LC high resolution MS/MS analysis of chemical compounds in rat plasma after oral administration of Nao Mai Tong and its individual herbs

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1 Received: 18 June 2016 Revised: 6 Deceme 2016 Accepted: 13 Deceme 2016 DOI /mc.3920 RESEARCH ARTICLE LC high esolution MS/MS analysis of chemical compounds in at plasma afte oal administation of Nao Mai Tong and its individual hes Yueying Rong 1,2,3 Suxiang Feng 4 Chunwei Wu 1,2,3 Shumei Wang 1,2,3 Shengwang Liang 1,2,3 Dongyun Liu 1,2,3 1 School of Taditional Chinese Medicine, Guangdong Phamaceutical Univesity, Guangzhou, People's Repulic of China 2 Key Laoatoy of Digital Quality Evaluation of Chinese Mateia Medica of State Administation of TCM, Guangzhou, People's Repulic of China 3 Engineeing Technology Reseach Cente fo Chinese Mateia Medica Quality of the Univesities of Guangdong Povince, Guangzhou, People's Repulic of China 4 Henan Univesity of Taditional Chinese Medicine, Henan Povincial Collaoative Innovation Cente fo Respiatoy Disease Diagnosis and Teatment, and Chinese Medicine Reseach and Development, Zhengzhou, People's Repulic of China Coespondence Shumei Wang, Engineeing Technology Reseach Cente fo Chinese Mateia Medica Quality of the Univesities of Guangdong Povince, Guangzhou , People's Repulic of China @qq.com Astact Nao Mai Tong (NMT), a Chinese heal fomula, is used fo teating ischemia ceeal apoplexy. To discove the components of NMT with potential in vivo ioactivity and investigate the diffeences etween NMT and its single he components, this study igoously compaed plasma samples collected fom ats dosed with NMT and single he extacts at diffeent times though LC high esolution MS/MS and data pocessing (Metwoks). The plasma of the NMT goup contained a total of 66 identified compounds (25 pototypes and 41 metaolites), including anthaquinones, titepenoid saponins, isoflavones and phthalides. Additionally, glucuonidation, sulfation and cysteine conjugation wee the majo eactions though which the compounds in NMT wee metaolized. The compaison of the goups evealed two metaolites that wee only detected in the plasma fom the NMT dosed goup, wheeas seven pototype ingedients (chysophanol 8 O glucoside, ginsenoside Rf, Rg2, Rh1, F1, F2 and chikusetsusaponin IVa) and 12 metaolites (two novel titepenoid saponins) wee only discoveed in the plasma samples fom the single he dosed goups. Moeove, the tends in the chemical compounds detected pesented maked diffeences etween NMT dosed at plasma and plasma samples fom the single he dosed goups. The aove data indicate that pesciption compatiility affects the assimilation and elimination of ingedients and povides useful infomation fo futhe phamacokinetic studies. KEYWORDS HPLC MS/MS, identification, metaolites, Nao Mai Tong 1 INTRODUCTION NMT (Nao Mai Tong), a Chinese medicinal fomula, contains Rheum palmatum L. (hua), Panax ginseng C.A. Mey (ginseng), Pueaia loata (Willd.) Ohwi (pueaia) and Ligusticum chuanxiong Hot (Ligusticum wallichiii). NMT has een used fo teating ischemia ceeal apoplexy in clinical pactice fo decades. A clinical epot (Li & Guo, 2005) demonstated that NMT could significantly impove the health condition of patients with acute ceeal infaction. A phamacological study (Ren, Li, Feng, & Lu, 2004) indicated that Aeviations used: Aa, aainoside; C max, concentation maximum; Glc, glucoside; NMT, Nao Mai Tong; Rha, hamnoside; TCM, taditional Chinese medicine; Xyl, xyloside. NMT exets a potective effect against ischemia ceeal apoplexy and epefusion injuy, potentially though inhiition of the expession of tumo necosis facto α, vascula cell adhesion molecule 1 and intecellula adhesion molecule 1 (Li, Gao, Zhou, & Liu, 2006) and inceases in the gene and potein expession of the angiopoietin Tie system (Li et al., 2013). To optimize the dose of the NMT fomula, Chen et al. (2012) analyzed the changes in plasma metaolites in ats fed diffeent doses of NMT though Metaonomics and comined the esults with hematoxylin eosin staining of at ain tissue iopsies. Administation of the modeate dose of NMT (3.0 g/kg/day) fo 5 days yielded effective potection. A phytochemical investigation of the NMT fomula showed that its main constituents included anthaquinones, isoflavones and titepenoid saponins (Wang, Li, Liang, Gao, & Li, 2009). Biomedical Chomatogaphy 2017;e wileyonlineliay.com/jounal/mc Copyight 2016 John Wiley & Sons, Ltd. 1of13

2 2of13 RONG ET AL. Of note, a vast nume of chemical compounds in TCM can ind multiple tagets, which poaly contiutes to the theapeutic and synegistic effects of TCM (Nomile, 2003; Van De Geef et al., 2007). Fom a phamacokinetic point of view, only the compounds asoed into the lood can show ioactivity (Wang, 2002). Theefoe, a systematic analysis of the chemical constituents of NMT detected in vivo afte oal administation is necessay. Howeve, numeous isoaic endogenous compounds ostuct metaolite detection. Futhemoe, without sensitive analytical appoaches, the in vivo detection of ingedients pesent at low levels is a challenge. LC HR MS/MS, a sensitive and eliale analytical appoach, can apidly sepaate multiple components and povide accuate masses of the pecusos and fagment ions. Thus, this technique is helpful fo distinguishing metaolites fom complex iological matices y calculating the elemental composition of elated ions (Guo, Zhang, Elmoe, & Vishwanathan, 2013; Lu et al., 2016; Zhu, Zhang, & Humpheys, 2011). Moeove, fomula compatiility, one of the most impotant chaacteistics of TCM, is often applied to induce synegistic effects o elieve advese eactions. Though he compatiility, the in vito o in vivo ioactive constituents of he pais might diffe fom those of the single hes (Jin et al., 2016). Thus, at plasma samples collected fom the NMT dosed goup and fou single he dosed goups at diffeent time points wee analyzed y LC HR MS/MS to sceen fo potential active constituents and investigate the diffeences in chemical compounds and tends in vivo. Consideing the enomous amount of MS data, we used datapocessing methods suppoted y eliale softwae, such as ackgound sutaction and component detection of dugs and thei metaolites, to apidly sceen potential chemical components fom lage ackgound signal ions. Using data mining tools and compaisons with efeence sustances and elated liteatue data, 25 pototypes and 41 elated metaolites wee identified o tentatively chaacteized in the NMT dosed goup, wheeas one pototype, fou metaolites, two metaolites, five pototypes and six metaolites wee discoveed only in the hua, pueaia, L. wallichiii and ginseng dosed goups, espectively. 2 EXPERIMENTS 2.1 Mateials and chemicals Rheum palmatum L. (140310), Panax ginseng C.A. Mey. (140225), Pueaia loata (Willd.) Ohwi. (140114) and Ligusticum chuanxiong Hot. (140301) wee puchased fom Zisun Chinese Phamaceutical Co. Ltd (Guangzhou, China) and wee identified y Pofesso Jizhu Liu (Guangzhou, China). Aloe emodin ( ), hein ( ), emodin ( ), pueain ( ), daidzin ( ), feulic acid ( ), ginsenoside Re ( ), ginsenoside Rg1 ( ) and ginsenoside R1 ( ) with a puity of 98% wee puchased fom the National Institute fo Food and Dug Contol (Beijing, China). Daidzein (MUST ), ginsenoside Rc (MUST ), ginsenoside Rf (MUST ), ginsenoside R2 (MUST ), ginsenoside Rd (MUST ), ginsenoside Rg3 (MUST ) and ligustilide (MUST ) wee puchased fom Chengdu Must Bio technology Co. Ltd (Chengdu, China). 3 Hydoxypueain (130527), 3 methoxypueain ( ), senkyunolide I (140226), senkyunolide A (140827), utylphthalide (140825) and chysophanol 8 O glucoside (150114) wee puchased fom Chengdu Chom Biotech Co. Ltd (Chengdu, China). The puity of the aove mentioned efeence sustances was >95%. HPLC gade methanol, acetonitile and fomic acid wee otained fom Fishe Scientific (Fai Lawn, NJ, USA). Ethanol (AR, 95%) was otained fom Tianjin Fuyu Fine Chemical Co. Ltd (Tianjin, China). Wate was puified using a Millipoe Milli Q puification system (Bedfod, MA, USA). 2.2 Pepaation of NMT NMT was pepaed y mixing hua, ginseng, pueaia and L. wallichiii at a atio of 9:9:6:6. This atio was ased on decades of clinical expeience y Pofesso Jiansheng Li (Henan Univesity of Taditional Chinese Medicine, Henan, China) on teating ischemia ceeal apoplexy. The mixtue of hes wee extacted twice (each time fo 1 h) in 60% ethanol (100 g/1000 ml) at 90 C. The extacts wee comined, filteed and concentated to 2 g/ml to otain the solution of NMT used fo oal administation solution. Rhua solution (0.6 g/ml), ginseng solution (0.6 g/ml), pueaia solution (0.4 g/ml) and L. wallichiii solution (0.4 g/ml) wee otained using the potocol simila to that used to pepae the NMT solution. 2.3 Animals and teatment The animal welfae and expeimental pocedues wee pefomed stictly accoding to the Guide fo the Cae and Use of Laoatoy Animals (US National Reseach Council, 1996) and the guidelines of the Committee on the Cae and Use of Laoatoy Animals of China. Male Wista ats weighing g wee maintained with a 12 h light/ 12 h dak cycle and a tempeatue of 20 ± 2 C fo 7 days efoe the expeiments. The ats wee andomly divided into six goups: the contol goup (n=6); the NMT dosed goup (n=6); the hua dosed goup (n=6); the ginseng dosed goup (n=6); the pueaia dosed goup (n=6); and the L. wallichiii dosed goup (n=6). The dosed ats wee oally administeed the espective solution at 10 ml/kg/day fo five consecutive days. 2.4 Plasma sample collection and pepaation All ats wee fasted ovenight with fee access to wate efoe the fifth administation. Blood samples wee collected via the caudal vein into hepainized tues at 0.5, 1.5, 4, 8, 12 and 24 h afte the fifth administation. The plasma was otained y centifugation at 5000 pm and 4 C fo 30 min. The sample pepaation potocol was optimized, and the optimized pepaation potocol was the following: 2000 μl of methanol and 80 μl of 0.1% fomic acid wee added to 400 μl of plasma; the mixtue was then mixed y votexing (fo 1 min) and shaking (fo 10 min); and the samples wee susequently centifuged at 15,000 pm and 4 C fo 30 min. The supenatant was tansfeed and evapoated unde nitogen gas at

3 RONG ET AL. 3of13 oom tempeatue. The esidue was dissolved in 100 μl of 60% methanol (v/v) containing 0.1% fomic acid and then centifuged twice at 15,000 pm and 4 C fo 30 min. Finally, a 10 μl aliquot was injected into the HPLC MS/MS system. Metwoks softwae. The esults demonstated that 0.1% fomic acid was optimal, and 60% methanol containing 0.1% fomic acid was selected as the dissolvent. Backgound sutaction was useful fo optimizing sample peteatment. 2.5 Instuction and analytical conditions The LC system consisted of a Dionex Ultimate 3000 UHPLC system (Themo Fishe Scientific, USA) equipped with a quatenay pump, an online degasse, an autosample and a column oven. The samples wee sepaated on a Venusil XBP C 18 column ( mm, 5 μm, Agela Technologies, Tianjin, China) at 30 C y passing though the moile phase, which was composed of 0.1% (v/v) fomic acid in wate (A) and acetonitile (B). The following linea gadient pogam at a flow ate of 1 ml/min was used: 0 5 min, 10% B; 5 15 min, 10 14% B; min, 14 20% B; min, 20 30% B; min, 30 40% B; min, 40 55% B; min, 55 68% B; and min, 68 85% B. A high esolution Q Exactive mass spectomete (Themo Fishe Scientific, Bemen, Gemany), specifically a quadupole oitap hyid mass spectomete coupled with a heated electospay ionization souce, was opeated in oth the positive and the negative ion modes. The paametes of the mass spectomete wee set as follows: spay voltage, 2.8 and 3.5 kv fo the negative and positive modes, espectively; capillay tempeatue, 350 C; auxiliay gas flow ate, 10 L/min; auxiliay gas heate tempeatue, 200 C; scan ange, m/z 100 to m/z 1500; and stepped nomalized collision enegy (NCE), 20 ev, 35 ev. XCaliu 3.0 softwae (Themo Fishe Scientific, USA) was used fo contolling the instument and data acquisition and analysis. Q Exactive 2.0 SP 2 (Themo Fishe Scientific, USA) was used fo contolling the mass spectomete. Backgound sutaction and component detection wee pefomed using Metwoks 1.3 SP4 softwae (Themo Fishe Scientific, USA). 3 RESULTS 3.1 Optimization of sample peteatment and chomatogaphic sepaation To optimize the elution system, a methanol 0.1% fomic acid aqueous solution and an acetonitile 0.1% fomic acid aqueous solution wee compaed, and the latte displayed efficient sepaation. Based on ou pevious study, the addition of a cetain amount of fomic acid (V 0.1% HCOOH :V plasma, 1:5) to at plasma fo potein pecipitation y methanol could incease the peak intensity of the compounds detected y the mass detecto. Additionally, solutions with diffeent atios of methanol to 0.1% fomic acid wee used to dissolve the esidues, which could affect the detection of the ingedients of the dug. Theefoe, diffeent concentations (0.1, 0.2 and 0.4%) of fomic acid and diffeent atios (V CH3OH :V 0.1% HCOOH, 40:60, 60:40 and 80:20) of the dissolvent wee analyzed to detect the optimal values. The chemical noise fom the iological matix makes identification difficult though visual compaison of the chomatogam. Theefoe, using the MS data fom the lank goup as a contol, the aw sample data was sujected to ackgound sutaction using 3.2 Data mining method Without assistance fom data pocessing tools, the detemination of multiple pototypes and metaolites of TCM fom mass chomatogaphic data is time consuming and complicated. In this study, the component detection pocessing tool in Metwoks was adopted to detemine the ions of possile pototypes and elated metaolites in NMT. The following analysis pocess was utilized: fist, a selected sample file and a contol file fo component detection wee selected. The analytes, including the name, fomula, mass and polaity wee then specified and selected fom the epoted chemical constituents of the NMT decoction (Rong et al., 2016). Thid, eactions such as glucuonidation, deglycosylation, sulfation and hydoxylation wee set as known modifications ased on elated findings concening the metaolism of Radix et Rhizoma Rhei (Shia et al., 2009; Zhang et al., 2015), ginsenosides (Wang et al., 2007; Yang, Xu, Liu, & Su, 2009), Pueaia loata (Willd.) Ohwi (Miao et al., 2013; Zhou et al., 2013) and Ligusticum chuanxiong Hot (Zuo et al., 2011) and phase I and II common factoy lists offeed y the softwae. Fouth, use taces wee defined, and the pocessing paametes (e.g. theshold >10,000, mass toleance <5 ppm, mass ange m/z 150 to m/z 1500) wee set. Finally, candidates of pecuso ions wee otained. Calculation of neutal losses etween fagment ions and thei elemental composition in the MS/MS spectum of the candidates and compaisons with the fagment pattens of the paent chemical compounds evealed that some candidates wee potentially elated to metaolites of NMT. Most assigned metaolites, such as anthaquinones, isoflavones, phthalides and phenolic acids, wee sceened out using the aove descied appoaches, wheeas othes wee discoveed though a seach of the MS/MS lists and an analysis of whethe thei fagment pathways wee consistent with those of potential paent components. 3.3 Analysis of constituents of NMT in at plasma Both the negative and positive ion modes wee adopted fo the detection of constituents in at plasma samples. Most compounds, with the exception of some isoflavones and phthalides, showed ovious signals in the negative mode. In total, of the 66 constituents (Tale 1 and Figue 1), 25 anthaquinones, 11 titepenoid saponins, 17 isoflavones, one pueoside, nine phthalides and thee phenolic acids wee identified o tentatively distinguished y compaing thei MS data and etention times with those of efeence compounds and single dug goups o liteatue data. Most chemical components displayed stong signals at 0.5 and 1.5 h afte oal administation. Specifically, the majoity of the metaolites and ginsenosides also showed intense signals at 8 and 12 h in addition to 0.5 and 1.5 h. The chemical constituents wee gadually tansfomed and exceted fom the ody, and the signals detected with the mass detecto theefoe ecame weake o even disappeaed ove time.

4 4of13 RONG ET AL. TABLE 1 LC high esolution MS/MS analysis of chemical compounds in plasma afte oal administation of Nao Mai Tong and its individual hes No. tr (min) Compounds Fomula Pecuso ions (m/z) MS/MS Resouce Pathway 0.5 h 1.5 h 4 h 8 h 12 h 24 h Feulic acid GlcA C16H18O [M H] ( ) , , c GlcA Pueain GlcA C27H28O [M H] ( ) , , g GlcA Pueain sulfate C21H20O12S [M H] ( ) , , , , n.a. SUL Hydoxypueain sulfate C21H20O13S [M H] ( ) , , , g SUL Feulic acid sulfate C10H10O7S [M H] ( ) , , c SUL Daidzein sulfate GlcA C21H18O13S [M H] ( ) , g SUL + GlcA Daidzin sulfate C21H20O12S [M H] ( ) , , g SUL Methoxypueain sulfate C22H22O13S [M H] ( ) , , , g SUL Pueain a C21H20O [M + H] + ( ) , [M H] (+) , , , , Pueain Xyl C26H28O [M + H] + ( ) , , [M H] (+) , , , Methoxypueain a C22H22O [M + H] + ( ) , (+) , [M H] , , Pueain Api C26H28O [M + H] + ( ) , , [M H] (+) , , 381, 0965, g g g g Genistein O Glc sulfate C21H20O13S [M H] ( ) , , g SUL Aloe emodin diglca C27H26O [M H] ( ) , d diglca Methoxypueain C22H22O [M H] ( ) , , g Daidzein GlcA C21H18O [M + H] + ( ) (+) g GlcA [M H] Aloe emodin GlcA sulfate C21H18O14S [M H] ( ) , , d GlcA + SUL Aloe emodin GlcA C21H18O [M H] ( ) , d GlcA Feulic acid a C10H10O [M + H] + (+) , , c Rhein GlcA C21H16O [M H] ( ) , , d GlcA Aloe emodin sulfate C15H10O8S [M H] ( ) , d SUL Senkyunolide F GlcA C18H22O [M H] ( ) , , c GlcA Emodin diglca C27H26O [M H] ( ) , , d diglca Daidzein sulfate C15H10O7S [M + H] + (+) g SUL Senkyunolide J/N cysteine C15H23O5NS [M + H] + (+) , , , , , , c Cys Chysophanol O Glc GlcA C27H28O [M H] ( ) , , D GlcA (Continues)

5 RONG ET AL. 5of13 TABLE 1 (Continued) No. tr (min) Compounds Fomula Pecuso ions (m/z) MS/MS Resouce Pathway 0.5 h 1.5 h 4 h 8 h 12 h 24 h Chysophanol diglca C27H26O [M H] ( ) , d diglca Aloe emodin sulfate C15H10O8S [M H] ( ) , , d SUL Emodin diglca C27H26O [M H] ( ) , , , d diglca Hydoxyl hein GlcA C21H16O [M H] ( ) , d SUL Senkyunolide I cysteine C15H21O5NS [M + H] + (+) , , , , , c Cys Rhein GlcA C21H16O [M H] ( ) , , , Senkyunolide F sulfate C12H14O6S [M + H] + d GlcA [M H] (+) , , c SUL Methyl aloeemodin diglca C21H34O [M H] ( ) , , n.a. diglca Physcion diglca C28H28O [M H] ( ) , d diglca Senkyunolide I a C12H16O [M + Na] + (+) , , , , c G Rg1 a C42H72O [M + HCOO] ( ) , G Re a C48H82O [M + HCOO] ( ) , , , , Dehydoxylaccaic acid D GlcA C22H18O [M H] ( ) , d GlcA Fomononetin GlcA C22H20O [M H] ( ) , g GlcA Pueol B GlcA C24H24O [M H] ( ) , , g GlcA Daidzein a C15H10O [M + H] + (+) g Emodin GlcA C21H18O [M H] ( ) , , d GlcA Chysophanol sulfate C15H10O7S [M H] ( ) , d SUL Emodin sulfate C15H10O8S [M H] ( ) d SUL Chysophanol GlcA C21H18O [M H] ( ) d GlcA GRa3/isome C59H100O [M + 2HCOO] 2 ( ) , , , [M + HCOO] Physcion GlcA C22H20O [M H] ( ) , , d GlcA G Ra2 C 58 H 98 O [M + 2HCOO] 2 ( ) , , , [M + HCOO] , Physcion GlcA C22H20O [M H] ( ) , , d GlcA Emodin GlcA C21H18O [M H] ( ) , d GlcA G Ra3/isome C59H100O [M + 2HCOO] 2 ( ) , , , [M + HCOO] , , G R1 a C54H92O [M + HCOO] ( ) , , , , (Continues)

6 6of13 RONG ET AL. TABLE 1 (Continued) No. tr (min) Compounds Fomula Pecuso ions (m/z) MS/MS Resouce Pathway 0.5 h 1.5 h 4 h 8 h 12 h 24 h Emodin sulfate C15H10O8S [M H] ( ) d SUL G Rc a C53H90O [M + HCOO] ( ) , , , , , , G Ra1 C58H98O [M + 2HCOO] 2 ( ) , , , [M + HCOO] , , , G Ro C48H76O [M H] ( ) , , , , , G R2 a C53H91O [M + HCOO] ( ) , , , , , , G Rd a C48H82O [M + HCOO] ( ) , , , Emodin sulfate C15H10O8S [M H] ( ) d SUL Rhein a C15H8O [M H] ( ) , d Senkyunolide A a C12H16O [M + H] + (+) , , c Butylphthalide a C12H14O [M + H] + (+) , , c Emodin a C15H10O [M H] ( ) , , d Neocindilide C12H18O [M + H] + (+) , , c Ligustilide a C12H14O [M + H] + (+) , , c C Senkyunolide F cysteine C15H19O4NS [M + H] + (+) , , , , , , c Cys C Butyl 6/7 hydoxy 4,5,6,7 tetahydophthalide C12H18O [M + H] + (+) , , , c Hydolysis D Chysophanol 8 O Glc a C21H20O [M H] ( ) , d G Methoxydaidzein GlcA C22H20O [M + H] + (+) , , g GlcA G Dihydodaidzein GlcA C21H20O [M + H] + (+) , g GlcA + Hydogen G Methoxydaidzein sulfate C16H12O8S [M + H] + (+) , , g SUL G Biochanin A GlcA C22H20O [M + H] + (+) , g GlcA R G Rf C42H72O [M + HCOO] ( ) , , R G Rg2 a C42H72O [M + HCOO] ( ) , , R G Rh1 C36H62O [M + HCOO] ( ) R G F1 C36H62O [M + HCOO] ( ) , R Unknown C45H92O [M + HCOO] ( ) [M H], [M H Xyl/Aa], [M H 2Xyl/Aa], deglc (Continues)

7 RONG ET AL. 7of13 TABLE 1 (Continued) No. tr (min) Compounds Fomula Pecuso ions (m/z) MS/MS Resouce Pathway 0.5 h 1.5 h 4 h 8 h 12 h 24 h [M H 2Xyl/Aa Glc], , , R Unkonwn GlcA C36H60O [M H] ( ) [M H], [M H CH2O3], [M H GlcA] GlcA + deglc R G F2 C42H72O [M + HCOO] ( ) , R Chikusetsusaponin Iva C42H66O [M H] ( ) , , , , , , R Ginsenoside Mc/Mx/Y C41H70O [M + HCOO] ( ) , deglc R Unknown C30H50O [M H] ( ) [M H], [M H CO2], [M H CO2 C5H10] deglc R G C K C36H62O [M + HCOO] ( ) , deglc R Unknown C30H50O [M H] ( ) [M H], [M H CO2], [M H CO2 C5H10] deglc a Confimed y efeence standads;, pototype; n.a., unknown; OH, hydoxylation; Methyl, methylation; GlcA, glucuonidation; diglca, diglucuonidation; deglc, deglycosylation; SUL, sulfation; Cys, cysteine; Api, apioside; Glc, glucose; Rha, hamnose; Xyl, xylose; d, Rheum palmatum L;, Panax ginseng C.A.Mey.; c, Ligusticum chuanxiong Hot.; g, Pueaia loata (Willd.) Ohwi. When ionic stength < , it was set as ; when ionic stength was n 10 5 and < n 10 5 < , it was set as n+ ; when ionic stength was n 10 6 and < n 10 6 < , it was set as n++ ; when ionic stength was n 10 7 and < n 10 7 < ,it was set as n+++ ; when ionic stength was n 10 8 and < n 10 8 < , it was set as n++++.

8 8of13 RONG ET AL. FIGURE 1 Extacted ion chomatogams of identified components in Nao Mai Tong (NMT) dosed goup and single he dosed goups. (a) Components with weak signals; () components with intense signals Among the metaolites detected in the analysis of the NMTdosed goup, 25, 11, 16 and 12 chemical constituents wee also identified in the hua, ginseng, pueaia and L. wallichiii dosed goups, espectively, as detemined though a compaison of the etention ehavios and mass spectum data of the plasma fom the NMT dosed goup with those otained fo the plasma samples fom the individualhe dosed goups and NMT extact. Howeve, peaks 3 and 34 wee not oseved in the chomatogams of the individual he dosed goups and the NMT extact and ae thus likely to e a esult of the comined decoction of the cude hes. Accoding to thei chaacteistic ions and exact masses, these peaks wee deduced to e metaolites and denoted pueain sulfate and methyl aloe emodin Oglucoside O glucuonide, espectively. Metaolite discimination was ased on etention ehavios, fagmentation pathways of thei paent components and masses of neutal losses coesponding to metaolite eactions, such as SO 3 (79.96 Da) to sulfation, C 6 H 8 O 6 ( Da) to glucuonidation and C 3 H 7 NO 2 S ( Da) to cysteine conjugation (Levsen et al., 2005; Pasad, Gag, Takwani, & Singh, 2011). 3.4 Analysis of constituents of hua The analysis of MS fagments and elated efeences (Jin et al., 2007; Liu et al., 2014; Zhang et al., 2015; Zhu et al., 2015) evealed that the 25 constituents of hua wee anthaquinones, specifically two pototypes (peaks 61 and 64) and 23 metaolites (including chysophanol O glucoside glucuonide). The elated liteatue epots that pola anthaquinone glycosides ae not oseved in at plasma (Shia et al., 2009; Wu, Yan, Yao, Zhan, & Wang, 2014). In this study, chysophanol 8 O glucoside (identified using a efeence sustance, as shown in Figs 1 and 2 in the Suppoting Infomation) was detected in the plasma fom the hua dosed goup at 0.5 h with a weak signal, and its elated metaolite (chysophanol O glucoside glucuonide, as shown in Figs 3 and 4 in the Suppoting Infomation) was found in the plasma samples fom the NMT and hua dosed goups at 0.5 h. Othe anthaquinone glycosides have not yet een discoveed in plasma, and thei content in plasma might e too low to detect y mass spectomety. In the plasma fom the NMT and hua dosed goups, the contents of hein and emodin deceased apidly fom 0.5 to 4 h afte administation, suggesting that these metaolites wee asoed and eliminated apidly (as shown in Tale 1 and Fig. 5 in the Suppoting Infomation). Inteestingly, most metaolites (including metaolites that undewent sulfation, glucuonidation and diglucuonidation) displayed stong ion signals at 0.5 and 8 h. It is possile that the metaolites wee eliminated slowly and stoed afte fou doses, esulting in stong signals at the ealy time points, and fee anthaquinones and glycosides wee tansfomed apidly into coesponding metaolites afte the second dose. Peak 61 gave a depotonated molecula ion [M H] at m/z and yielded ions at m/z [M H C 2 H 2 ] and [M H CO 2 ]. Based on exact masses, MS fagments and efeence sustances, peak 61 was assigned to hein. Emodin (peak 64) was distinguished in a simila manne. The identification of anthaquinone elated metaolites fom hua was mainly ased on diagnostic ions fom pecuso compounds; fo example, the ions at m/z and wee assigned to aloe emodin, the ions at m/z

9 RONG ET AL. 9of and wee assigned to chysophanol; the ions at m/z and wee assigned to emodin; the ions at m/z and wee assigned to physcion; and the ions at m/z , and wee assigned to hein (Liu et al., 2016). Finally, five aloe emodin elated metaolites (peaks 14, 17 18, 21 and 28), fou chysophanol elated metaolites (peaks 26 27, 44 and 46), seven emodin elated metaolites (peaks 23, 29, 43, 45, 51, 54 and 60), thee physcion elated metaolites (peaks 35, 48 and 50), thee hein elated metaolites (peaks 20, 30 and 32) and one 6 dehydoxylaccaic acid D elated metaolite (peak 39) wee distinguished in at plasma afte administation. Howeve, this identification appoach did not wok fo metaolites geneated though a seies of complicated metaolic pathways. Peak 30, a metaolite of glucuonidation with [M H] ion at m/z (calculated as C 21 H 16 O 13 ), geneated deglucuonidated ions at m/z ( Da) and ( Da). Based on the aove descied analysis, peak 30 was identified as the hydoxylated and glucuonidated poduct of hein, and this identification was futhe confimed in anothe study (Dahms et al., 1997). Howeve, many unknown metaolites fom hua still need to e confimed. 3.5 Analysis of constituents of ginseng Two potopanaxatiol ginsenosides (peaks 37 and 38), eight potopanaxadiol ginsenosides (peaks 47, 49, 52, 53, 55, 56, 58 and 59) and one oleanolic acid ginsenoside (peak 57) wee detected in at plasma afte oal administation of the NMT and ginseng decoctions, and all of these ae pototype components, as detemined though a compaison with data otained fom the NMT extact. In addition, six pototypes (ginsenosides Rf, Rg2, Rh1, F1 and F2 and chikusetsusaponin IVa) and six metaolites (ginsenosides Mc/Mx/Y and C K and two novel components) wee found only in the plasma fom the ginseng dosed goup. The main metaolic pathway consisted of deglycosylation and glucuonidation. Futhemoe, the content of titepenoid saponins in the plasma fom the NMT dosed goup emained stale fom 10 to 24 h (Fig. 6 in the Suppoting Infomation), ut the amount of these compounds in the plasma fom the ginsengdosed goup eached a peak at 8 h and declined quickly. The stategy fo disciminating common titepenoid saponins was the following (Fig. 7 in the Suppoting Infomation). Fist, the chaacteistic poduct ions of aglycone in the MS/MS spectum wee used to deduce the stuctual type; fo example, a fagment ion at m/z in the negative mode was efeed to as potopanaxadiol type, wheeas that at m/z was defined as the potopanaxatiol type and that m/z was classified as the oleanolic acid type. Second, suga moieties linked to the aglycone poduced neutal losses and ich low mass fagment ions in high enegy collisions, which could indicate the types, numes and linkages of the sugas. Fo example, the neutal loss mass of Da and low mass ions at m/z and coesponded to one glucoside (Glc), and a neutal loss mass of Da and low mass ions at m/z and coesponded to one aainoside (Aa) o xyloside (Xyl). In addition, a neutal loss mass of Da and low mass ions of m/z and coesponded to one hamnoside (Rha), wheeas low mass ions of m/z and , of m/z and , of m/z and , and of m/z coesponded to two Glcs, Glc Rha, Glc Xyl/Aa and Aa/Xyl Aa/Xyl, espectively (Qiu et al., 2015). Finally, thei molecula fomulas, which wee calculated accoding to the exact masses of thei pecuso ions (<±5 ppm), wee seached in the SciFinde dataase, and the stuctual fomulas otained fom the seach esults wee compaed with the aove stuctual deductions ased on expeimental data. Following the aove descied stategy, peaks 37, 38, 47, 49, 52, 53, 55 59, R1 R4, R7 R9 and R11 wee identified as Rg1, Re, Ra3/ isome, Ra2, Ra3/isome, R1, Rc, Ra1, Ro, R2, Rd, Rf, Rg2, Rh1, F1, F2, chikusetsusaponin IVa, Mc/Mx/Y and C K, espectively. Most of the sustances, including Rg1, Re, R1, Rg2, Rc, R2 and Rd, wee futhe confimed y efeence sustances. In the moile phase system consisting of CH 3 CN 0.1% HCOOH, many ginsenosides exhiited intensive [M + 2HCOO] 2 pecusos, which is useful fo distinguishing components at low amounts. Peaks R5, R6, R10 and R12 wee identified as metaolites fom ginsenosides accoding to a mass defect window fom 0.3 to 0.6 Da and fagmentation pathways simila to those of ginsenosides. Peak R5, a metaolite of deglycosylation, yielded an adduct ion [M + HCOO] at m/z (C 45 H 92 O 26 ) and aundant poduct ions at m/z [M H], [M H Xyl/Aa], [M H 2Xyl/Aa], [M H 2Xyl/Aa Glc], , which could e deduced as two Aa/Xyl units and two Glc units, , which could coespond to two Glcs linked with each othe, and , which could e consideed one Aa/Xyl connected to one Glc. Peak R6 with a depotonated ion at m/z geneated ions at m/z [M H CH 2 O 3 ] and [M H C 6 H 8 O 6 ], implying that R6 was a glucuonidated metaolite. Howeve, ecause R5 and R6 wee not ecoded in the SciFinde dataase, they ae potential novel components. Peaks R10 and R12, a pai of isomes with the same ions at m/z [M H], yielded ions at m/z [M H CO 2 ] and [M H CO 2 C 5 H 10 ] and wee tentatively disciminated as metaolites of titepenoid saponins. 3.6 Analysis of constituents of pueaia In total, six pototypes (pueain, pueain xyloside, 3 methoxypueain, pueain apioside, methoxypueain and daidzein), thee isoflavone C glycoside elated metaolites (peaks 2, 4 and 8), two isoflavone O glycoside elated metaolites (peaks 7 and 13), fou isoflavone aglycone elated metaolites (peaks 6, 16, 24 and 40), and one pueoside elated metaolite (peak 41) wee tentatively chaacteized in the plasma of NMT and pueaia dosed ats, and the detailed analysis is shown in Tale 1. In addition, fou metaolites (methoxydaidzein glucuonide, dihydodaidzein glucuonide, methoxydaidzein sulfate and iochanin A glucuonide) wee detected only in pueaia dosed at plasma. With espect to isoflavone, the metaolic pathways contained glucuonidation and sulfation eactions. Most pototypes in the plasma fom the NMT and pueaia dosed goups showed stong ion signals at ~0.5 and 8 h and ~1.5 h, espectively (as shown in Tale 1 and Fig. 8 in the Suppoting Infomation). Additionally, thei elimination in the plasma fom the NMT dosed

10 10 of 13 RONG ET AL. goup was slowe than that oseved in the plasma fom the pueaiadosed goup, indicating that the comination of cude hes contiuted to an extension of the theapeutic time peiod. Owing to the small nume of coesponding metaolites, it is difficult to discen the diffeences etween the NMT dosed and pueaia dosed goups. Isoflavone C glycosides yielded moe aundant neutal losses in the MS/MS spectum than isoflavone O glycosides, such as C 4 H 8 O 4 (120 Da) and C 5 H 8 O 5 (148 Da), in the negative ion mode. With espect to isoflavone O glycosides, only C 6 H 10 O 5 (162 Da) pesented a chaacteistic neutal loss. Peaks 2, 4, 8 12 and 15 showed aundant diagnostic neutal losses of 120 Da (C 4 H 8 O 4 ) and 148 Da (C 5 H 8 O 5 )in the negative mode and wee thus tentatively identified as isoflavone C glycosides. Peak 2 ([M H] ion at m/z ) displayed a mass Da highe than peak 9 ([M H] ion at m/z ), and these peaks pesented a seies of the same fagmentation ions at m/z , and Thus, peak 2 was assigned as a glucuonidated metaolite of peak 9, which was confimed as pueain ased on data fom standads. Theefoe, peak 2 was deduced as pueain O glucuonide ased on the liteatue (Yan et al., 2013). Similaly, peaks 4, 8, and 15 wee identified as 3 hydoxypueain sulfate, 3 methoxypueain 7/4 O sulfate, pueain xyloside, 3 methoxy pueain, pueain apioside and 3 methoxy pueain isome, espectively. Peaks 7 and 13 wee suggested to e isoflavone O glycosideelated metaolites. The depotonated ion [M H] of daidzin sulfate (peak 7) at m/z yielded poduct ions at m/z [M H SO 3 ], [M H SO 3 C 6 H 10 O 5 ] and [M 2H SO 3 C 6 H 10 O 5 ]. Peak 13 displayed a ase peak at m/z [M H] (molecula fomula was calculated as C 21 H 20 O 13 S) and yielded poduct ions at m/z , , and in the MS/MS specta fom the NMT and pueaia dosed goups. Accoding to neutal losses of and Da, peak 13 was tentatively assigned as genistein O glucoside sulfate. Few efeences have epoted isoflavone O glycoside elated metaolites in at plasma. 3.7 Analysis of constituents of L. wallichiii Nine phthalides (five pototypes and fou elated metaolites) and thee phenolic acids (one pototype and two elated metaolites) wee discoveed in the plasma fom the NMT and L. wallichiii dosed goups. In addition, two metaolites (senkyunolide F cysteine and 3 utyl 6/7 hydoxy 4,5,6,7 tetahydophthalide) wee only detected in the L. wallichiii dosed goup. Pototypes and metaolites fom L. wallichiii displayed stong signals at 0.5 h afte oal administation in the plasma fom the NMT and L. wallichiii dosed goups. Ove time, these signals declined apidly o even disappeaed, indicating that these phthalides wee tansfomed o exceted apidly. Howeve, some diffeences wee still detected. In the plasma fom the NMT dosed goup, the pototypes and thei elated metaolites exhiited a slowe elimination (Fig. 9 in the Suppoting Infomation) and showed two peaks at 0.5 h and 8 h. Phthalides, which wee easily oseved in the ESI +, showed neutal losses of H 2 O (18 Da) and CO (28 Da) in the MS/MS spectum. Five pototypes, namely feulic acid (peak 19), senkyunolide I (peak 36), senkyunolide A (peak 62), utylphthalide (peak 63) and ligustilide (peak 66), wee confimed y elated efeence sustances. Neocindilide (peak 65), which was shown to e a pototype component though an analysis of the NMT extact, was identified y compaing the MS/MS data and etention ehavio with a elated efeence (Zuo et al., 2011). The metaolic eactions of the elated metaolites included glucuonidation, sulfation and cysteine conjugation. Peak 31, with a molecula fomula of C 15 H 21 O 5 NS (m/z [M + H] + ), poduced a seies of fagment ions at m/z , , , and Accoding to neutal losses of and Da, peak 31 was assigned as a cysteine conjugate. Based on a pevious epot (Zuo et al., 2011), peak 31 was chaacteized as senkyunolide I cysteine. The detailed analysis of the othe metaolites is listed in Tale 1. Peak C1 was oseved as an adduct ion [M + H] + at m/z (which is Da highe than that of senkyunolide F), which poduces neutal ions simila to those otained with peak 31. Theefoe, peak C1 was tentatively assigned as senkyunolide F cysteine. The pecuso ion of peak C2 at m/z was highe than that of senkyunolide A and yielded ions at m/z , , and in the MS/MS spectum. Accodingly, peak C2 was tentatively identified as 3 utyl 6/7 hydoxy 4,5,6,7 tetahydophthalide. 4 DISCUSSION Using LC HR MS/MS and data mining methods, aundant chemical components of NMT and its individual hes wee tentatively assigned ased on MS fagmentation ions and elution times. The identified chemical components could e soted into five goups, specifically anthaquinones, titepenoid saponins, isoflavones, phthalides and phenolic acids. Compaed with data fom the NMT extact (Rong et al., 2016), we discoveed that aloe emodin, chysophanol, physcion and anthaquinone glycosides (except fo chysophanol 8 O glucoside) wee not detected in the plasma fom dosed ats. Howeve, thei sulfated and glucuonidated metaolites wee still detected in plasma. It is possile that the low content of fee anthaquinone in the administeed solution esulted in asoption, wheeas anthaquinone glycosides wee apidly tansfomed into elated metaolites in vivo, which might account fo the aove descied phenomenon. Remakaly, chysophanol 8 O glucoside was only detected in the plasma fom the hua dosed goup, ut the coesponding liteatue (Shia et al., 2009; Wu et al., 2014) suggests that anthaquinone glycosides ae too hydophilic to pemeate though the enteocyte memane. The discovey of anthaquinone glycosides pesent at low levels in complex plasma samples might e difficult. Moeove, the pototype compounds of 3 hydoxypueain and daidzin wee also not detected in vivo, ut thei coesponding metaolites wee found in plasma. It is possile that these wee metaolized at a high ate, ut this finding equies futhe eseach. Additionally, acylation ginsenosides, some isoflavone glycosides (such as 3 methyoxy pueain O glucoside), and phthalide dimes, which ae included in cetain amounts in cude dugs, wee not asoed in plasma. The aove descied findings agee with the esults of elated investigations (Jiang et al., 2009; Miao

11 RONG ET AL. 11 of 13 et al., 2013; Zuo et al., 2011). The aove mentioned compounds might e easily hydolyzed o not easily asoed into the ody. Many compounds fom hua, ginseng, pueaia and L. wallichiii pesented diffeent asoption tends and slowe elimination ates in the plasma fom the NMT dosed goup compaed with the plasma fom the single he goups. This finding indicates that pesciption compatiility in TCM could influence the maintenance of the lood concentation of the dug at a stale level while affecting the asoption of a few ingedients. Moeove, many studies (Li et al., 2015; Zheng et al., 2011) have also poven that pesciption compatiility in TCM could significantly affect the in vivo phamacokinetics. Additionally, thee wee some ovious diffeences in the constituents identified in the plasma samples fom the NMT dosed and single he dosed goups, and these might e attiuted to compatiility effects of othe hes to diminish possile advese eactions (Jin et al., 2016). Howeve, this hypothesis equies futhe investigation. Metaolites o components oseved in the lood might exhiit phamacological ioactivities. Pevious studies have epoted that hua aglycone contains phamacodynamic ingedients fo the teatment of ischemia ceeovascula disease. Emodin exets neuopotective effects against glutamate induced apoptosis in ceeal ischemia (Ahn et al., 2016). Chysophanol possesses anti inflammation activity to potect mice fom ceeal ischemic injuy (Zhang et al., 2014). Additionally, ginsenosides, such as Rg1, Re, Rd, and R1, can epotedly act as neuopotectants though vaious mechanisms, including inducing the expession of TIPE 2 (Zhou et al., 2016), amelioating lipid peoxidation (Zhou, Cao, & Dou, 2006), activating the PI3K/ Akt pathway (Liu et al., 2015) and potecting the integity of the lood ain aie (Chen et al., 2015). Accodingly, 3 methoxypueain and daidzein could potect ats against ceeal ischemia epefusion injuy (Aas et al., 2015; Liu et al., 2010). Moeove, feulic acid (Cheng et al., 2010), ligustilide (Peng et al., 2013) and senkyunolide I (Hu, Duan, Liang, & Wang, 2015) fom L. wallichiii ae active anti ceeal ischemia sustances. Futue phamacokinetics eseach on NMT is equied fo detemining whethe the asoed constituents can e maintained at effective concentations ove time in lood. 5 CONCLUSION In this study, LC HR MS/MS, a apid and sensitive analytical technique, was adopted to detemine the exogenous ingedients in at plasma afte the oal administation of a Nao Mai Tong fomula and its individual hes. Using data pocessing tools, 25 pototypes (two anthaquinones, 11 titepenoid saponins, six isoflavones, five phthalides and one phenolic acid) and 41 elated metaolites (twenty fou anthaquinones, ten isoflavones, one pueoside, fou phthalides and two phenolic acids) wee identified in plasma afte NMT administation and chaacteized y analyzing thei etention ehavios, exact masses and mass fagments. The aove mentioned five ingedients, namely anthaquinones, titepenoid saponins, isoflavones, phthalides and phenolic acids, could e potentially active components, and thei main metaolic pathways wee glucuonidation, sulfation, cysteine conjugation and hydoxylation. In addition, seven pototype ingedients (chysophanol 8 O glucoside and six titepenoid saponins) and 12 metaolites (two possily novel titepenoid saponins) wee only discoveed in the plasma samples fom the fou single he dosed goups. Many of the components identified in the plasma fom the single he dosed goups showed diffeent tends fom that detected in the plasma fom the NMT dosed ats fom 0.5 to 24 h. Theefoe, a complex pesciption such as NMT should e analyzed y comining its individual he components to eveal the esulting metaolic pocesses. This epot povides the fist analysis of the chemical compounds in at plasma samples collected at diffeent time points afte the oal administation of NMT. This study could povide data of chemical components fo analytical studies on the phamacodynamics and phamacokinetics of NMT. ACKNOWLEDGEMENTS This study was financially suppoted y the National Natual Science Foundation of China (nos and ). CONFLICT OF INTEREST The authos have declaed no conflict of inteests. REFERENCES Ahn, S. M., Kim, H. N., Kim, Y. R., Choi, Y. W., Kim, C. M., Shin, H. K., & Choi, B. T. (2016). Emodin fom Polygonum multifloum amelioates oxidative toxicity in HT22 cells and deficits in photothomotic ischemia. Jounal of Ethnophamacology, 188, Aas, A. B., Guven, M., Akman, T., Ozkan, A., Sen, H. M., Duz, U., Cosa, M. (2015). Neuopotective effects of daidzein on focal ceeal ischemia injuy in ats. Neual Regeneation Reseach, 10, Chen, X., Yang, Y. X., Wang, S. M., Wang, Z. H., Su, Z. B., Li, J. S., & Liang, S. W. (2012). Study on effects of extact in Naomaitong fomula on ceeal ischemia epefusion model ased on NMR metaolomics. 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