Journal of Parmaceticl an Biomedical Analysis 97 (2014) 166-177 Contents lists available at ScienceDirect, Journal of Pharmaceutical and Biomedical Analysis i journal homepage: www.elsevier.com/locate/jpba Identification of known chemicals and their metabolites from Alpinia oxyphylla fruit extract in rat plasma using liquid chromatography/tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring Qo Feng Chen*", Hai-Long Li*, Yin-Feng Tan*, Yong-Hui Li*», Wei-Yong Lai*, Wei-Wei Guan*®, Jun-Qing Zhang*"*, Yuan-Sheng Zhao‘, Zhen-Miao Qin®” Schoo of hemacy Hain Medical Uses, ako 57101, china 285.0 assy] fovas Mocéetoos. — Tzalpinin aan 285.0-+242.0 Coiechen ta. /Juml of Pharmacetical and Biomedical Analyse 97 (2014) 166-177 1m Enzymatic hydrolysis ” M_KaeG 477.19301.1 von 75 ° vensve2 M_KaeG Yichi extract fess 4771-03011 + esate vtec Kaempferide Yizhi extract fe " sont-2860 The precursor-to-product ion paits used for selected reaction monitoring of glucuronidated yakuchinone A (M.YA-G), sulfated yakuchinone A (MLYA-S), glucuronidated yakuchinone B (M.YB-C), sulfated yakuchinone B (M.YB-S), glucuronidated oxyphyllacinol (M.Oxy-C), sulfated oxyphyllacinol (M.Oxy-S), glucuronidated fectochrysin (M-Tec-G), sulfated tectochrysin (M-Tec-S), glu curonidated apigenin-4’,7-dimethylether (M.APi-G), sulfated apigenin-4’,7-dimethylether (M.Api-S), glucuronidated izalpinin (M.lza-G), Sullated izalpinin (M4225), izalpinin diglucuronide (M.1za-2G), izalpinin disulfate (M.t2a-25), sulfated izalpin gla- curonide (M-lza-G-S), glucuronidated chrysin (M.Chr-G), sulfated chrysin (M.Chr-S), chrysin diglucuronide (M.Chr-2G), chrysin dlisulfate (M.Chr-25), sulfated chrysin glucuronide (M.Chr-C-5), slucuronidated Kaempferide (M-Kae-G), sulfated kaempferide (M.Kae-S), kaempferide diglucuronide (M.Kae-2G), kaempferide disulfate (M-Kae-25), sulfated kaempferide glucuronide (M_Kae- 8), glucuronidated keempherol (M_Kmf-G), sulfated kaempherol (M.krnf-S) were m/z 489.2312, 393.2»3132, 4872->3112, 3012-3112, 4913-43153, 3953-3153, 445.1+269.1, 349152691, 4752-42992, 3792-2982, 461.0+285.0, 3650-42850, 637.0-+2850, 445,0-+285.0, 541.0285. 4811255.1, 335.1-5255.1, 6071-2551, 415.1255. SUL1255.1, 477.1-5301.1, 381.3011, 653.1301, 4611-0301.1, 557.1-0301.1, 463242872 and 3672-2872 respectively 3, Results and discussion 3.1, Parent chemicals from Yizhi extract in ra plasma After p.o. administration of Yizhi extract to rats, the parent chemicals of tectochrysin, chrysin, apigenin-4,7-dimethylether Fig. 1a), nootkatone (Fig. 1b),yakuchinone A/8 and oxyphyllacinol were detected (Fig 1c). On the contrast, the free forms of zalpinin and kaempferide were almost undetectable (Fig. 1b) Alter p.o. ingestion of Suoquan pills or Suoquan capsules, the parent drugs of Yizhi flavonoids except for tectochrysin were almost undetectable, but the nootkatone, yakuchinone A and oxy- phyllacinol were measured in the rat plasma samples (Fig, 2) 3.2. Total flavonoids and diarylheptanoids from Yizhi extract in rat plasma after hydrockloric acid treatment The Yizhi flavonoids and diarylheptanoids contain phenolic hydroxyl groups (OH) in their chemical structures. This func tional group is often catalyzed by conjugative enzymes, such as UbP-glucuronyltransferase and sulfotransferase, to form phase ll ‘metabolites. These phase Il reactions resultin the synthesis of more hydrophilic compounds that increase their elimination efficiency via normal renal and intestinal pathways. In the current study, ‘we found that the systemic exposure to the parent chemicals of Yizhi flavonoids and diarylheptanoids were very low, or absent. ‘Therefore, the total drug concentration in the plasma matrix can be used to substantiate the systemic exposure to these chemicals after p.o. administration. This strategy needs to convert all conju- gated metabolites tothe parent drug forms via acidic or enzymatic hydrolysis. ‘After p.o. medication of Yizhi extract to rats, all the Yizhi flavonoids could be measured inrat plasma sample treatedby acidic hydrolysis (Fig. 3a and b). The yakuchinone A and oxyphyllacinol could also be detected (Fig. 3¢). This was not true to yakuchinone B. Meanwhile, the nootkatone could not be clearly determined because of the obvious degeneration (Fig. 3b). Therefore, we hypothesized that this sesquiterpene and the other chemicals might be lable during the process of hydrochloric acid treatment. Subsequently, we studied the stability of these Yizhi chemicals under harsh pH and temperature conditions. Our results revealed that incubation of these molecules with an equivalent volume of 4M hydrochloric acid at 80°C for only O.5h resulted in at least ‘40-60% degradation, Moreover, the concentration of hydrachloric acid, ie, 2M,1M,05 Mand0.2M, was further tested and theresults showed that the incubation conditions led to obvious degradation forall analytes. Thus, the suitability ofthe acidichydrolysis method for the simultaneous quantification of these compounds was not ood.4 Chm et a Jour of Pharmacia and Bem! Anas 97 (2014) 166-177 3.3, Phase If conjugation metabolites of Yzhi flavonoids and diarytheptanoids in vat plasma Glucuronidated or sulfated metabolites, diglicuronide,disul- fate and sulfated diglucuronide metabolites of Yizhi flavonoids and diarylheptanoids were monitored using LC-MS/MS with selected ‘multiple reaction monitoring. Our results revealed thatthe dsul- fate, sulfated metabolites and sulfated diglicuronide metabolites were not measured. The major phase metabolites for these chemn- ieals were glucuronidated forms. These metabolites were absent aller enzymatic hydrolysis by B-glucuronidase.As shown in Fig. 4a and b, glucuronidated tectochrysin and chrysin were obviousiy detected with 2 retention time (Rt) at 530min and 4:55 min, respectively. After enzymatic hydrolysis by Beglicuronidase, these peaks disappeared; meanwhile, the aglycone tectochrysin (Rt 05min) and chrysin (RC 5:98min) Were librated from their conjugated forms. The peaks of Rt 5.35 min(Fig 4a) and Rt4.09 min, Rt 5.54min (Fig. 4b) all came from the controlled biological matrix. As for apigenin-4',7-dimethylether with a 5-OH, we proposed its glucuronidated metabolite appeared at Rt 5.55min (Fig. 4c). Izalpinin has two hydroxyl groups (3-OH and S-OH) and these posi- tions may be catalyzed by UDP-glucuronyltransferase theoretically In this study, we found two peaks (Rt 4.62min and Re 5.61 min) ‘occurring in the precipitated plasma samples (Fig. dd), The latter peak (Rt 5.61 min) might be the major glucuronidated metabo- lite because of steric hindrance between 3-OH and 2-phenyl group of izalpinin. Additionally, the peaks (Rt 4.27 min and Rt 6.13 min) present in enzymatic hydrolysis samples both originated from con- trolled matrix. Although there are three hydroxyl groups in the chemical structure of kaempferide, we only found one peak (Rt 5.11 min; Fig. 4e) standing for the glucuronidated kaempferide ‘metabolite, However, the aglycone kaempferide (Rt 6.16 min) was Yakuchinone B 028 3ILIU7O ‘A Methanol precipitation (Blank) Methanol precipitation (.v. Yakuchinone B) ba voy) M43 —-Yakuchinone A 313,29136.9 ox 880, Mox0013<¢ Oayphyllacinol 315,39137.0 Mexia. M_YB.G 487.2—9311.2 le ool LI LL clad bo Max. 608.7 ep. Lay Man ist8 Te. M_Oxy-G Fig. Representative chromatograms ridenicationetyakuchinone Bandits metabolicreductian metabolerinat plasma after methane precipitation andensymati Feros treatment (Theat received ant, boas nection of ykehinane Briton a2 mek"chen ta. /Jurl of Pharmacetical and Biomedical Anais 97 (2014) 166-177 18 Enzymatic hydrolysis, (Blank) Enzymatic hydrolysis (i.v. Yakuchinone B) Yakuchinone B S217.0 Yakuchinone A 313.2-7136.9 Oxyphyllacinol 315.3 9137.0 i 00. cp nad Meestenres M_YB.G 178 487.2—9311.2 aye nn eae M_YAG Ng 119 489.293 131.2 LI ao m ve nae mao ML O1y-G net a fans 491.3-9315.3 ip not the major hydrolysis product after B-glucuronidase treatment, The chemical structure of Rt 5.18 min temained unknown. Compared withthe parent diarylheptanoids, the glucuronidated forms of yakuchinone A/B and oxyphyllacinol were the main ‘metabolites. This data are only shown in Supplemental Fig. $12-c Yakuchinone B sulphate and oxyphyllacinol sulphate were not ‘observed inthe rat plasma afterp.o. administration of Yizhi extract, as well as Suoquan capsules and Suoquan pills. These results were then confirmed in another rat studies in which the animals ceived, an Ly. bolus administration of injectable yakuchinone B solution However, this was not true to the yakuchinone A after an iv. bolus injection of yakuchinone A solution to rats. Aminor peak of M.YA-S. (Rt6:74min) was observed (Fig. 2a). This peak did not disappear alter enzymatic hydrolysis (Fig, S2b) because of, at least in part, the very poor sulfatase activity (Le, 75UmL-!) and non-optimal incubation PH condition. Similarly, the diaryiheptanoid curcumin sulphate both in rodents and humans have also been reported 3) {zalpinin, chrysin and kaempferide have two or three hydroxyl groups; therefore, diglucuronide metabolites may be produced in theory after p.o. administration of Yizhi extract to rats. Our results revealed that some peaks occurred after methanol precipi- tation treatment in the selected reaction monitoring ion channels, ie, 637.0-+285,0, 607.1-+255.1 and 653.1201, standing for Mlza-2G, M.Chr-2G and MKae-2G, respectively, The retention time of major peak was Rt 4.36 min, Rt 3.63 min and Rt 3.63 min, respectively (left panels of Fig. S3a-c). These peaks vanished completely after B-glucuronidase incubation (right panels of Fig. S3a-c), Furthermore, the peaks of Rt 428min and Rt 6.14min (Fig, $3a), Rt 4.09 min and Rt 5.54 min (Fig. S3b) were all from the controlled blank samples. Further work would be required to char= acterize the chemical structures of these proposed diglucutonide176 Chen et a Journal of Pharmaceutical and Bm! Anas 97 (2014) 166-177 cot 0 3, Dov oor ° orca coe Fig. 6. Proposed metabolic pathways ofthe yakuchinone Bin plasma fllvang iv -minstation of mjectable yakuchinone solution rats metabolites. In addition, aglycone kaempferol (Rt 4.80 min) was also observed in the enzyme-treated plasma samples, in place of methanol-precipitated ones, after po. medication (Fig, Sa). The free kaempterol was not measured, This metabolite originated from the kaempferide via O-demethylation reaction and then catalyzed by UDP-glucuronyltransferase to form glucuronidated kaemplerol metabolites (Fig. 4b). Demethylation of tectochrysin to form chrysin has been described previously [24]. Thus, we could not exclude possibilty ofthe total plasma chrysin partly coming from tectochnysin. In addition, demethylated intermediates of apigenin- 4.,7-dimethylether and izalpinin were transformed immediately to their glucuronidated metabolites (Fig. S4c and d). Chrysin sulta- tion oF glucuronidation have been reported both in human liver samples [24], in human liver cell lines {25}, human intestinal cell, lines [26], as well asin recombinant human sulfotransferase pro- teins 27) and rt hepatocytes (28). In this study, we have identified chrysin glucuronidation metabolite; however, we could not make sue chrysin formed sulfate because of multiple peaks appearing in the 335.1-+255.1 ion channel for M.Chr-5 (Fig. S4e), 43.4. Biotransformation of Vizhi diarylheptanoids via metabolic reduction in rats Yakuchinone A, B and oxyphyllacinol are analogs of diarythep- tanoids. The chemical difference between yakuchinone B and A is that aC1-C2 double bonds displaced by aC1—C2 single bond in the latter compound. A free hydroxyl in the C3 position af the oxyphyl- Jacinol; when this group is oxidized to a ketone group, the chemical structure is transformed to yakuchinone A In this study. we have found that the glucuronidated forms were major metabolites for these diarylheptanoids. However, we suspected that biotransfor- mation among these chemicals via metabolic reduction would happen in rats receiving po. or iv. medication. As shown in Fig, 2a and b, the oxyphyllacinol (Rt 6.89 min) in rat plasma was detected after an iv. bolus administration of yakuchinone A soli tion, Meanwhile, the metabolites of M.YA-G (Rt 6.03 min) and M.YA-S (Rt 6.74 min) were also measured. In another rat study. where the rats received an iv. bolus medication of yakuchinone B solution. the yakuchinone B (Rt 10.25 min), yakuchinone A (Rt 10.11 min), oxyphyllacinol (Rt 10.12 min), as well as M.YB-G (Rt ‘9.64 min) and M.YA-G (Rt 9.65 min) were finely identified (Fig. 5a and b), Overall, these studies have confirmed our hypothesis that the metabolic reduction did occur. Yakuchinone B was reduced {o yakuchinone A: yakuchinone A then be reduced to oxyphyl- lacinol. Meanwhile, these products were rapidly transformed to slucuronidated metabolites. The proposed metabolic pathways of yakuchinone A/B and oxyphyllacinol in rats are summarized in Fig.6 ‘Cuscummin has been reported to undergo metabolic reduction to dihydrocurcumin, tetrahydrocurcumin and hexabydrocurcuminby endogenous reductase systems in a stepwise manner and subse- quently glucuronidated by UDP-glucuronosyl transferase [29,30] Subsequent report reveals that curcumin is a poor substrate of human microsomal reducing enzymes; in contrast, human gut cytosol reduced curcumin easily. Furthermore, equine alcohol dehydrogenase was found to catalyze the reduction of curcumin to hhexahydrocurcumin [31 Inthe current study, our results indicated that significantlevels of reduction products of yakuchinone B and A were detected, Therefore, itis probable that a variety of ubiquitous and nonspecific oxido-reductases including alcohol dehydrogenase reduce yakuchinone B and thus contribute to the formation of yakuchinone B reduction products in vivo. This suggestion needs confirmation by further research 4. Conclusions In conclusion, low or trace plasma level of parent chemicals were measured after p.o, administration of Yizhi extract, Suo- quan capsules and Suoguan pills to rats. Yizhi flavonoids and diarylheptanoids formed mainly monoglucuronide metabolites. Diglucuronide metabolites for chrysin, izalpinin and kaempferide were also detected. Demethylation of kaempferide to form kaempferol was observed but free kaempferol was undetectable because of rapid glucuronidation. Metabolic reduction of Yizhi diarylheptanoids occurred in rats. Yakuchinone B was reduced to yakuchinone A; yakuchinone A then be reduced to oxyphylla- cinol in a stepwise manner and subsequently glucuronidated by UDP-glucuronosyl transferase. Further research is needed to char acterize the UDP-glicuronosyl transferase and reductase involved. in the biotransformation of Yizhi chemicals. Competing interests There are no competing interests to declare, ‘Acknowledgements We ate grateful to Dr. Li Li from Shanghai Institute of Materia Medica, China and Dr, Dan-Dan Tian from The Chinese University of Hong Kong, China for valuable comments and suggestions regard- ing this article. We are also grateful to Dr. Chen Cheng and Dr. Mei-Juan Li from Shanghai Institute of Materia Medica for screening, relevant articles from reference lists. This work was supported by Grants 812189 and813196 fromthe Natural Science Fund of Hainan Province, Grants ZDZX2013008- 2 and ZDZX2013008-3 from the Hainan Science and Technology Major Project and Grant 2011 BA101B07 from the National Science & Technology Pillar Program during the 12th Five-Year Plan Period. of China. The work was also financially supported by Grant HY2012- 013 from the Hainan Medical University for Young scholars. ‘Appendix A. 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