the same protonated molecular ion at m/z 331.08 (C17 H14 O7 , retention times 4.35 and 5.52 min), which was 16 Da (O) higher than the protonated molecule of Ir. Reduced and monomethylated metabolite of Ir or hydroxylated metabolite of Ir might be the metabolic pathway. However, mass error was 0.7 mDa (317.0818–317.0811 Da) for hydroxylation but 37 mDa (331.1182–331.0811 Da) for reduced and monomethylated metabolism and, so, hydroxylation could be the metabolic pathway. Therefore, M10 and M10-1 were identified as hydroxylated metabolites of Ir. 3.2.3. Hydroxylated and sulfate metabolite (M16) M16 with a protonated molecular ion at m/z 411.04 (C17 H14 O10 S, retention time 4.16 min) was 80 Da (SO3 ) higher than the protonated molecule of M10. The characteristic fragment ions at m/z 331 (M+H−SO3 ) indicated that M16 was a hydroxylated, sulfate metabolite of Ir. 3.2.4. Sulfate metabolites (M14 and M14-1) M14 and M14-1 exhibited the same protonated molecular ion at m/z 395.04 (C17 H14 O9 S, retention times 3.63 and 5.48 min), which was 80 Da (SO3 ) higher than the protonated molecular ion of Ir. The diagnostic fragment ion at m/z 315 indicated that M14 and M14-1 were sulfate metabolites of Ir. 3.2.5. Dehydroxylated metabolites (M6 and M6-1) M6 and M6-1 with m/z 299.09 (C17 H14 O5 , retention times 4.65 and 5.95 min) were 16 Da (O) less than the protonated molecular ion of Ir. So, M6 and M6-1 were identified as dehydroxylated metabolites of Ir. 3.2.6. Glucuronide metabolites (M21, M21-1, and M21-2) These metabolites had the same protonated molecular ion at m/z 491.12 (C23 H22 O12 , retention times 2.60, 4.86 and 5.17 min), which was 176 Da (C6 H8 O6 ) higher than the protonated molecular ion of Ir. Diagnostic losses of 176 Da was corresponding to glucuronide conjugates. In the high energy channel, they all exhibited the same fragment ion at m/z 315 and they were identified as glucuronide metabolites of Ir. Among them, we were able to identify M21-2 as irisolidone-7-O-glucuronide (Ir-7G) by comparing the retention time and MS spectra with those of an authentic standard. 3.2.7. Diglucuronide metabolite (M24) M24 with a molecular ion at m/z 667.15 (C29 H30 O18 , retention time 5.07 min) was 352 Da (2C6 H8 O6 ) higher than the protonated molecular ion of Ir. The diagnostic fragment ions at m/z 491 (M+H−C6 H8 O6 ) and m/z 315 (M+H−2C6 H8 O6 ) indicated that M24 was a diglucuronide metabolite of Ir. 3.2.8. Demethyl metabolites (M7, M7-1, and M7-2) Metabolites M7, M7-1, and M7-2 with m/z 301.07 (C16 H12 O6 , retention times 5.16, 5.25, and 5.78 min) were 14 Da (CH2 ) lower than the protonated molecular ion of Ir, and the characteristic fragment ions were similar to those of Te, indicating that they were demethyl metabolites of Ir. The compounds eluted at 5.16 min (M7) and 5.25 min (M7-1) were identified as isotectorigenin and Te, respectively, by comparing with authentic standards. M7-2 with a similar polarity to Ir may have a similar structure to Ir, as described in Section 3.1, and the diagnostic fragment ion at m/z 132.06 indicated a hydroxymethylene conjugate at the B-ring 4 site (Fig. 2), and so M7-2 was identified as 6-hydroxybiochanin A (6-OH-BiA).

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G. Zhang et al. / J. Chromatogr. B 941 (2013) 1–9

Fig. 2. The proposed fragmentation pathway of irisolidone, obtained at collision energy of 20–40 eV.

Fig. 3. Metabolic profile of irisolidone in rats plasma, bile, urine, and feces after a single oral administration of 100 mg/kg.

G. Zhang et al. / J. Chromatogr. B 941 (2013) 1–9

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Table 1 Empirical formula, observed and calculated mass/charge ratios, double bond equivalents (DBE), and mass errors of the principal fragment ions observed in the product ion mass spectrum of [irisolidone + H]+ (m/z 315). No.

Predicted formula

Calculated mass (Da)

Observed mass (Da)

DBE

Error (mDa)

Error (ppm)

1 2 3 4 5 6 7 8 9 10 11

C16 H12 O6 + C15 H9 O6 + C15 H11 O5 + C14 H9 O5 + C15 H10 O4 + C14 H9 O4 + C13 H9 O4 + C13 H9 O3 + C7 H4 O5 + C6 H4 O4 + C9 H8 O+

300.0634 285.0399 271.0606 257.0450 254.0579 241.0501 229.0501 213.0552 168.0059 140.0110 132.0575

300.0629 285.0389 271.0597 257.0445 254.0579 241.0486 229.0500 213.0552 168.0053 140.0105 132.0572

11.0 11.5 10.5 10.5 11.0 10.5 9.5 9.5 6.0 5.0 6.0

0.5 1.0 0.9 0.5 0.0 1.5 0.1 0.0 0.6 0.5 0.3

1.7 2.8 4.4 1.9 2.4 6.2 2.2 0.0 3.6 2.1 2.3

3.2.9. Demethoxylation metabolites (M4 and M4-1) The metabolites M4 and M4-1 exhibited the same protonated molecular ion at m/z 285.08 (retention times 4.78 and 6.49 min), which represented a loss of 30 Da (OCH2 ) when compared with the protonated molecular ion of Ir. They were identified as demethoxylation metabolites of Ir. The demethoxylation could take place at the C-6 or C-4 position of Ir. M4-1 was identified as biochanin A (BiA) by comparing with authentic standard and, so, M4 could tentatively be identified as 5,7-dihydroxy-6-methoxy-3-phenyl-isoflavone. 3.2.10. Demethoxylation and glucuronide metabolite (M18) The metabolite M18 had a molecular ion at m/z 461.11 (retention time 4.87 min), which represented an increase of 176 Da (C6 H8 O6 ) when compared with the protonated molecule of M4 and, so, M18 was identified as the demethoxylation and glucuronide metabolite of Ir. The fragment ion at m/z 285 supports this hypothesis. The

glucoside is probably conjugated at the 7-O-site, compared with over 95% of Ir being transformed into Ir-7G in bile. However, the structure could not be fully confirmed in the present study. 3.2.11. Demethyl and sulfate metabolites (M12, M12-1, M12-2, and M12-3) M12, M12-1, M12-2, and M12-3 exhibited the same protonated molecular ion at m/z 381.03 (C16 H12 O9 S, retention times 3.67, 3.8, 3.84, and 5.37 min), which was 80 Da (SO3 ) higher than the protonated molecule of M7. They all showed the same fragment ion at m/z 301, and so they could be identified as demethyl and sulfate metabolites of Ir. M12-1, M12-2, and M12-3 were identified as sulfate metabolites, tectorigenin-7-O-sulfate (Te-7S), tectorigenin-4 -O-sulfate (Te-4 S) and 6-hydroxybiochanin A-6sulfate (6-OH-BiA-6S), respectively, by comparing with authentic standards.

Fig. 4. Metabolic pathways of irisolidone in vivo in rats after a single oral administration of 100 mg/kg. (1) Demethylation; (2) demethoxylation; (3) dehydroxylation; (4) decarbonylation; (5) reduction; (6) hydroxylation; (7) sulfation; and (8) glucuronidation.

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Table 2 UHPLC/Q-TOF MS analysis of irisolidone and its metabolites in rat plasma, bile urine, and feces. No.

Calculated mass (Da)

Observed mass (Da) Molecular/main fragment ionsa

Mass errorb (mDa)

Metabolite description

Formula

Matrixc

3.81 5.73 3.65 5.18 5.05 4.78 6.49 4.10 4.65 5.95 5.16 5.25 5.78 2.97 5.10 6.61 4.86 5.14 6.49 4.73 4.35 5.52 3.70 3.67 3.80 3.84 5.37 3.55 3.63 5.48 3.99 4.16 2.07 4.87 1.49 2.05 3.13 2.86 3.42 5.10 2.60 4.86 5.17 1.67 3.77 5.07

M1 M1-1 M2 M2-1 M3 M4 M4-1 M5 M6 M6-1 M7 M7-1 M7-2 M8 M8-1 M0 M0-1 M0-2 M0-3 M9 M10 M10-1 M11 M12 M12-1 M12-2 M12-3 M13 M14 M14-1 M15 M16 M17 M18 M19 M19-1 M19-2 M20 M20-1 M20-2 M21 M21-1 M21-2 M22 M23 M24

261.0763 261.0763 271.0606 271.0606 273.0763 285.0763 285.0763 287.0556 299.0919 299.0919 301.0712 301.0712 301.0712 303.0869 303.0869 315.0869 315.0869 315.0869 315.0869 317.0661 331.0818 331.0818 367.0124 381.0280 381.0280 381.0280 381.0280 383.0437 395.0437 395.0437 397.0229 411.0386 460.9836 461.1084 463.0877 463.0877 463.0877 477.1033 477.1033 477.1033 491.1190 491.1190 491.1190 557.0601 653.1354 667.1510

261.0764, 243.0657 261.0756, 243.0651 271.0599 271.0606, 253.0524, 215.0711, 197.0600, 169.0652, 153.0190 273.0779 285.0745 285.0762, 269.0450 287.0552, 269.0447 299.0922, 241.0978 299.0922, 285.0754 301.0706 301.0710, 287.0505 301.0707, 132.0562 303.0875 303.0864 315.0866, 301.0665, 285.0395 315.0865, 300.0627, 285.0746 315.0858, 300.0651, 285.0757 315.0872, 301.0685, 285.0762 317.0661, 301.0720 331.0797, 231.0778 331.0811, 301.0731 367.0126, 287.0546 381.0290, 301.0704 381.0276, 301.0711 381.0274, 301.0710 381.0283, 301.0707 383.0442, 303.0868 395.0428, 315.0887 395.0421, 315.0860 397.0231, 317.0666 411.0378, 331.0810 460.9829, 381.0284, 301.0719 461.1085, 285.0748 463.0889, 287.0561 463.0860, 287.0531 463.0862, 287.0551 477.1034, 301.0712 477.1037, 301.0707 477.1034, 301.0705 491.1172, 315.0883, 491.1165, 315.0861, 491.1190, 315.0862, 176.0747 557.0585, 477.1090, 381.0291, 301.0766 653.1351, 477.1011, 301.0713 667.1506, 491.1161, 315.0868

−0.10 0.70 0.70 0.00 −1.60 1.80 0.10 0.40 −0.30 −0.30 0.60 0.20 0.50 −0.60 0.50 0.30 0.40 1.10 −0.30 0.00 2.10 0.70 −0.20 −1.00 0.40 0.60 −0.30 −0.50 0.90 1.60 −0.20 0.80 0.70 −0.10 −1.20 1.70 1.50 −0.10 −0.40 −0.10 1.80 2.50 0.00 1.60 0.30 0.40

Didemethylation + decarbonylation + reduction Didemethylation + decarbonylation + reduction Demethylation + demethoxylation Demethylation + demethoxylation Demethylation + demethoxylation + reduction Demethoxylation Demethoxylation Didemethylation Dehydroxylation Dehydroxylation Demethylation Demethylation Demethylation Demethylation + reduction Demethylation + reduction Parent Parent isomer Parent isomer Parent isomer Demethylation + hydroxylation Hydroxylation Hydroxylation Didemethylation + sulfation Demethylation + sulfation Demethylation + sulfation Demethylation + sulfation Demethylation + sulfation Demethylation + reduction + sulfation Sulfation Sulfation Demethylation + hydroxylation + sulfation Hydroxylation + sulfation Demethylation + disulfation Demethoxylation + glucuronidation Didemethylation + glucuronidation Didemethylation + glucuronidation Didemethylation + glucuronidation Demethylation + glucuronidation Demethylation + glucuronidation Demethylation + glucuronidation Glucuronidation Glucuronidation Glucuronidation Demethylation + glucuronidation + sulfation Demethylation + diglucuronidation Diglucuronidation

C14 H12 O5 C14 H12 O5 C15 H10 O5 C15 H10 O5 C15 H12 O5 C16 H12 O5 C16 H12 O5 C15 H10 O6 C17 H14 O5 C17 H14 O5 C16 H12 O6 C16 H12 O6 C16 H12 O6 C16 H14 O6 C16 H14 O6 C17 H14 O6 C17 H14 O6 C17 H14 O6 C17 H14 O6 C16 H12 O7 C17 H14 O7 C17 H14 O7 C15 H10 O9 S C16 H12 O9 S C16 H12 O9 S C16 H12 O9 S C16 H12 O9 S C16 H14 O9 S C17 H14 O9 S C17 H14 O9 S C16 H12 O10 S C17 H14 O10 S C16 H12 O12 S2 C22 H20 O11 C21 H18 O12 C21 H18 O12 C21 H18 O12 C22 H20 O12 C22 H20 O12 C22 H20 O12 C23 H22 O12 C23 H22 O12 C23 H22 O12 C22 H20 O15 S C28 H28 O18 C29 H30 O18

F F F U,F F U P,F,U U,F B U,F U P,U,B,F U,F U U P,U,B,F P,F F U U U U,F U U P,U,B U P,U U B P,B U U U,P B U U P,U P,U,B U U,B,P U,B U P,U,B P,U,B P,U,B U

a b c

Only characteristic fragment ions were shown. Indicate the mass error of molecular ions. U, urine samples, B, bile samples, P, plasma samples, and F, fecal samples.

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RT (min)

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3.2.12. Demethyl and disulfated metabolite (M17) M17 with m/z 460.98 (C16 H12 O12 S2 , retention time 2.07 min) was 160 Da (2SO3 ) higher than the protonated molecule of M7. According to its fragment ions at m/z 381 (M+H−SO3 ) and m/z 301 (M+H−2SO3 ), M17 could be identified as the demethyl and disulfated metabolite of Ir. The most likely structure is tectorigenin7-O-sulfate-4 -O-sulfate (Te-7S-4 S). 3.2.13. Demethyl and glucuronide metabolites (M20, M20-1, and M20-2) M20, M20-1, and M20-2 showed the same protonated molecular ion at m/z 477.10 (C22 H20 O12 , retention times 2.86, 3.42 and 5.10 min), which was 176 Da (C6 H8 O6 ) higher than the protonated molecule of M7, suggesting that they were demethyl and glucuronide metabolites of Ir. These metabolites showed the same fragment ion at m/z 301, which indicated that they had a similar basic carbon skeleton. The glucuronide conjugate position may be at the 7-O- or 4 -O-site of M7. Compared with authentic standards, M20 was identified as tectorigenin-7-O-glucuronide (Te-7G). Another two demethyl and glucuronide metabolites may involve the 6-O- or 7-O-site of 6-OH-BiA, which was the isomer of Te. The retention time of M20-2 was much longer than those three, so M20-2 could tentatively be identified as biochanin A6-O-glucuronide (BiA-6G) by comparing its retention time with biochanin A-6-O-sulfate (BiA-6S) relative to Te-7S [12]. Further investigation is needed to determine the complete structure. 3.2.14. Demethyl and diglucuronide metabolite (M23) M23 exhibited the same protonated molecular ion at m/z 653.14 (C28 H28 O18 , retention time 3.77 min), which was 352 Da (2C6 H8 O6 ) higher than the protonated molecule of M7. The diagnostic increase of 352 Da corresponded to the diglucuronide conjugate metabolite. The fragment ions at m/z 477 (M+H−C6 H8 O6 ) and m/z 301 (M+H−2C6 H8 O6 ) can support our identification. For these reasons, M23 was identified as the demethyl and diglucuronide metabolite of Ir. 3.2.15. Demethyl, glucuronide, and sulfate metabolite (M22) M22 exhibited a protonated molecular ion at m/z 557.06 (C22 H20 O15 S, retention time 1.67 min) and was 256 Da (C6 H8 O6 +SO3 ) higher than the protonated molecule of M7, therefore, M22 was identified as the demethyl, glucuronide, and sulfate metabolite of Ir. M22 generated fragment ions at m/z 381 (M+H−C6 H8 O6 ), m/z 477 (M+H−SO3 ), and 301 (M+H−C6 H8 O6 −SO3 ), which supports our identification. M22 was identified as tectorigenin-7-O-glucuronide-4 -O-sulfate (Te-7G4 S) by comparing its retention time and MS spectrum with that of an authentic standard. 3.2.16. Demethyl and reduced metabolites (M8, M8-1) M8 and M8-1 exhibited the same protonated molecular ion at m/z 303.09 (C16 H14 O6 , retention times 2.97 and 5.10 min), which was 2 Da (2H) higher than the protonated molecule of M7, suggesting that they were demethyl and reduced metabolites of Ir. The reduction of M8 and M8-1 most probably happened at the 2,3-double bond, furthermore, isoflavone glycitein could be converted to 6-OH-dihydrodaidzein in rats [14], which also support our speculation. Demethylation may have occurred at the 6-O- or 4 -O-site, as we described in Section 3.2.8, where a metabolite with a methyl group at the 6-O-site was eluted before the methyl group at the 4 -O-site, and so, we tentatively identified M8 and M8-1 as 2,3-dihydro-5, 7-dihydroxy-3-(4-hydroxyphenyl)-6-methoxy-isoflavone and 2,3-dihydro-5,6,7-trihydroxy-3-(4-methoxyphenyl)-isoflavone, respectively.

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3.2.17. Demethyl, reduced, and sulfate metabolite (M13) M13 with m/z 383.04 (C16 H14 O9 S, retention times 3.55 min) was 80 Da (SO3 ) higher than the protonated ion of M8, generating a fragment ion at m/z 303.087 and, thus, we identified M13 as the demethyl, reduced, and sulfate metabolite of Ir. The sulfation may be at the 7-O-site. We believe that the reduction happened at the 2,3-double bond or the carbonyl group in the C-ring, and named them M13-a and M13-b, respectively. The calculated 1octanol/water partition coefficient (CLog P) of these metabolites were calculated using ChemBioDraw Ver.11.0 (Cambridge-Soft, Cambridge, MA, USA) and a value of 2.81 was obtained for M13a and a value of 1.06 was obtained for M13-b. Because the CLog P value of Te-7G-4 S was 2.2, and its retention time was 1.67 min, far less than the retention time of M13 (3.55 min), the reduction may have occurred at the 2,3-double bond. Furthermore, isoflavone glycitein encountered similar reaction in vivo [14], which also supported our hypothesis. 3.2.18. Demethyl and hydroxylated metabolite (M9) M9 with an m/z of 317.07 (C16 H12 O7 , retention time 4.73 min) was 16 Da (O) higher than the protonated M7. Demethyl and hydroxylated metabolite of Ir or reduced metabolite of Ir was the probable metabolite description of M9. While mass error was 0 mDa (317.0661–317.0661 Da) for demethyl and hydroxylated metabolism but 36 mDa (317.1025–317.0661 Da) for reduction and, so, reduction could not be the metabolic pathway of M9, and we identified M9 as demethyl and hydroxylated metabolite of Ir. 3.2.19. Demethyl, hydroxylated, and sulfate metabolite (M15) M15 with an m/z of 397.02 (C16 H12 O10 S, retention time 3.99 min) was 80 Da (SO3 ) higher than the protonated molecule of M9. M15 generated a fragment ion at m/z 317.07, and we could identify M15 as the demethyl, hydroxylated, and sulfate metabolite of Ir. 3.2.20. Didemethyl metabolite (M5) M5 with an m/z of 287.06 (C15 H10 O6 , retention time 4.11 min) was 28 Da (2CH2 , 315–287) less than the protonated molecular ion of Ir. Decarbonylation or didemethylation might be the possible metabolic pathway. By comparison with calculated and observed mass, the mass error of decarbonylation and didemethylation was 37 mDa (287.00919–287.0552 Da) and 0.4 mDa (287.0556–287.0552 Da), respectively. Therefore, M5 was identified as a didemethyl metabolite of Ir. There are only two CH3 groups in the structural formula of Ir (Fig. 1), so M5 was tentatively identified as 6-hydroxygenistein. 3.2.21. Demethyl and demethoxylation metabolites (M2 and M2-1) M2 and M2-1 had the same protonated molecular ion at m/z 271.06 (C15 H10 O5 , retention times 3.65 and 5.18 min) was was 44 Da (CH2 + CH2 + O) lower than the protonated molecular ion of Ir. They were identified as demethyl and demethoxylation metabolites of Ir. The calculated 1-octanol/water partition coefficient (CLog P) value of these metabolites obtained using ChemBioDraw Ver.11.0 was 2.41 for M2-a and 2.50 for M2-b. Furthermore, the product ion mass spectrum of M2-1 in positive mode was similar to that of genistein [13], and they both produced the same fragment ions at m/z 253, 243, 215, 197, 169, and 153 and, thus, M2 and M2-1 were tentatively identified as 5,6,7-trihydroxy-3-phenylisoflavone and genistein, respectively. 3.2.22. Demethyl, demethoxylated, and reduced metabolite (M3) A metabolite with an m/z of 273.08 (C15 H12 O5 , retention time 5.05 min) was only found in feces, and it was 42 Da (CH2 + OCH2 –2H) lower than the protonated molecular ion of Ir.

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M3 was tentatively be identified as a demethyl, demethoxylated, and reduced metabolite of Ir. As described in Section 3.2.17, the reduction was at the 2,3-double bond in the C-ring.

Table 3 Metabolic pathways and relative content of 25 kinds of metabolites of irisolidone in rat plasma, bile, urine, and feces. M+H

3.2.23. Didemethyl, decarbonylated, and reduced metabolites (M1 and M1-1) M1 and M1-1 with the same protonated molecular ion at m/z 261.08 (C14 H12 O5 , retention times 3.81 and 5.73 min) were 54 Da (CH2 + CH2 + CO–2H) lower than the protonated molecular ion of Ir. They were identified as didemethyl, decarbonylated, and reduced metabolites of Ir. Both of them exhibited the same fragment ions at m/z 243 (261–18), suggesting a molecular loss of H2 O. The most likely reduction site is the 2,3-double bond in the C-ring, and decarbonylation may also happen at the C-ring, although the structure could not be fully confirmed in the present study. Such metabolites could only be detected in feces, and we believe that Ir underwent didemethylation, decarbonylation, and reduced reaction, and was transformed into M1 and/or M1-1 by intestinal bacteria, and these metabolites were not significantly absorbed into the blood stream or were unstable in blood. 3.2.24. Didemethyl and glucuronide metabolites (M19, M19-1 and M19-2) M19, M19-1 and M19-2 exhibited the same protonated molecular ion at m/z 463.09 (C21 H18 O12 , retention times 1.49, 2.05 and 3.13 min), which was 176 Da (C6 H8 O6 , 463–287) higher than the protonated molecule of M5, suggesting that they were didemethyl and glucuronide metabolites of Ir. 3.2.25. Didemethyl and sulfate metabolite (M11) M11 had an m/z of 367.01 (C15 H10 O9 S, retention time 3.7 min) which was 80 Da (SO3 ) higher than the protonated molecule of M5 and, so, M11 was identified as the didemethyl and sulfate metabolite of Ir. The fragment ion at m/z 287 supported our hypothesis. The conjugation most likely happed at the C-7 or C-4 hydroxyl group and, so, M11 was tentatively identified as 6-hydroxygenistein-7S or 6-hydroxygenistein-4 S, although the structure could not be fully confirmed in the present study. In conclusion, metabolites M0, M0-3, M4-1, M7, M7-1, M121, M12-2, M12-3, M20, M21-2, and M22 were identified as Ir, 5,7-dihydroxy-8,4 -dimethoxyisoflavone, BiA, isotectorigenin, Te, Te-7S, Te-4 S, 6-OH-BiA-6S, Te-7G, Ir-7G, and Te-7G-4 S, respectively, by comparison with authentic standards. Metabolites M2, M2-1, M4, M5, M7-2, M8, M8-1, M17, M20-2 were speculated to be 5,6,7-trihydroxy-3-phenyl-isoflavone, genistein, 5,7dihydroxy-6-methoxy-3-phenyl-isoflavone, 6-hydroxygenistein, 2,3-dihydro-5,7-dihydroxy-3-(4-hydroxyphenyl)6-OH-BiA, 6-methoxy-isoflavoe, 2,3-dihydro-5,6,7-trihydroxy-3-(4methoxyphenyl)-isoflavone, Te-7S-4 S, and BiA-6G, respectively, from their polarity, fragmentation patterns, etc. Only the metabolic pathways of other metabolites were identified, while their structures could not be confirmed in the present study, and some metabolites may even be new, although the metabolism of isoflavones has been comprehensively studied for many years. 3.3. Relative content of metabolites in plasma, bile, urine, and feces The peak area of each metabolite was also obtained automatically using the MetaboLynxTM software (Fig. 3), and the metabolites with the same m/z are summarized together, as shown in Table 3. The relative content of each kind of metabolite was indicated as the percentage of their peak areas relative to the total peak areas in each biological sample (plasma, urine, bile, and feces). Over 65% of Ir was transformed into Ir-7G in blood, while only 2% of Ir remained unchanged. Over 95% of Ir was transformed into Ir-7G in bile, which

261.08 271.06 273.08 285.08 287.06 299.09 301.07 303.09 315.09 317.07 331.08 367.01 381.03 383.04 395.04 397.02 411.04 460.98 461.11 463.09 477.10 491.12 557.06 653.14 667.15

Relative content (%) Urine

Feces

Bile

Plasma

0.00 0.83 0.00 13.12 3.67 0.62 27.72 1.64 27.11 0.18 0.25 0.07 13.54 0.29 0.00 0.25 0.28 0.34 0.00 0.54 3.42 5.11 0.27 0.62 0.14

0.06 0.43 0.05 4.67 7.87 0.46 5.20 0.00 80.73 0.00 0.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.17 0.29 0.00 0.47 0.00 0.00 0.00 0.11 0.00 0.36 0.00 0.00 0.00 0.78 0.00 0.48 96.87 0.46 0.00 0.00

0.00 0.00 0.00 2.22 0.00 0.00 3.04 0.00 2.07 0.00 0.00 0.00 2.48 0.00 1.76 0.00 0.00 0.81 0.00 1.35 16.97 66.6 0.64 0.54 0.00

was in accordance with the blood data, indicating a marked hepatic first-pass effect. About 28% of Ir and its isomers were excreted in urine as the unchanged form. As to feces, 80% of Ir was excreted in unchanged form. 3.4. Result of analysis The UHPLC/Q-TOF MS method was employed for rapid analysis of the metabolic profile of Ir in rats. Some isoflavones were easily broken into fragments in the cone, especially when the cone voltage was set at a relative high value. In this study, the cone voltage varied from 15 to 50 eV, and the signal of the metabolites exhibited the highest sensitivity when the cone voltage was set at 40 eV. With the help of the MetabolynxTM software, abundant potential metabolites were identified. Also, a lot of false positives were abandoned by carefully setting the parameters, such as Elemental Composition, MS traces, etc., and by checking the given m/z as molecular or fragment ion. Because of the low content of metabolites in biological samples, especially in plasma, the sensitivity mode was selected to acquire data, in which the signal was 10-fold higher than that in the resolution mode. Flavones were mostly detected in negative ion mode [15,16], but the mass signal gave a higher response in positive ion mode than that in negative ion mode when 0.2% formic acid was added to mobile phase; accordingly, the positive ion mode was used in our study. A total of 46 metabolites including the parent compound Ir were identified in plasma, bile, urine, and feces samples compared with blank samples (Fig. 3). Our results indicated that decarbonylation, reduction, demethylation, demethoxylation, dehydroxylation, hydroxylation, sulfation, and glucuronidation were the major metabolic pathways of Ir in vivo (Fig. 4). Bai et al. have identified 4 metabolites in plasma and 12 metabolites in urine after oral administration of KA to rats [12,17]. Nine of the metabolites were also detected in the present study, except tectoridin, daidzein and equol, indicating that KA was initially hydrolyzed to Ir by intestinal bacteria [3,6], and then absorbed into blood, and the metabolites of KA and Ir were similar after oral administration. Because of the higher sensitivity of the UHPLC/Q-TOF MS method, some new metabolites

G. Zhang et al. / J. Chromatogr. B 941 (2013) 1–9

at low concentrations were detected in this study, while the accurate structures of most metabolites could not be confirmed only by MS. Only phase I metabolites were found in feces, indicating that Ir was initially transformed by intestinal bacteria, and then Ir and these phase I metabolites were absorbed into the blood through the small intestine, undergoing phase II metabolism, such as sulfation and glucuronidation. The parent drug was the most important excretion form with 28% in the urine and 80% in the feces. As a polyhydroxyl compound, very little Ir was detected in plasma (2%), and it was mainly in the form of phase II metabolites. The metabolite Ir7G (more than 65% in plasma, more than 95% in bile) was the major metabolite of Ir in vivo. So, we were able to infer that Ir undergoes extensive first-pass metabolism in the gut epithelium or liver after absorption, followed by conjugation with glucuronic acid in the intestinal epithelium and liver by UGT resulting in conjugates that are excreted in the bile, urine and feces. In general, the role of phase II metabolism in vivo is drug detoxification by means of conjugation of phase I metabolites with endogenous substances to increase their water solubility, and to reduce or abolish their biological activity [18]. However, several studies have shown that some conjugated isoflavone metabolites exhibit some physiological and pharmacological effects, such as the inhibitory effect of daidzein-4 ,7-disulfate on sterol sulfatase in hamster liver microsomes [19], the stimulatory effect of daidzein7-glucuronide-4 -sulfate on growth of MCF-7 cells [20], and the hypotensive and vasodilator effects of daidzein sulfates in rats [21]. The metabolic pathways and the main metabolites, especially the conjugated metabolites should not be ignored, and the bioactivities of these conjugated metabolites need further investigation. 4. Conclusion In this paper, the UHPLC/Q-TOF MS method was employed for rapid analysis of the metabolic profile of Ir in rat plasma, bile, urine, and feces, after administration of a single dose, assisted by automatic data analysis software (MetaboLynxTM ). In total, 46 metabolites, including the parent compound irisolidone, were identified in rats, and 11 of them were identified by comparison with authentic standards, and 9 structures were inferred from fragment ion or polarity data. Decarbonylation, reduction, demethylation, demethoxylation, dehydroxylation, hydroxylation, sulfation, and glucuronidation were the identified metabolic

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pathways of Ir in rats. The relative content of the metabolites in biological samples provides us with the major metabolic pathways. To our knowledge, this is the first time the metabolic profile of Ir has been studied in vivo, and it provides evidence for the possible metabolic ways of isoflavones. Acknowledgments This work was financially supported by the Research Fund for the Doctoral Program of Higher Education, China (No. 200801630007), the 2008 Scientific Technology Plan Project from Science and Technology Department of Liaoning Province, China (No. 20082260233), the central financial support to the local provincial key disciplines of special fund, and distinguished professor fund of Liaoning province of 2011. References [1] E. Sjostrom, Wood Chemistry Fundamentals and Applications, Academic Press Inc., New York, London, 1981. [2] E.C. Bate-Smith, in: W.E. Hillis (Ed.), Wood Extractives and their Significance to the Pulp and Paper Industries, Academic Press Inc., New York, London, 1962. [3] Y.O. Han, M.J. Han, S.H. Park, D.H. Kim, J. Pharmacol. Sci. 93 (2003) 331. [4] K. Yamaki, D.H. Kim, N. Ryu, Y.P. Kim, K.H. Shin, K. Ohuchi, Planta Med. 68 (2002) 97. [5] S.W. Min, D.H. Kim, Biol. Pharm. Bull. 30 (2007) 1965. [6] J.E. Shin, E.A. Bae, Y.C. Lee, J.Y. Ma, D.H. Kim, Biol. Pharm. Bull. 29 (2006) 1202. [7] J.S. Park, M.S. Woo, D.H. Kim, J.W. Hyun, W.K. Kim, J.C. Lee, H.S. Kim, J. Pharmacol. Exp. Ther. 320 (2007) 1237. [8] K.A. Kang, R. Zhang, M.J. Piao, D.O. Ko, Z.H. Wang, B.J. Kim, J.W. Park, H.S. Kim, D.H. Kime, J.W. Hyuna, Bioorg. Med. Chem. 16 (2008) 1133. [9] S.Y. Kim, D.H. Kim, J.W. Hyun, J.W. Henson, H.S. Kim, Biochem. Biophys. Res. Commun. 344 (2006) 3. [10] S.Y. Kim, E.J. Lee, M.S. Woo, J.S. Jung, J.W. Hyun, S.W. Min, D.H. Kim, H.S. Kim, Biochem. Biophys. Res. Commun. 366 (2008) 493. [11] E.A. Bae, M.J. Han, D.H. Kim, Planta Med. 67 (2001) 161. [12] X. Bai, J. Qu, J. Lu, Y. Kano, D. Yuan, J. Chromatogr. B 879 (2011) 395. [13] R.E. March, X.S. Miao, C.D. Metcalfe, Int. J. Mass Spectrom. 232 (2004) 171. [14] C.E. Rufer, R. Maul, E. Donauer, E.J. Fabian, S.E. Kulling, Mol. Nutr. Food Res. 51 (2007) 813. [15] P. Shi, Q. He, Y. Song, H. Qu, Y. Cheng, Anal. Chim. Acta 598 (2007) 110. [16] H. Zhang, X. Yang, J. Pharm. Biomed. Anal. 49 (2009) 843. [17] X. Bai, Y. Xie, J. Liu, J. Qu, Y. Kano, D. Yuan, Drug Metab. Dispos. 38 (2010) 281. [18] D. Liska, Altern. Med. Rev. 3 (1998) 187. [19] C.K. Wong, W.M. Keung, Biochem. Biophys. Res. Commun. 233 (1997) 579. [20] J. Kinjo, R. Tsuchihashi, K. Morito, T. Hirose, T. Aomori, T. Nagao, H. Okabe, T. Nohara, Y. Masamune, Biol. Pharm. Bull. 27 (2004) 185. [21] Y.X. Cao, X.J. Yang, J. Liu, K.X. Li, Basic Clin. Pharmacol. 99 (2006) 425.