Accepted Manuscript

Title: In vitro metabolism of alisol A and its metabolites’identification using high-performance liquidchromatography–mass spectrometry

Author: Yue Yu Zhenzhen Liu Ping Ju Yuanyuan ZhangLunhui Zhang Kaishun Bi Xiaohui Chen

PII: S1570-0232(13)00511-4DOI: http://dx.doi.org/doi:10.1016/j.jchromb.2013.09.029Reference: CHROMB 18556

To appear in: Journal of Chromatography B

Received date: 31-3-2013Revised date: 16-9-2013Accepted date: 20-9-2013

Please cite this article as: Y. Yu, Z. Liu, P. Ju, Y. Zhang, L. Zhang, K. Bi, X. Chen, Invitro metabolism of alisol A and its metabolites’ identification using high-performanceliquid chromatography–mass spectrometry, Journal of Chromatography B (2013),http://dx.doi.org/10.1016/j.jchromb.2013.09.029

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

Page 1 of 21

Accep

ted

Man

uscr

ipt

1

In vitro metabolism of alisol A and its metabolites’ identification using

high-performance liquid chromatography–mass spectrometry

Yue Yua,1 , Zhenzhen Liua, Ping Jua, Yuanyuan Zhanga, Lunhui Zhanga, Kaishun Bia,

Xiaohui Chena,

School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,

Shenyang 110016, China

1 Zhejiang Jingxin Pharmaceutical Co., Ltd, No.800 Xinchang East Road, Yulin subdistrict, Xinchang County, Zhejiang Corresponding author. Tel.: +86 24 23986259; Fax: +86 24 23986259E-mail address: cxh_syphu@yahoo.com.cn

Page 2 of 21

Accep

ted

Man

uscr

ipt

2

Abstract

A liquid chromatography–mass spectrometry (LC-MS) method was developed

and successfully applied to the study on the enzyme kinetics of alisol A in rat liver

microsomes (RLM) and human liver microsomes (HLM) incubation systems, and

employed for semi-quantitative determination of each metabolite of alisol A. The

metabolites of alisol A in RLM, HLM and human recombinant CYP3A4 enzyme

incubation systems were identified by high-performance liquid

chromatography–quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS). A

total of 3 and 6 oxidative metabolites were found in RLM and HLM incubation

systems, respectively. 3 metabolites found in both incubation systems were identified.

The exact position of hydroxylation for the metabolites M1 and M2 could not be

determined. Chemical inhibitors of cytochrome P450 (CYP450) and individual human

recombinant CYP450 enzyme were used to identify the CYP450 isozymes involved

in the formation of each metabolite of alisol A. The result indicated that the formation

of each metabolite of alisol A was mainly catalyzed by CYP3A4 enzyme.

Keywords: Alisol A; metabolism; HPLC-QTOF MS; rat liver microsomes; human

liver microsomes

1. Introduction

Alisol A is one of the major active triterpenes isolated from Rhizoma Alismatis

(RA), a famous Traditional Chinese Medicine widely used for diuretic, hypolipidemic,

anti-nephrolithic, anti-atherosclerotic, anti-inflammatory and anti-diabetic purposes in

China for more than 1,000 years [1,2]. However, our previous research has shown that

Page 3 of 21

Accep

ted

Man

uscr

ipt

3

the prolonged use of RA may damage the renal proximal convoluted tubules [3]. In

order to clarify the toxic substance in RA, the cytotoxic effect of alisol A on renal

epithelial cells (HKC) was investigated in our previous research using aristolochic

acid (a known nephrotoxic substance) as a positive control. The IC50 values of alisol A

in HKC cells following incubation for 24, 48, and 72 h, were significantly lower than

those of aristolochic acid, respectively, which indicated that alisol A has considerable

cytotoxic effect on the HKC cells. Based on these results, much attention has been

paid to the pharmacokinetic study of these protostane-type triterpenes [4]. There were

fewer papers about the metabolism of alisol A. In particular, metabolism studies

include the determination of the specific enzymes responsible for breaking down the

drug, the kinetic parameters of enzyme interactions, and the products of the reactions

[5,6]. Understanding the metabolism of alisol A in vitro is important for predicting in

vivo clearance and assessing potentially toxic or biologically active metabolites.

In this study, an LC-MS method was developed and applied to the study on the

enzyme kinetics of alisol A in RLM and HLM incubation systems. In addition, an

HPLC-QTOF MS method was applied to identify the structure of alisol A and its

metabolites. Chemical inhibitors of CYP450 and individual human recombinant

CYP450 enzyme were used to identify the CYP450 isozymes involved in the

formation of each metabolite of alisol A.

2. Materials and methods

2.1 Chemicals and reagents

Alisol A (purity > 98 %, calculated by peak area normalisation method (HPLC))

Page 4 of 21

Accep

ted

Man

uscr

ipt

4

was isolated in the author’s laboratory (Department of Pharmaceutical Analysis,

Shenyang Pharmaceutical University, Shenyang, People’s Republic of China) , and

the structure was characterized by spectral methods, including MS, 1H- and 13C-NMR

spectra. The data were consistent with those reported in literatures [7–9]. Diazepam,

Quercetin, α-Naphthoflavone and Ketoconazole were purchased from the National

Institute for Control of Pharmaceutical and Biological Products (Beijing, China).

Sulfaphenazolum and Methoxsalen were purchased form Sigma (St. Louis, MO,

USA). Reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) was

obtained from Roche (Basel, Switzerland). Acetonitrile (HPLC grade) were obtained

from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid (HPLC grade) was

purchased from Kermel Chemical Reagent Co., Ltd. (Tianjin, China). All other

chemicals were of analytical grade and obtained from common commercial source.

Distilled water, prepared with demineralized water, was used throughout the study.

Rat liver microsomes were prepared by the procedure of Omura and Sato [10] in

the author’s laboratory (Department of Pharmaceutical Analysis, Shenyang

Pharmaceutical University, Shenyang, People’s Republic of China). Human liver

microsomes and individual human recombinant CYP450 enzyme were purchased

from BD Biosciences (Bedford, MA, USA).

2.2 Instrumentation and chromatographic conditions

The assay was performed on Shimadzu 2010 liquid chromatography-mass

spectrometry (Japan) equipped with electrospray ionization (ESI) interface. Liquid

chromatographic separations of the analytes were performed by Kromasil C18 column

Page 5 of 21

Accep

ted

Man

uscr

ipt

5

(150 × 4.6 mm, 5 μm) with the mobile phase of acetonitrile-(containing 0.1% of

formic acid) water (60:40, v/v) at a flow rate of 0.8 ml/min (25% of the eluent was

splitted into the inlet of the mass spectrometer) in a run time of 15 min. The source

conditions were as follows: nebulizing gas 1.5 L/min; drying gas 1.5 L/min; CDL

temperature 250 �; heat block temperature 200 �; detector voltage 1.75 kV. The

injection volume was 20 μL.

The structure identification was performed on a Bruker Daltonics microTOF-Q

mass spectrometer (Billerica, MA, USA). The following parameters were employed:

the capillary voltage was set at 4,500 V. Nebulizing gas (N2) preesure was 1.2 Bar,

drying gas (N2) flow rate was 8.0 L/min and gas temperature was 180 �. Argon was

employed as the collision gas. The scan range was set at m/z 50–1000. Mass

spectrometer was equipped with an electrospray ionization (ESI) source.

2.3 Incubation conditions

The incubation conditions of the experiment were established and controlled to

provide a reproducible and linear rate of the metabolism in vitro. A typical reaction

mixture in the final volume of 250 μL contained 100 mmol/L of Tris–HCl buffer (pH

7.4), 5 mmol/L of MgCl2, 0.5 g/L of rat microsomal protein (or 0.2 g/L of individual

human microsomal protein) and 10.03 μmol/L of alisol A. The incubation mixture was

pre-incubated at 37 � for 3 min and reactions were initiated by adding 25 μL NADPH

(10 mmol/L), then incubated at 37 � in a water bath shaker. The disappearance rate of

alisol A rose following microsomal protein concentration (0.2–2.0 g/L for rat

microsomal protein concentration or 0.1–1.0 g/L for individual human microsomal

Page 6 of 21

Accep

ted

Man

uscr

ipt

6

protein concentration) and time (5–120 min). The reaction was terminated by adding

250 μL of ice-cold acetonitrile, then vortexed and centrifuged at 15000 rpm for 7 min

to remove precipitated protein. 20 μL of supernatant solution was used for analysis.

2.4 Chemical inhibition study

The effect of specific CYP inhibitors [11–15] such as α-naphthoflavone

(CYP1A2), methoxsalen (CYP2A6), ketoconazole (CYP3A5), quercetin (CYP2C8),

sulfaphenazolum (CYP2C9) and quinine (CYP2D6) on alisol A metabolism was

investigated in rat and human liver microsomes. Each inhibitor was tested in three

randomly selected rat liver samples, and the concentration range of inhibitors was

0–10 μmol/L except ketoconazole (0–4 μmol/L). Incubation conditions have been

described above and various concentrations of the different inhibitors were added in a

final volume of 0.25 mL. The organic solvent did not exceed 1% (v/v) in the

incubation mixture. Incubations without inhibitor were regarded as controls. The

metabolism of alisol A was analyzed by LC-MS and expressed as alisol A

disappearance rate.

2.5 Human recombinant CYP3A4 enzyme study

On the basis of the experiment with inhibitors, P450 enzymes shown to be

involved in the metabolism of alisol A were further examined using CYP3A4

recombinant system. All incubations in each experiment were performed in triple.

3. Results and discussion

3.1 Metabolite identification

Page 7 of 21

Accep

ted

Man

uscr

ipt

7

The extracted ion chromatograms for the positive ion electro-spray LC-MS analysis

of an incubation of 10.03 μmol/L of alisol A with rat and human liver microsomes are

shown in Fig.1 and Fig.2. A total of 3 and 6 oxidative metabolites were found in RLM

and HLM incubation systems, respectively. 3 metabolites found in both incubation

systems were identified by HPLC-QTOF MS. The retention time and the associated

information used in the identification are summarized in Table 1. The proposed

structures of alisol A and its metabolites M1–M3 are shown in Fig.3. Due to the

limitative fragmentation information, the structures of metabolites M4–M6 need to be

investigated in future.

3.1.1 Alisol A

The compound eluting at 11.3 min possessed the same molecule ion, full scan

MS/MS spectrum and chromatographic behavior with authentic alisol A. Therefore, it

was identified as unchanged alisol A. Alisol A showed strong sodium adduct [M+Na]+

at m/z 513.3567, [M+H−H2O]+ at m/z 473.3638 in positive mode and strong

[M+HCOO]− at m/z 535.3617 in negative mode, and MS/MS spectrum of them

provided a number of characteristic fragment ions listed in Table 1, which are useful

fragmental information in metabolite identification. The possible MS/MS

fragmentation mechanism of alisol A in positive and negative ion mode were shown

in Fig.4 and Fig.5, respectively.

3.1.2 M1

Metabolite M1 showed sodium adduct [M+Na]+ at m/z 529.3479, [M+H]+ at m/z

507.3657 in positive mode and [M+HCOO]− at m/z 551.3595 in negative mode,

Page 8 of 21

Accep

ted

Man

uscr

ipt

8

indicating an increase of 16 Da from alisol A, which suggested that M1 was a

hydroxylation metabolite. The MS/MS product ion at m/z 457 in negative mode was

48 Da lower than [M−H]− at m/z 505, suggesting the loss of HCHO and H2O. The

fragment ion at m/z 345 in positive mode was possibly formed by loss of the side

chain CH2C(OH)CH(OH)C(CH3)2OH and HCHO from parent ion [M+H]+ at m/z 507.

According to the loss of HCHO, hydroxylation might occur at the primary carbon. In

addition, the main fragment ions of M1 at m/z 381, 363, 337 were 2 Da lower than

those in MS/MS spectra of alisol A at m/z 383 ([M+H−H2O−CH2(OH)C(CH3)2OH]+),

m/z 365 ([M+H−2H2O−CH2(OH)C(CH3)2OH]+), and m/z 339

([M+H−H2O−CH3CH(OH)CH(OH)C(CH3)2OH]+), respectively. Therefore, the two

primary carbons of C-26 and C-27 were not hydroxylated. The fragment ions of M1 at

m/z 229 and 217 were also 2 Da lower than those in MS/MS spectra of alisol A at m/z

231 and 219, respectively, suggesting that one of the four primary carbons (C-18，

C-19，C-28 and C-29) on the A/B rings was hydroxylated. According to the

conformation of alisol A, C-28 and C-29 might be hydroxylated more easily than

C-18 and C-19. In addition, the carbonyl (C-3) made the loss of HCHO more easily

by the hydrogen rearrangement at the C-28-OH (or C-29-OH). According what stated

above, M1 most likely corresponds to the C-28 (or C-29)-hydroxy metabolite of alisol

A.

3.1.3 M2

Metabolite M2 showed sodium adduct [M+Na]+ at m/z 527.3326, [M+H]+ at m/z

505.3525 in positive mode and [M+HCOO]− at m/z 549.3402 in negative mode,

Page 9 of 21

Accep

ted

Man

uscr

ipt

9

indicating an increase of 14 Da from alisol A, which suggested that M2 was a

hydroxylation and carbonylation metabolite. The fragment ions of M2 at m/z 199, 217,

229 were the same as those of M1, indicating that C-28 (or C-29) was hydroxylated

and not carbonylated, and C-11-OH was also not carbonylated. The MS/MS product

ion at m/z 385 in negative mode was possibly formed by loss of side chain

HCOCH(OH)C(CH3)2OH, or HCOC(CH3)2OH and HCHO from the parent ion

[M−H]− at m/z 503, which indicated that the carbonylation occurred at C-23-OH.

Therefore, M2 was deduced as C-28 (or C-29)-hydroxy and C-23-OH carbonylation

metabolite of alisol A.

3.1.4 M3

Metabolite M3 showed sodium adduct [M+Na]+ at m/z 511.3379, [M+H−H2O]+

at m/z 471.3451 in positive mode and [M+HCOO]− at m/z 533.3472 in negative mode,

indicating a loss of 2 Da from alisol A, which suggested that M3 was a C-OH

carbonylation metabolite. However, C-25-OH couldn’t be carbonylated, for it was a

tertiary alcohol. Therefore, the carbonylation should occurred at C-11, C-23 and C-24.

The fragment ions of M3 at m/z 205, 219 and 231 were as same as those of alisol A,

indicating that the C-11-OH was not carbonylated. The main fragment ion at m/z 381

was 2 Da lower than [M+H−H2O−CH2(OH)C(CH3)2OH]+ of alisol A at m/z 383,

which indicated that C-24-OH was not carbonylated. Therefore, M3 was deduced as

C-23-OH carbonylation metabolite of alisol A.

3.2 Enzyme Kinetic Studies

Biotransformation of alisol A was found to be linear up to 0.5 mg/mL of

Page 10 of 21

Accep

ted

Man

uscr

ipt

10

microsomal protein and 15 min of incubation in RLM, and 0.2 mg/mL microsomal

protein and 10 min of incubation in HLM. The concentration/time-dependent overall

clearance of alisol A proceeded with a Km of 15.65 ± 2.33 µmol/L and a maximal rate

of 2.195 ± 0.156 nmol/min/mg protein in RLM, and 9.98 ± 3.85 µmol/L and 2.067 ±

0.419 nmol/min/mg protein in HLM.

3.3 Influence of P450 inhibitors on alisol A metabolism

To determine which isoform of CYPs involved in the alisol A metabolism, the

effect of specific CYP inhibitors on alisol A metabolism at 10.03 μmol/L was

investigated and shown in Fig.6. CYP3A4 inhibitor ketoconazole resulted in

significant decrease in metabolism of alisol A. It was suggested that CYP3A

contribute majorly to the metabolism of alisol A in vitro.

3.4 Influence of human recombinant CYP3A4 enzyme on alisol A metabolism

The percentage of parent compound disappearance relative to that in the

respective control incubations showed the metabolism of alisol A to be catalyzed by

CYP3A4 enzyme. A total of 6 metabolites were found and as same as the metabolites

in HLM, which indicatied that CYP3A4 was the main enzyme for catalyzing the

metabolism of alisol A.

3.5 Discussion

Our previous study indicated that alisol A has considerable cytotoxic effect on

the HKC cells. However, the detailed metabolism of alisol A and the cytochrome

P450 isoforms involved had not been clarified. It is important to understand the

metabolism of alisol A to identify the roles of CYP isoforms and metabolites of alisol

Page 11 of 21

Accep

ted

Man

uscr

ipt

11

A. In this study, we evaluated the metabolism of alisol A with an in vitro incubation of

liver microsomes, chemical inhibition method and CYP3A4 enzyme.

In chemical inhibition assay, six major CYP isoforms (CYP1A2, CYP2A6,

CYP2A5, CYP2C8, CYP2C9 and CYP2D6) in rat and human liver microsomes were

investigated, and results showed that CYP3A may be mainly involved in the

metabolism of alisol A in vitro. It was suggested that there would be several metabolic

pathways of alisol A and produce several metabolites. The distinct effect of different

metabolites, as a result of individual variation of CYPs may cause the individual

difference of alisol A toxic response.

An in vitro metabolism of alisol A was investigated and several metabolites of

alisol A were detected and characterized by LC-MS analysis. Comparing with the

control, three metabolites were found in the incubation with RLM, and six metabolites

were found in the incubation with HLM and CYP3A4 at the present of NADPH.

4. Conclusion

In summary, characterization of metabolites and cytochrome P450 isoforms

involved in the metabolism of alisol A were investigated in vitro metabolic

experiment. Alisol A was biotransformed into at least three and six metabolites in rat

and human liver microsomes, respectively. Chemical inhibitors of CYP450 and

individual human recombinant CYP450 enzyme study indicated that the formation of

each metabolite of alisol A was mainly catalyzed by CYP3A4 enzyme. The structure

of alisol A is so complex and contains so many similar moieties that it would be

extremely difficult to unambiguously identify metabolites solely using mass

Page 12 of 21

Accep

ted

Man

uscr

ipt

12

spectrometry. For example, the exact position of hydroxylation for the metabolites M1

and M2 remain unknown. Hence, the exact structure identification of metabolites

needs more information, especially the NMR data. In order to correlate the

involvement of the metabolites detected in this study and link the metabolism of alisol

A to its toxicity, research in vivo is needed.

Acknowledgements

This work was supported by National Natural Science Foundation of China

(No.20875064).

References

[1] W.F. Fong, C. Wang, G.Y. Zhu, C.H. Leung, M.S. Yang, H.Y. Cheung,

Phytomedicine 14 (2007) 160-165.

[2] H. Matsuda, N. Tomohiro, M. Yoshikawa, M. Kubo, Biol. Pharm. Bull. 21 (1998)

1317-1321.

[3] Y. Yu, C. Ma, K.S. Bi, G. G. Yang, P. Xie, J. Wang, X.H. Chen, Rapid. Commun.

Mass Spectrom. 25 (2011) 2633-2640.

[4] Y. Yu, Q. Li, K.S. Bi, P. Xie, G.G. Yang, X.H. Chen, Anal. Bioanal. Chem. 399

(2011) 1363-1369.

[5] J.H. Lin, A.Y. Lu, Pharmacol. Rev. 49 (1997) 403-449.

[6] R. Kostiainen, T. Kotiaho, T. Kuuranne, S. Auriola, J. Mass Spectrom. 38 (2003)

357-372.

[7] G.P. Peng, G.Y. Zhu, F.C. Lou, Nat. Prod. Res. Develop. 14 (2002) 5-8.

Page 13 of 21

Accep

ted

Man

uscr

ipt

13

[8] T. Murata, Y. Imai, T. Hirata, M. Miyamoto, Chem. Pharm. Bull. 18 (1970)

1347-1353.

[9] T. Murata, M. Miyamoto, Chem. Pharm. Bull. 18 (1970) 1354-1361.

[10] T. Omura, R. Sato, J. Biol. Chem. 239 (1964) 2370-2378.

[11] V.M. Lakshmi, T.V. Zenser, B.B. Davis, Drug Metab. Dispos. 25 (1997) 481-488.

[12] T. Hasegawa, K. Hara, T. Kenmochi, S. Hata, Drug Metab. Dispos. 22 (1994)

916-921.

[13] H.J. Chung, Y.H. Choi, S.H. Kim, M.G. Lee, J. Pharm. Pharmacol. 58 (2006)

449-457.

[14] Y. Jiang, C.L. Kuo, S.J. Pernecky, W.N. Piper, Biochem. Biophys. Res. Commun.

246 (1998) 578-583.

[15] L. Yu, D.J. Waxman, Drug Metab. Dispos. 24 (1996) 1254-1262.

Page 14 of 21

Accep

ted

Man

uscr

ipt

Highlights

1. The metabolites of alisol A were identified by HPLC-QTOF MS.

2. A total of 3 oxidative metabolites were found and identified in RLM incubation

systems.

3. A total of 6 oxidative metabolites were found and 3 of which were identified in

HLM incubation systems.

4. The formation of each metabolite of alisol A was mainly catalyzed by CYP3A4

enzyme.

*Highlights (for review)

Page 15 of 21

Accep

ted

Man

uscr

ipt

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

2.5

5.0(x10,000)

473.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

0.5

1.0

(x10,000)

529.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

0.5

1.0

(x10,000)

527.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.0

0.0

2.5

5.0(x1,000)

471.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

2.5

(x10,000)

473.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

2.5

(x10,000)

529.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

0.5

1.0

(x10,000)

527.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

0.5

1.0

(x10,000)

471.40 (1.00)

(a) (b)

M0 M0

M1

M2

M3

Fig.1 Extracted ion chromatograms of alisol A and the metabolites in RLM samples: (a) the sample incubated with deactivated microsomes (b) the sample after

incubation with 10.03 µmol/L alisol A for 20 min. (M0: alisol A; M1–M3: metabolites of alisol A)

Figure

Page 16 of 21

Accep

ted

Man

uscr

ipt

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

1.0

2.0(x10,000)

473.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

0.5

1.0

(x10,000)

529.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

2.5

(x1,000)

527.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

1.0

(x1,000)

471.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

0.5

1.0

1.5(x10,000)

473.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

2.5

5.0

7.5(x1,000)

527.40 (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

0.5

1.0

(x10,000)

471.40 (1.00)

M0 M0

M2

M3

M6

(a) (b)

0.0 2.5 5.0 7.5 10.0 12.5 15.00.0

2.5

(x10,000)

529.40 (1.00)

M1

M5M4

Fig. 2 Extracted ion chromatograms of alisol A and the metabolites in HLM samples: (a) the sample incubated with deactivated microsomes (b) the sample after

incubation with 10.03 µmol/L alisol A for 20 min. (M0: alisol A; M1–M6: metabolites of alisol A)

Figure

Page 17 of 21

Accep

ted

Man

uscr

ipt

O

HO

OH

OH

H

Alisol A

HO

O

O

OH

OH

H

HO

OH

2928

26

272524

23

2221

20

1918

17

16

15

14

1312

11

10

9

8

7

654

3

2

1

30

M2

O

HO

OH

OH

H

HO

OHM1

O

O

OH

OH

H

HO

M3

Fig.3 Chemical structures of alisol A and the identified metabolites (M1–M3)

Figure

Page 18 of 21

Accep

ted

Man

uscr

ipt

O

HO

OH

OH

H

m/z 473

- H2O

O

OH

OH

H

m/z 455

O

OH

H

m/z 437

- 2H2O

O

H

m/z 419

- 3H2O

O

HO

OH

H

m/z 415

O-

O

OH

H

m/z 397

- H2O

O

H

m/z 383

O

- OHHO

O

H

m/z 365

- H2O - OHHO

O

H

m/z 339

O

OH

OH

O

H

m/z 231

O

m/z 205

H

m/z 347

H+

H+

H+

H+

H+

O-

H+

H+

H+

H+

H+

H+

- 2H2O

(A)(B)

(C)(D)

-

Fig.4 The possible MS/MS fragmentation mechanism of alisol A in positive ion mode.

Figure

Page 19 of 21

Accep

ted

Man

uscr

ipt

O

HO

OH

OH

H

m/z 535 [M+HCOO]-

O

OH

OH

H

m/z 471

O

OH

H

m/z 453

M - 2H2O

O

OH

H

m/z 435

O-

O

OH

H

m/z 413

M - H2O

O

H

m/z 395

O

H

m/z 377

M- H

2 O

-

O

H

m/z 353

HO

OH

OH

-

HCOO-

O

H

m/z 371

O

OH

OHM -

HO

O

HO

OH

OH

H

m/z 489

H

HO

HO

H

OH

H

M - 3H2O

H

HO

H

O-M - 2H2O

OHH H

O-M - 3H2OHO

H

H

O

OH

OHM - H2O -

O

H

m/z 339

H

M - H2O

Fig. 5 The possible MS/MS fragmentation mechanism of alisol A in negative ion mode.

Figure

Page 20 of 21

Accep

ted

Man

uscr

ipt

0

10

20

30

40

50

60

70

80

90F

orm

ati

on

of

M1

(%C

on

trol)

Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

0

10

20

30

40

50

60

70

80

90

Form

ati

on

of

M2

(%C

on

trol)

Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

0

20

40

60

80

100

120

Fo

rmati

on

of

M3

(%C

on

tro

l)

Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

0

10

20

30

40

50

60

70

80

90

100

Fo

rmati

on

of

M1

(%C

on

tro

l)

Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

0

10

20

30

40

50

60

70

80

90

Fo

rmati

on

of

M2

(%C

on

tro

l)Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

0

10

20

30

40

50

60

70

80

90

100

Fo

rmati

on

of

M3

(%C

on

tro

l)

Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

0

20

40

60

80

100

120

Form

ati

on

of

M4

(%C

on

trol)

Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

0

20

40

60

80

100

120

Form

ati

on

of

M5

(%C

on

trol)

Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

0

10

20

30

40

50

60

70

80

Fo

rmati

on

of

M6

(%C

on

tro

l)

Naph Methox Sul Quer Qui Ket

Inhibitor Low Middle High

Fig. 6 Effects of specific inhibitors on the formation of metabolites M1, M2 and M3 of alisol A in RLM (a) and HLM (b); M4, M5 and M6 in HLM (c).

(n =3; ■, ■ and ■ represent low, medium, and high concentrations of inhibitors)

(a)

(b)

(c)

Figure

Page 21 of 21

Accep

ted

Man

uscr

ipt

Table 1 Alisol A and the metabolites identified in RLM and HLM samples using HPLC- QTOF MS after incubation with 10.03 µmol/L alisol A for 20 min.

tR

(min)

Positive/negative

ion mode (+/−) Calculated Measured Formula

|Error|

(ppm) MS/MS fragments

M0 11.3

+

529.3290[M+K]+

513.3550[M+Na]+

473.3625[M+H−H2O]+

529.3284[M+K]+

513.3567[M+Na]+

473.3638[M+H−H2O]+

C30H50KO5

C30H50NaO5

C30H49O4

1.1

3.3

2.7

(473)a 455 437 415 397 383 365 341 339

323 231 219 205

− 535.3640[M+HCOO]

−

525.3352[M+Cl]−

535.3617[M+HCOO]−

525.3327[M+Cl]−

C31H51O7

C30H48ClO6

4.4

4.8

(535)a 489 471 453 435 413 395 377 371

353 339

M1 4.3

+ 529.3500[M+Na]

+

507.3680[M+H]+

529.3479[M+Na]+

507.3657[M+H]+

C30H50NaO6

C30H51O6

4.0

4.6

(507)a 471 453 435 417 399 381 369 363

355 351 345 339 337 321 229 217 199

− 551.3589[M+HCOO]

−

541.3301[M+Cl]−

551.3595[M+HCOO]−

541.3316[M+Cl]−

C31H51O8

C30H50ClO6

1.0

2.8

(551)a 505 487 457 445 427 409 391 379

367 353 337

M2 5.5

+

543.3082[M+K]+

527.3343[M+Na]+

505.3524[M+H]+

543.3077[M+K]+

527.3326[M+Na]+

505.3525[M+H]+

C30H48KO6

C30H48NaO6

C30H49O6

0.9

3.2

0.2

(505)a 487 469 451 439 433 411 385 351

339 337 321 229 217 199

−

549.3433[M+HCOO]−

539.3145[M+Cl]−

503.3378[M−H]−

549.3402[M+HCOO]−

539.3159[M+Cl]−

503.3354[M−H]−

C31H49O8

C30H48ClO6

C30H47O6

5.6

2.6

4.8

(549)a 503 485 473 445 427 415 409 407

397 385 379 377 323

M3 13.8

+ 511.3394[M+Na]

+

471.3469[M+H−H2O]+

511.3379[M+Na]+

471.3451[M+H−H2O]+

C30H48NaO5

C30H47O4

2.9

3.8

(471)a 453 435 413 395 381 353 341 339

253 231 219 205

− 533.3484[M+HCOO]

−

523.3196[M+Cl]−

533.3472[M+HCOO]−

523.3188[M+Cl]−

C31H49O7

C30H48ClO6

2.2

1.5

(533)a 487 469 429 411 399 393 391 353

311

M4 3.5

+ 529.3500[M+Na]+ 529.3466[M+Na]

+ C30H50NaO6 6.4 /

− 551.3589[M+HCOO]

−

541.3301[M+Cl]−

551.3574[M+HCOO]−

541.3323[M+Cl]−

C31H51O8

C30H50ClO6

2.7

4.1 (551)

a 505 487 469 427 393 367 351

M5 3.7

+ 529.3500[M+Na]+ 529.3479[M+Na]

+ C30H50NaO6 4.0 /

− 551.3589[M+HCOO]

−

541.3301[M+Cl]−

551.3563 [M+HCOO]−

541.3324[M+Cl]−

C31H51O8

C30H50ClO6

4.7

4.2 (551)

a 505 487 469 451 429 393 367 351

M6 5.1

+ 527.3343[M+Na]

+

505.3524[M+H]+

527.3314 [M+Na]+

505.3497[M+H]+

C30H48NaO6

C30H49O6

5.5

5.3 /

− 549.3433[M+HCOO]

−

539.3145[M+Cl]−

549.3398[M+HCOO]−

539.3116[M+Cl]−

C31H49O8

C30H48ClO6

6.4

5.4

(549)a 503 445 427 409 397 379 377 371

323 a m/z of parent ions.

Tables