Accepted Manuscript

Title: Chemical and metabolic analysis of Achyranthes

bidentate saponins with intestinal microflora-mediated
biotransformation by ultra-performance liquid
chromatography-quadrupole time-of-flight mass spectrometry
coupled with metabolism platform

Authors: Jun Fu, Hong Wu, Huan Wu, Ran Deng, Feng Li

PII: S0731-7085(18)32853-X
DOI: https://doi.org/10.1016/j.jpba.2019.03.041
Reference: PBA 12554

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 20 December 2018

Revised date: 13 March 2019
Accepted date: 18 March 2019

Please cite this article as: Fu J, Wu H, Wu H, Deng R, Li F, Chemical and metabolic

analysis of Achyranthes bidentate saponins with intestinal microflora-mediated
biotransformation by ultra-performance liquid chromatography-quadrupole time-of-
flight mass spectrometry coupled with metabolism platform, Journal of Pharmaceutical
and Biomedical Analysis (2019), https://doi.org/10.1016/j.jpba.2019.03.041

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Chemical and metabolic analysis of Achyranthes bidentate saponins with intestinal

microflora-mediated biotransformation by ultra-performance liquid

chromatography-quadrupole time-of-flight mass spectrometry coupled with

metabolism platform

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Jun Fua,b,c, Hong Wua,b,c*, Huan Wua,b,c*, Ran Denga,b,c, Feng Lia,b,c

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Affiliation

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a. Anhui University of Chinese Medicine, Hefei 230012, China.
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b. Key Laboratory of Xin'an Medicine, Ministry of Education, Anhui Province Key
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Laboratory of R&D of Chinese Medicine, Hefei 230012, China.
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c. Anhui Province Key Laboratory of Chinese Medicinal Formula, Hefei 230012,

China
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* Corresponding author: Hong Wu, Anhui University of Chinese Medicine, Qianjiang

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Road No.01, Hefei 230012, China. Tel.: +86 551 6812 9166; fax: +86 551 6812 9166.
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E-mail address: wuhongprof@aliyun.com

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* Corresponding author: Huan Wu, Anhui University of Chinese Medicine, Meishan

Road No.103, Hefei 230038, China. Tel.: +86 551 65169051. E-mail address:

wuhuancpu@163.com

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Graphical abstracr

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Research highlights:
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 A total of 40 triterpene saponins were identified from the Achyranthes bidentate

(AB) extract.
 39 metabolites of AB triterpene saponins transformed by intestinal microflora in
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vitro were characterized.

 Deglycosylation, glycosylation, oxidation and dehydrogenation were the main
metabolic pathways.
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 Metabolic position of the oleanane-type triterpenoid saponins biotransformed by

intestinal microflora was proposed.
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Abstract
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Achyranthes bidentate (AB) is a typical traditional Chinese medicine (TCM) that has
been widely used in clinical practices for more than a thousand years. Modern
pharmacological studies have shown that triterpene saponins are the main
pharmacological active ingredients in AB. Meanwhile, the poor oral bioavailability of
triterpene saponins in AB indicates that these ingredients are probably metabolized by
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intestinal microflora before absorption. In this work, an integrated analysis based on
ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry
(UPLC-Q-TOF/MS) combined with a metabolism platform was developed to identify
the chemical constituents and intestinal metabolic profiles of triterpene saponins in AB.
As a result, a total of 40 triterpene saponins (including thirty-eight oleanane-type, one
hederagenin-type and one machaerinate-type triterpene saponin) were identified from
the AB extract. Moreover, 39 biotransformation products mediated by intestinal

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microflora were characterized, which mainly underwent four metabolic reactions

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including deglycosylation, glycosylation, oxidation and dehydrogenation. To our

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knowledge, the in vitro metabolites of AB through intestinal microflora metabolism,

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especially triterpene saponins, have not been studied previously. The obtained results
could be helpful for the further evaluation of the pharmacokinetics and the

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pharmacological activity of triterpene saponins of AB in vivo.
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Keywords: Achyranthes bidentate, Triterpene saponins, Intestinal microflora, UPLC-
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Q-TOF/MS, Metabolites
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Introduction
Achyranthes bidentate (AB) is generally known as “Niu Xi”, one of the most
important traditional Chinese medicines (TCM) [1]. The major ingredients of AB are
saponins, polysaccharides, ketosteroids, polypeptides and phytosterones [2-5]. Modern
pharmacological studies have shown that they exhibit different pharmacological
activities such as immuno-regulatory and anti-inflammatory activities [6-7]. Until now,
a total of more than 133 chemical constituents have been isolated and identified in AB

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[1]. Of these components, triterpene saponins [8-9] are the most abundant and effective

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constituents, and the triterpene saponins have been demonstrated to possess various

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activities including antitumor and anti-inflammatory activities. However, there is

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limited knowledge about the pharmacodynamics material basis of AB, and even worse,
the metabolic study of AB triterpene saponins is scarce. Thus, a comprehensive study

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on the chemical and metabolic profiles of AB is necessary.
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Recently, research on the biotransformation of saponins by intestinal microflora
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has become a focus of interest [10-13]. The low bioavailability of triterpene saponin
compounds by oral administration has become a bottleneck in the development of AB
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triterpene saponins. Since most Chinese medicine is administered orally, triterpene

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saponins are inevitably affected by the intestinal flora, resulting in changes in the
chemical structure of triterpenoid saponins and affecting their pharmacological
activities [14-16]. Therefore, by establishing an intestinal microflora metabolic system,
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we can better understand the metabolic characteristics of triterpene saponins in AB and

further investigate the potential pharmacodynamic substances based on intestinal
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microflora-mediated biotransformation.
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However, due to the complex composition of Chinese medicine and lack of

standard products, metabolic research on TCM has always been a very challenging task.
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With the development of sophisticated instruments and analytical techniques, ultra-

performance liquid chromatography coupled with quadrupole time-of-flight mass
spectrometry (UPLC-Q-TOF/MS) has been widely used in chemical characterization
and metabolites identification due to its high selectivity and resolution [17]. UPLC-Q-
TOF/MSE (E represents collision energies) allows identification of metabolites using
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parallel scans at low energy or at high energy. Thus, accurate precursor and fragment
ions are collected in a single run, which is helpful for the structural elucidation of
multiple components in plant extracts [18-19]. Meanwhile, some powerful data
processing platforms (UNIFI, MetaboLynx, MassHunter, etc.) have been successfully
applied to quickly profile the chemical structures and metabolites from complex
compounds [20-23]. Therefore, in this study, an integrated analysis based on UPLC-Q-
TOF/MSE coupled with a metabolism platform was successfully applied for chemical

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constituents and intestinal metabolic profiles of triterpene saponins in AB, and a total

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of 40 triterpene saponins were identified. Moreover 39 metabolites were tentatively

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identified, three representative triterpene saponins were chosen to investigate the

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metabolic characteristics and the metabolic pathway was also proposed. The results of
the study could be helpful to gain a better understanding of the metabolic mechanism

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of triterpene saponins in vitro and may provide suggestions for further study in vivo.
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2 Materials and methods
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2.1 Materials and reagents

The roots of Achyranthes bidentate were purchased from the Henan Province of
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China. The samples were identified by Prof. Jinsong Liu (Anhui University of Chinese
Medicine, Heifei, China). The voucher specimens (20170623) were deposited at the
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herbarium of Anhui University of Chinese Medicine, Heifei, China. Chikusetsusaponin

V (CHB170310), Chikusetsusaponin IV (CHB180524), Chikusetsusaponin IVa
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(CHB170227) and 28-desglucosylchikusetsusaponin IVa (CHB180307) were

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purchased from Mansite Biotech Co., Ltd. (Chengdu, China). The purity of each
standard compound was determined to be >98%. LC-grade acetonitrile (ACN),
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methanol and formic acid were obtained from Tedia (Fairfield, OH, USA). Pure water
was prepared with the Millipore system (Millipore, MA, USA). Other reagents were of
analytical purity. Standard solutions were prepared in methanol at a concentration of
1.0 mg·mL−1, and stored in the refrigerator at 4 °C prior to use.

2.2 Sample preparation and extraction

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 The powder root of AB (100.0 g) was added to 1.0 L ethanol with a concentration
of 60% and was refluxed three times, each time for 0.5 h. The extracts were collected
together and concentrated as 0.1 g·mL−1 of raw herbs. 150 mL extract of AB was
purified with the D-101 macroreticular resin and washed with 400 mL distilled water.
It was subsequently eluted with 300 mL ethanol with a concentration of 70%. After
being evaporated to dryness (2.13 g), the residuals (10 mg) were dissolved with 10 mL
methanol. All sample solutions were filterd through 0.22 µm membrane filters before

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being injected into the UPLC.

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2.3 Preparation of the intestinal culture medium
Fresh feces collected from male Sprague-Dawley (SD) rats (200±20 g) were

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immediately mixed with cold physiological saline at a ratio of 1 g to 4 mL, and the
supernatant was obtained by centrifugation at 2000 r·min−1 for 10 min. 10 mL

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supernatant was added to 90 mL anaerobic culture medium, and then the intestinal
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bacteria culture medium was obtained. Anaerobic culture medium was prepared as
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follows: solution A (37.5 mL, 0.47% KH2PO4, 1.18% NaCl, 1.2% (NH4)2SO4, 0.12%
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CaCl2 and 0.25% MgSO4·H2O), K2PO4 (37.5 mL, 0.78%), Na2CO3 (50 mL, 8%); L-
cysteine (0.5 g), L-ascorbic acid (2 mL, 25%), eurythrol (1g), tryptone (1 g) and nutrient
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agar (1 g) were mixed together and diluted with distilled water to 1 L. The solution was
then adjusted to PH 7.5-8.0 with 2 M HCl [24].
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2.4 PCR analysis of intestinal microflora

A DNA kit (Omega Bio-tek, USA) was used to extract the DNA from the blank
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group and the experimental group according to the instructions of the kit. The V4
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variable [11] region of bacterial 16s rRNA gene was amplified with PCR (the
amplification conditions were: denaturation at 95 oC for 3 min, annealing at 55 oC for
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30 s, and elongation at 72 oC for 45 s, and then extension at 72 oC for 10 min), using

primers 515F 5'-GTGCCAGCMGCCGCGGTAA-3' and 806R 5'-
GGACTACHVGGGTWTCTAAT-3'. Three replicates were performed for each sample
amplification. Illumina MiSeq sequencing involved recovering the amplicon from a 2%
agarose gel, purifying according to the AxyPrep DNA Gel Extraction Kit instructions,
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and quantification by QuantiFluorTM-ST. The purified amplifiers were equally mixed
and paired-end sequenced according to the standard procedure of the Illumina MiSeq
sequencing platform.

2.5 In vitro biotransformation of three representative saponins by intestinal

bacteria
Three representative saponins were prepared by dissolving an accurately weighed

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amount in intestinal bacteria culture medium to obtain a final concentration of 1.03

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mg·mL−1 (Chikusetsusaponin V), 1.32 mg·mL−1 (Chikusetsusaponin IV) and 1.12

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mg·mL−1 (Chikusetsusaponin IVa), respectively. Each reference standard was

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incubated anaerobically at 37 oC for 0, 1, 2, 4, 6, 8, 10, 12 and 24 h. The incubation
system was terminated by the addition of an equal volume of cold methanol. The
mixture was vortex-extracted for 2 min and centrifuged at 10000 r·min−1 for 10 min.
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Then, the supernatant was filtered through 0.22 µm filter membranes for analysis.
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2.6 In vitro biotransformation of triterpene saponins in AB by intestinal bacteria
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Take 10 mL of the intestinal bacteria culture medium and added 10 mg of AB

extract in parallel with 9 parts. The incubation system was carried out at 37 oC under
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anaerobic conditions for 0, 1, 2, 4, 6, 8, 10, 12 and 24 h. The incubation system was

terminated by the addition of an equal volume of cold methanol. The blank was the
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incubation solution without AB extract and the 0 h incubation solution served as the
control. The mixture was vortex-extracted for 2 min and centrifuged at 10000 r·min−1
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for 10 min. Then, the supernatant was filtered through 0.22 µm filter membranes for
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analysis.

2.7 UPLC-Q-TOF/MS analysis

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By adopting Waters ACQUITY UPLC incorporated with a thermostatically

controlled column compartment, an autoplate-sampler, an online degasser and a binary
pump, the chromatographic analysis was performed. The analytical column
(Phenomenex C18 column, 2.1mm × 100 mm, 1.6 µm, USA) was maintained at 30 oC.
The flow rate was kept at 0.2 mL·min−1, and the sample injection volume was 2 µL.
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The mobile phases were 0.1% (v/v) formic acid in water (A) and ACN (B). The gradient
was as follows: 0-10 min (5-30% B), 10-11 min (30% B), 11-12 min (30-45% B), 12-
12.5 min (45-47% B), 12.5-15 min (47-48% B), 15-16 min (48-50% B), 16-19 min (50-
70% B), 19-22 min (70-85% B), 22-24 min (85-95% B), 24-27 min (95% B), 27-28
min (95-5% B) and 28-30 min (5% B).
The detection was conducted with a Xevo G2-XS QTOF mass spectrometer
(Waters, Milford, USA) with a LockSpray capable-electrospray interface (ESI). The

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MS parameters were presented as follows: a capillary voltage of -2.5 kV, source offset

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of 80 V, sampling cone of 40 V, source temperature of 120 oC, desolvation temperature

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of 350 oC, cone gas flow rate of 50 L· h−1 and desolvation gas (N2) flow rate of 600

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L· h−1. It was set to the negative mode and the mass range was set to m/z 50-1200. Mass
accuracy was kept at a concentration of 200 ng·mL−1 using a lock spray with leucine

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enkephalin ([M-H]− =554.2615) and a flow rate of 10 μL·min−1 via a lockspray
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interface. The scan time for the lock mass was set to 0.5 s with an interval of 60 s. In
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MSE mode, the MS data were collected at a constant collision energy setting of 6 eV
during low-energy MS mode for precursor ion data. On that basis, in the high-energy
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MSE mode, the collision energy dynamically ranged from 80 eV to 100 eV for optimal
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fragmentation data. The system was controlled using MassLynx V 4.1 software (Waters
Co., USA).
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3 Results and discussion

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3.1 The analysis of the identification chemical constituents and metabolites of AB

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saponins
As mentioned in the introduction, an integrated analysis based on UPLC-Q-
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TOF/MSE combined with a metabolism platform was applied for chemical constituents
and intestinal metabolic profiles of AB triterpene saponins. First, a self-building
chemical database was established by searching the mass spectrometry database
(Database of TCM chemical component, Chemspider, SciFinder and Mass bank) and
related literature. All compounds were listed in an excel spreadsheet, including

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compound names, formulas and exact molecular weights of the compounds. In addition,
the MOL files (ChemBioDraw Ultra 14.0, USA) of all compound structures were also
saved in the same folder. Second, 4 reference standards were used to summarize the
retention time and fragmentation behaviours. The standards were divided into two types
based on the substituent groups linked at C-3 and/or C-28. Then, the characteristic
fragment ions and chromatographic behaviours were obtained. Third, chemical
components in AB were screened by UNIFI. Oleanolic acid (C30H48O3) was set as the

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common fragment; a margin of error no more than 5 ppm for identified compounds was

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allowed; negative adducts, including +COOH and -H were selected. After being

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processed, the most rational formulas were screened by self-building chemical and

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unknown compounds were searched in an online database. Then, 3 representative
saponins were chosen to investigate the major metabolites and metabolic pathways.

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Last, diagnostic ion extraction, neutral loss (NL), mass defect filter (MDF) and
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manually elucidation were also used to enrich the metabolites. As depicted in Fig. 1,
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the identification of chemical compounds and metabolites became easier, and the
UNIFI metabolism platform combine with multiple data mining techniques could be
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applied to detect the metabolites with a lower intensity, which is helpful to predict
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metabolites.

3.2 Fragmentation behaviours of four reference substances

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The fragmentation behaviours of triterpene saponins in AB were initially

investigated by four oleanane-type reference substances (28-
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desglucosylchikusetsusaponin IVa, chikusetsusaponin IVa, chikusetsusaponin IV and

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chikusetsusaponin V).
28-Desglucosylchikusetsusaponin IVa exhibited a quasi-molecular ion at m/z
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631.3837 [M−H]− (collision energy was 6 eV). The main fragment ions of the mass
spectra (collision ramp energy was 80-100 eV) were observed at m/z 455.3509,
175.0243 and 113.0240. Among these fragments, m/z at 455.3509 [M−H−GluA
(C6H8O6)]− and 175.0243 were the direct fragmentation products of 28-
Desglucosylchikusetsusaponin IVa. Fragment ions at m/z 175.0243 and 113.0240
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indicated the presence of glucuronic acid (GluA). The proposed fragmentation pathway
was described in Fig. 2 A.
Chikusetsusaponin IVa produced the [M−H]− ion at m/z 793.4402, which produced
the fragment ions at m/z 631.3837, 613.3747, 569.3859, 455.3508, 175.0238 and
113.0235. The ion at m/z 631.3837 corresponded to a loss of –Glc from the ion at m/z
793.4416. The ions at m/z 613.3747 and 569.3859 resulted from 631.3837 by loss of
H2O and CO2. The ions at m/z 455.3508 corresponded to oleanolic acid, which was

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generated by the loss of a -GluA from the ion at m/z 631.3837. (Fig. 2 B).

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Chikusetsusaponin IV yielded a deprotonated molecule [M-H] － ion at m/z

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925.4816. In a high energy MSE scan, the product ions at m/z 793.4344 [M-H-

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Arabinose (Ara)]−, 631.3865 [M-H-Ara-Glc]− and 455.3508 [M-H-Ara-Glc-GluA]−
were produced by the continuous loss of a sugar moiety from C-28 and C-3 of oleanolic

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acid. The fragment ion at m/z 613.3696 was obtained by the loss of H2O from 631.3865,
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and the further fragment ion at m/z 569.3859 yielded from the cleavage of CO2. (Fig. 2
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C).
Chikusetsusaponin V showed a quasi-molecular ion [M−H]− at m/z 955.4922. The
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main fragment ions of the mass spectra were observed at m/z 793.4402 [M−H−Glc]−,
731.4390 [M−H−Glc−H2O−CO2]−, 613.3747 [M−H−2Glc−H2O]−,
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569.3859
[M−H−2Glc−H2O−CO2]− and 455.3508 [M−H−2Glc−GluA]−. m/z at 455.3508
[M−H−2Glc−GluA]− was the characteristic adduct ion of oleanolic acid (Fig. 2 D).
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According to the fragmentation behaviours of the four reference substances,

mono-substituted sugar was substituted at the C-3 position of oleanolic acid or di-
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substituted sugars at the C-3 and C-28 positions. Interestingly, we observed that di-
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substitution outputted a representative doubly charged adduct ion [M+HCOOH−2H]2−

(Fig. S1). The substituent at the C-28 position was easier to remove than the C-3
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substituent, pentose was easier to eliminate than hexose. All triterpenoid saponins of
AB have the same diagnostic and characteristic fragment ion at 455.35, which is the
adduct ion of oleanolic acid. A previous study [25] reported that the development of
LC-MS methods for determination of triterpene is hampered by its poor fragmentation
during collision-induced dissociation (CID). In this study, the chemical structure of
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oleanolic acid is relatively firm and not easy to crack under the collision energy when
ramped from 80 eV to 100 eV. In addition, the fragmental ions such as 176 Da (GluA),
162 Da (Glc), or 132 Da (Ara) were also observed in the mass spectra (collision ramp
energy was 80-100 eV) of the reference substances. It also found that the sugar residue
ions had high abundance in the range of m/z 100-400. GluA and Glc were the most
common substitution at the C-3 and C-28 positions, as shown in Fig. S2. At the high
energy scan, m/z 175.0231, 157.0131 and 113.0237 indicated the presence of -GluA,

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while -Glu showed the characteristic ions at m/z 179.0555, 161.0439, 131.0324,

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119.0320, 113.0327 and 101.0299.

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3.3 Characterization of the triterpene saponins in AB
After data acquisition, chemical components in AB were automatically and fast

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screened by UNIFI based on a self-built database (containing the compound name,
accurate m/z, molecular formula and the structure of AB triterpene saponins derived
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from the literature and online databases). As a result, 40 triterpene saponins were
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rapidly characterized, including thirty-eight oleanane-type triterpene saponins (13
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mono-substituted and 25 di-substituted), one hederagenin-type and one machaerinate-

type triterpene saponin. The detailed MS data, including retention time, accurate mass,
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formula, ppm error and fragment ions are summarized in Table S1. The structures are
shown in Fig. S3.
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3.4 The genus composition of intensitinal microfloa

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The histograms of the intestinal microbial community revealed the species and
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their relative abundance (Fig. 3). Bacteroides, Bifidobacterium and Lachnoclostridium

were higher in the blank group, while Pediococcus was increased significantly in the
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experimental group. AB saponin metabolism in the experimental group produced a

large number of sugars to promote the growth of bacteria and thus accelerated the
metabolism of saponins. The operational taxonomic unit (OUT) was generated using
UPARSE7.1 software. By comparing the blank group with the experimental group OTU,
there was no significant difference in the composition of intestinal flora between the

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groups.

3.5 Characterization of three representative compounds related metabolites and

metabolic pathways in vitro
Due to the complexity of the composition of Chinese herbal medicines, it is
difficult to obtain standard substances. Therefore, considering the chemical structure
and contents, three representative compounds were used to investigate the metabolites

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and metabolic pathways. Data acquisition was conducted by UPLC-Q-TOF/MSE in the

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negative ion mode, and then the MSE raw data were processed with the UNIFI platform.

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We set up the metabolic reactions and corresponding parameters before analysis, MDF,

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NL and common fragments were also applied for confirmation of the metabolites. As a
result, the metabolites were tentatively identified, and these metabolites all exhibited

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similar fragmentation behaviours. Thereby, by setting these parameters, metabolites
were easily screened.
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3.5.1 Metabolites of Chikusetsusaponin IVa
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A total of 6 metabolites (IVa-M (1-6)) of chikusetsusaponin IVa were identified

(Fig. 4). Among them, deglycosylation was the main metabolic pathway. Detailed
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information of metabolites is shown in Table 1. IVa-M0 was eluted at 14.51 min with
a [M-H]－ ion at m/z 793.4390, which produced the fragment ions at m/z 631.3853,
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613.3747, 569.3810, 455.3508, 175.0238 and 113.0235. The ion at m/z 631.3853
corresponded to a loss of –Glc from the ion at m/z 793.4390. The ions at m/z 613.3747
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and 569.3810 resulted from 631.3853 by loss of H2O and CO2. The ions at m/z 455.3508
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corresponded to oleanolic acid, which was generated by loss of a -GluA from the ion at
m/z 631.3837.
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IVa-M3, IVa-M4 and IVa-M6 were formed by deglycosylation. IVa-M3 was

eluted at 19.16 min and yielded a deprotonated molecule [M-H]－ ion at m/z 631.3853
(C36H56O9), which was 162 (C6H10O5) Da lower than the deprotonated ion of IVa-M0.
IVa-M4 was observed at the retention time of 19.96 min and exhibited an [M+HCOO]
－
ion at m/z 663.4119, which indicated a -GluA cleavage at C-3. We found that when

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C-3 was the hydroxyl radical, [M+HCOO] － was produced. IVa-M6 (24.51 min,
C30H48O3) was formed by cleavage of -GluA and -Glc of IVa-M0.
IVa-M2 (11.40 min) showed an [M-H]－ ion at m/z 955.4928 (C48H76O19). Further
fragment ions at 793.4344 [M-H-Glc]－, 631.3853 [M-H-Glc-Glc]－, 569.3859 [M-H-
Glc-Glc-CO2] － and 455.3508 [M-Glc-Glc-GluA] － were generated which was
considered as the glycosylation conjugates of IVa-M0.
IVa-M1 (10.44 min) exhibited a deprotonated ion at m/z 809.4350 (C42H66O15),

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which was16 Da more than IVa-M0. The product ions at m/z 647.3792 and 471.3489

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were also produced by the cleavage of -Glc and -GluA at C-28 and C-3 of IVa-M0,

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suggesting the oxidation site was at the oleanolic acid skeleton instead of the glycosyl

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substituents.
IVa-M5 (21.34 min) showed the [M-H]－ ion at 453.3358 (C30H46O3), which was

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2 Da less than IVa-M6, indicating that it was IVa-M6 dehydrogenation product, and
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the position of dehydrogenation was presumed to be at C-3 of oleanolic acid [26].
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3.5.2 Metabolites of Chikusetsusaponin IV
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A total of 12 metabolites (IV-M (1-12)) of chikusetsusaponin IV were identified

(Fig. 5). Detailed information on the metabolites are shown in Table 1. IV-M0 was
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eluted at 12.94 min and yielded a deprotonated molecule [M-H]－ ion at m/z 925.4803.
In the high energy MSE scan, the product ions at m/z 793.4402 [M-H-Ara]－, 631.3837
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[M-H-Ara-Glc] － , 613.3696 [M-H-Ara-Glc-H2O] － , 569.3810 [M-H-Ara-Glc-CO2] －

and 455.3508 [M-H-Ara-Glc-GluA] － were produced by continuous loss of a sugar
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moiety from C-28 and C-3 of oleanolic acid.

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IV-M4, IV-M6, IV-M7, IV-M9 and IV-M12 were produced by the cleavage of -
Ara, -GluA and -Glc at C-3 or/and C-28 of IV-M0, respectively. In the high energy MSE
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scan, these metabolites fragmentation behaviours were similar to IV-M0. IV-M2

showed the precursor ion [M-H]－ at m/z 809.4350 (C42H66O15), which was 16 Da more
than IV-M4. The product ions at m/z 647.3792 and 471.3444 were yielded in the same
manner as described above. In addition, IV-M1, IV-M5 and IV-M10 were oxidized
from IV-M0, IV-M6 and IV-M12, respectively. IV-M3 eluted at 11.37 min with an [M-
13
H] － ion at m/z 1087.5380 (C53H84O23), and further fragment ions at m/z 925.4504,
793.4337, 631.3904, 569.3908 and 455.3596 were produced, which indicated the
glycosylation reaction occurred at IV-M0. IV-M8 (21.34 min) showed a deprotonated
ion at m/z 661.3978 (C36H56O8), which was 2 Da lower than IV-M9, the high MSE scan
fragment ions of 453.3400 [M-H-Glc] － , indicating that it was a dehydrogenation
product of IV-M9. In addition, IV-M11 (23.13 min) also showed a deprotonated ion at
m/z 453.3440, which was the dehydrogenation product of IV-M12.

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3.5.3 Metabolites of Chikusetsusaponin V

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A total of 11 metabolites (V-M (1-11)) of chikusetsusaponin V were identified (Fig.

SC
6). Detailed information of metabolites are shown in Table 1. V-M0 was eluted at 11.44
min with a [M-H]－ ion at m/z 955.4931 and the high energy scan fragment ions at m/z

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793.4402 [M-H-Glc] － , 731.4390 [M-H-Glc-H2O-CO2] － , 613. 3747 [M-H-Glc-Glc-
H2O] － , 569.3859 [M-H-Glc-Glc-CO2] － , 455.3508 [M-H-Glc-Glc-GluA] － and
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113.0234 [GluA-H-H2O-CO2]－.
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V-M3, V-M5, V-M6, V-M7 and V-M11 were produced by cleavage of sugar
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moieties and glycolic acid at C-3 and/or C-28 of oleanolic acid. These metabolites
showed similar fragmentation behaviours to V-M0. It is worth noting that V-M3 and
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V-M5 are isomers, which were distinguished by comparing chromatographic

behaviours. In MS spectra, the deprotonated ion of V-M1 was at m/z 971.4852, which
PT

was 16 Da higher than V-M0. Moreover, the characteristic fragment ions at m/z
809.4408, 647.3792 and 471.3553 were found. Thus, it was identified as an oxidation
E

product of V-M0. In addition, V-M4 and V-M9 were oxidized from V-M3 and V-M11,
CC

respectively. V-M2 was observed at the retention time of 10.88 min and the
deprotonated molecule [M-H]－ ion at m/z 1117.5440, which was 162 Da more than V-
A

M0, and the further fragment ions were similar to V-M0, indicating that it was the
glycosylation derivative. V-M8 was observed at 21.33 min with an [M+HCOO]－ ion
at m/z 661.3978 (C36H56O8), which indicated a dehydrogenation product of V-M7
(19.96 min, C36H58O8). Likewise, V-M10 (23.13 min, 453.3355) was considered as the
dehydrogenation product of V-M11.
14
 In summary, the metabolite profiles of three representative saponins with rat
intestinal were investigated. The main metabolic pathway of oleanane-type triterpenoid
saponins are deglycosylation in rat intestines. Dehydrogenation, oxidation and
glycosylation were also found in this study. According to the MS/MS spectra of
dehydrogenation metabolites, the location of dehydrogenation was speculated to be at
C-3 of oleanolic acid instead of the substituent groups. Oxidation is also an important
metabolic pathway, and the location was presumed to be at the aglycones. The fragment

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ion at m/z 455.35 corresponded to the aglycone of oleanolic acid while the characteristic

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ions at m/z 453.33 and 471.34 were derived from dehydrogenation and oxidation of

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oleanolic acid. The degradation of the sugar moiety was observed by the losses of 176

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Da (GluA), 162 Da (Glc), and 132 Da (Ara). Additionally, other NL, including 44 Da
(CO2) and18 Da (H2O) were also observed.

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3.6 Identified metabolites (t=8 h) of AB triterpene saponins and proposed
N
metabolic pathways
A
The blank, control and 8 h incubation solution in the negative ion MSE mode are
M

shown in Fig. 7. All samples were prepared in parallel and analysed under the same
conditions. Based on the fragmentation behaviors summarized from the standards,
ED

common fragment ions, NL search and MDF were used to identify the metabolites. The
diagnostic ion extraction was used to identify the potential metabolites. After 8 h
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incubation with intestinal microflora, a total of 39 metabolites were detected (Table 2).
Among these metabolites, a common fragment ion of m/z 455.35 could be observed
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from the deglycosylation metabolites, since most saponins were based on oleanolic acid.
CC

Dehydrogenation and the oxidation product showed the fragment ions at m/z 453.33
and 471.34 respectively.
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According to the metabolic reaction of the representative standards, we set up four

metabolic reactions in UNIFI. Because of the similarity of the structures of these
compounds, these compounds may be transformed into each other. Therefore, this
experiment extracted the peak areas of 13 representative compounds with better
responses at different time points to describe the dynamic changes of the compounds.
15
As shown in Fig. 8, the compound did not change significantly in the first 4 h, and some
compounds began to decrease from 4 h to 6 h (e.g. Achyranthoside E, Zingibroside R1,
etc.), with Achyranthoside E as the most obvious. The peak area of most compounds
decreased between 6 and 8 h, indicating that the metabolic reaction was most intense at
this time. Most compounds decreased slowly after 8 h. In addition, some compounds
showed different trends. The Chikusetsusaponin-1 increased more than eight times at 6
h, and 28-deglucosyl-achyranthoside C and oleanolic acid increased to varying degrees.

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After 8 h, the growth of oleanolic acid increased significantly. Oleanolic acid and some

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mono-substituted compounds were predominant at 12 h. All compounds were found to

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be completely metabolized at 24 h. It can be found from the metabolism curve that the

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di-substituted compound can be metabolized into a mono-substituted compound in the
intestinal flora, so that the mono-substituted compound exhibits an "M" type curve.

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Deglycosylation is the main metabolic reaction. M3, M4, M12, M18, M19, M20,
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M22, M23, M26, M27, M28, M30, M32, M35, M36, M37, M38 and M39 were formed
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by the stepwise cleavage of -Glc and -GluA at C-3 and/or C-28 of the oleanolic acid.
Deglycosylation metabolites of triterpenoid saponins and their biotransformation
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characteristics in intestinal microflora are described in Fig. 9. And these metabolites

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exhibit the same fragmentation behaviours as prototypes. In addition, since some

compounds have a -C5H7O6 substituent at the C-3 of -GluA, we observed that these
compounds produce a metabolite that loses -C2H2O2. As an example, metabolite M10
PT

displayed a deprotonated molecule [M-H]－ ion at m/z 897.4468, which was proposed
to be 58 Da less than that of achyranthoside C. The high collision energy scan fragment
E

ions at m/z 793.4359 [M-H-C3H5O4]－, 631.3828 [M-H-C3H5O4-Glc]－, 569.3832 [M-H-

CC

－ －
C3H5O4-Glc-CO2] and 455.3519 [M-H-C3H5O4-Glc-GluA] were observed.
Therefore, it was identified as the elimination of a -C2H2O2 moiety from achyranthoside
A

C.
M5, M6, M8, M16, M21, M24, M25, M29 and M31 were eluted at 10.94 min,
11.17 min, 11.43 min, 14.98 min, 15.80 min, 16.86 min, 17.01 min, 19.17 min and
20.11 min, respectively. They all showed the same characteristic ions at m/z 471.3478
(C30H48O4), which was 16 Da more than oleanolic acid, indicating oxidation occurred
16
at aglycone. M37 was eluted at 21.26 min and the precursor ion [M-H] － at m/z
453.3372 (C30H46O3), which was 2 Da less than oleanolic acid. Therefore, M37 was
tentatively suggested to be the result of dehydrogenation of oleanolic acid.
M1, M2, M7, M13, M14 and M17 were all considered as glycosylation
conjugates. Taking M1 as an example, the deprotonated ion of M1 was at m/z
1089.5116 (C52H82O24), which was 162 Da more than Achyranthoside F-C2H2O2,
indicating it was a biotransformation component of Achyranthoside F after loss of the

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-C2H2O2 group. Moreover, the high MSE scan fragment ions at m/z 927.4591, 793.4376,

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631.3837, 569.3859 and 455.3526 were observed. Thus, it was identified as

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glycosylation of the metabolites. The proposed metabolic position is shown in Fig. 10.

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4. Conclusions

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In this study, an integrated analysis based on UPLC-Q-TOF/MSE combined with
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a metabolism platform was developed for chemical profiles and metabolites
A
characterization of triterpene saponins in AB. As a result, a total of 40 saponins (thirty-
eight oleanane-type triterpene saponins, one hederagenin-type and one machaerinate-
M

type triterpene saponin) were tentatively characterized. Furthermore, 39 metabolites

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were identified using this method. Deglycosylation, glycosylation, oxidation and

dehydrogenation were the main metabolic pathways of AB triterpene saponins in vitro.
It is worth noting that di-substituents could transform into mono-substituents by the
PT

deglycosylation of sugar moieties at the C-3 and/or C-28 position of oleanolic acid.
Nevertheless, an intestinal microflora metabolic system cannot completely simulate the
E

real metabolic environment. Therefore, triterpene saponins in AB in vivo should be

CC

studied further. Taken together, this study may lay the foundation for further effective
material basic research of triterpene saponins in AB.
A

Conflict of interest
The authors have declared that no conflict of interest.

Acknowledgments

17
Project was financially supported by grants from the National Natural Science
Foundation of China (No. 81473400; NO. 81874360), Exploratory Research Projects
of Anhui University of Chinese Medicine (NO. 2017HXTS37). We gratefully thank the
Scientific Research & Experiment Center and Key Laboratory of Xin’an Medicine of
Anhui University of Chinese Medicine for providing the facility support.

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18
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Fig. 1. Integrated analysis based on UPLC-Q-TOF/MS method combined with
metabolism platform was developed to identify the chemical constituents and

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metabolites of triterpene saponins in AB

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Fig. 2. Proposed fragmentation pathways of four reference standards of triterpenoid

saponins by UPLC-Q-TOF/MSE in the negative mode. A: 28-
Desglucosylchikusetsusaponin Iva; B: Chikusetsusaponin Iva; C: Chikusetsusaponin
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IV; D: Chikusetsusaponin V
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Fig. 3. The genus composition of intestinal microflora was analyzed by next generation sequencing. Blank group: intestinal microflora;
Experimental group: the intestinal microflora incubated with AB extract (n=3)

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Fig. 4. Extracted ion chromatograms (EICs) and MS/MS spectrum of the metabolites
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of Chikusetsusaponin IVa by UPLC-Q-TOF/MSE in the negative ion mode

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Fig. 5. EICs and MS/MS spectrum of the metabolites of Chikusetsusaponin IV by

UPLC-Q-TOF/MSE in the negative ion mode
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Fig. 5. (Continued)
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Fig. 6. EICs and MS/MS spectrum of the metabolites of Chikusetsusaponin V by

UPLC-Q-TOF/MSE in the negative ion mode
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Fig. 6. (Continued)
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Fig. 7. The total ion chromatograms (TIC) of intestinal microflora (A), AB extract
before biotransformed (t=0 h) (B) and AB extract after biotransformed by intestinal
microflora (t=8 h) (C) by UPLC-Q-TOF/MSE in negative mode

31
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Fig. 8. Dynamic variation curve of peak area of 13 representative compounds in AB
by intestinal microflora-mediated biotransformation at different time points (0, 1, 2, 4,
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6, 8, 10, 12, 24 h)
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Fig. 9. Deglycosylation metabolites of triterpenoid saponins and their biotransformation characteristics in intestinal microflora

33
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Fig. 10. The proposed metabolic position of the oleanane-type triterpenoid saponins
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biotransformed by intestinal microflora

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34
Table 1 UPLC-Q-TOF/MSE data in the negative ion mode of three reference standards
in AB biotransformed by intestinal microflora
NO. tR(min) Formula Adducts Observed PPM MS fragment
m/z
Chikusetsusaponin Iva
M0 14.51 C42H66O14 -H 793.4390 1.2 631.3853, 613.3747, 569.3810, 455.350
175.0238, 113.0235
M1 10.44 C42H66O15 -H 809.4350 2.5 647.3792, 471.3489
M2 11.40 C48H76O19 -H 955.4928 2.0 793.4344, 631.3853, 569.3859, 455.350
M3 19.16 C36H56O9 -H 631.3853 0.1 569.3859, 455.3552, 113.0234

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M4 19.96 C36H58O6 +HCOO 663.4119 1.6 455.3508

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M5 21.34 C30H46O3 -H 453.3356 -3.9 453.3356
M6 24.51 C30H48O3 -H 455.3508 -5.0 455.3508

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Chikusetsusaponin IV
M0 12.94 C47H74O18 -H 925.4803 0.1 793.4402, 631.3837, 613.3696, 569.381

SC
455.3508, 113.0232
M1 10.25 C47H74O19 -H 941.4787 3.7 779.4265, 647.3792, 471.3444
M2 10.44 C42H66O15 -H 809.4350 2.5 647.3792, 471.3489
M3 11.37 C53H84O23 -H
U
1087.5380 4.5 925.4504, 793.4337, 631.3904, 569.390
455.3596
N
M4 14.49 C42H66O14 -H 793.4402 2.7 631.3853, 613.3732, 569.3859, 455.355
A
113.0232
M5 14.74 C41H64O14 -H 779.4235 1.5 647.3792,471.2467
M

M6 18.49 C41H64O13 -H 763.4271 -0.3 631.3848, 613.3747, 569.3859, 455.350

113.0233
M7 19.16 C36H56O9 -H 631.3853 0.1 455.3508
ED

M8 21.34 C36H56O8 +HCOO 661.3978 3.9 453.3400

M9 19.96 C36H58O8 +HCOO 663.4110 0.3 455.3552
M10 22.44 C30H48O4 -H 471.3474 -1.2 471.3444
PT

M11 23.13 C30H46O3 -H 453.3355 -4.1 453.3440

M12 24.51 C30H48O3 -H 455.3552 4.6 455.3552
Chikusetsusaponin V
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M0 11.44 C48H76O19 -H 955.4931 2.4 793.4402, 731.4390, 613.3747, 569.385

455.3508, 113.0234
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M1 9.60 C48H76O20 -H 971.4852 -0.5 809.4408, 647.3792, 471.3533

M2 10.88 C54H86O24 -H 1117.5440 0.3 955.4971, 793.4402, 731.4390, 613.374
569.3859, 455.3552
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M3 14.49 C42H66O14 -H 793.4402 2.7 613.3747, 569.3859, 455.3508, 113.023

M4 14.64 C42H66O15 -H 809.4350 2.5 647.3792, 471.3489
M5 16.74 C42H66O14 -H 793.4391 1.3 631.3865, 569.3859, 455.3534, 113.024
M6 19.16 C36H56O9 -H 631.3853 0.1 455.3552
M7 19.96 C36H58O8 +HCOO 663.4119 1.6 455.3552
M8 21.33 C36H56O8 +HCOO 661.3957 0.7 453.3364
M9 22.44 C30H48O4 -H 471.3464 -3.3 471.3464
35
 M10 23.13 C30H46O3 -H 453.3376 0.4 453.3376
M11 24.51 C30H48O3 -H 455.3528 0.6 455.3528

Table 2 UPLC-Q-TOF/MSE data in the negative ion mode of AB triterpene saponins

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biotransformed by intestinal microflora

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MS
N tR(m Addu Observ PP
Formula fragme Identification
O. in) cts ed m/z M

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nt
927.45

SC
59,
793.43
Achyranthoside F-
C52H82 1089.5 - 76,
1 5.86
O24
-H
116 0.6 631.38
46, UC2H2O2(cleavage)+C6H10
O5
N
455.35
A
25
895.43
M

21,
Achyranthoside B-
C51H78 1057.4 - 733.38
2 6.15 -H C2H2O2(cleavage)+C6H10
O23 839 2.1 12,
ED

O5
455.35
21
747.39
PT

C42H62 805.40 - 59, AchyranthosideA-

3 6.96 -H
O15 17 6.3 455.35 C6H10O5(cleavage)
23
E

631.38
3-O-β-D-GluA-β-D-GluA-
CC

10.7 C42H64 807.41 - 36,

4 -H 28-O-β-D-Glc oleanolic
2 O15 61 1.4 455.35
acid-C6H10O5(cleavage)
16
647.38
A

10.9 C42H66 809.43 - 12,

5 -H Zingibroside R1+O
4 O15 27 0.2 471.34
76
809.43
11.1 C47H72 971.45 23,
6 -H 1.6 Achyranthoside G+O
7 O21 09 647.38
06,
36
 471.34
68
895..4
321,
Bidentatoside I-
11.3 C51H78 1057.4 - 733.38
7 -H C2H2O2(cleavage)+C6H10
5 O23 856 0.4 12,
O5
455.35
22
971.48
55,

T
809.43

IP
11.4 C53H82 1133.4 - 27,
8 -H Achyranthoside D+O
3 O26 992 2.6 647.38

R
06,
471.34

SC
65
28-
11.5 C36H56 647.37 - 471.34
9 -H Desglucosylchikusetsusap
1 O10 97 0.6 70
U
793.43
onin Iva+O
N
76,
14.0 C45H70 897.44 - 631.38 Achyranthoside C-
A
10 -H
1 O18 68 2.4 45, C2H2O2(cleavage)
M

455.35
22
793.43
ED

82,
14.4 C45H70 897.44 - 631.38 Achyranthoside G-
11 -H
3 O18 84 0.6 46, C2H2O2(cleavage)
PT

455.35
25
793.43
E

66,
631.38
CC

14.4 C47H72 955.45 43, Achyranthoside D-

12 -H 1.5
6 O20 59 569.38 C6H10O5(cleavage)
41,
A

455.35
26
909.44
83, AchyranthosideA-
14.5 C52H80 1071.5
13 -H 0.8 793.43 C2H2O2(cleavage)+C6H10
8 O23 026
76, O5
631.38
37
 43,
455.35
24
873.39
45,
793.43
Sulfachyranthoside B-
14.6 C48H76 1035.4 - 82,
14 -H C5H4O6(cleavage)+C6H10
6 O22S 470 0.6 631.38
O5
42,
455.35

T
21

IP
631.38
14.7 C45H68 895.43 49, Achyranthoside B-
15 -H 2.9

R
1 O18 59 455.35 C2H2O2(cleavage)
23

SC
809.43
25,
14.9 C47H72 971.45 647.37
16 -H 0.7 Achyranthoside C+O
8 O21 00 95,
U
471.34
N
65
631.38 28-deglucosyl-
A
14.9 C44H68 851.44 - 33, achyranthosideE-
17 -H
M

8 O16 22 1.4 455.35 C2H2O3(cleavage)+C6H10

21 O5
644.39
ED

27,
569.38
15.3 C42H66 777.43 - 53, Momordin IIa-
18 -H
PT

2 O13 85 5.8 455.35 C6H10O5(cleavage)

32,
113.02
E

42
649.35
CC

86, β-D-glucopyranosyl 3β-

631.38 [O-α-L-rhamnopyranosyl-
15.3 C41H64 811.41 48, (1 3)-O-β-D-
19 -H 3.1
A

5 O16 47 569.38 glucopyranuronosyloxy]m

41, achaerinate-
455.35 C6H10O4(cleavage)
24
β-D-glucopyranosyl 3β-
15.6 C35H56 +HC 681.38 473.32
20 0.1 [O-α-L-rhamnopyranosyl-
2 O10 OO 56 69
(1 3)-O-β-D-
38
 glucopyranuronosyloxy]m
achaerinate-
C12H18O10(cleavage)
809.39
56,
C47H70 969.43 647.37
21 15.8 -H 0.5 Achyranthoside B+O
O21 42 96,
471.34
70
631.38

T
46,

IP
16.6 C42H66 793.43 455.35 Achyranthoside G-
22 -H 0.6
7 O14 85 23, C5H6O6(cleavage)

R
113.02
42

SC
631.38
46,
613.37

16.6 C42H66 793.43

47,
U
569.38 Pseudoginsenoside RT1-
N
23 -H 1.3
9 O14 90 59, C5H8O4(cleavage)
455.35
A
08,
M

113.02
46
647.38
ED

16.8 C41H62 809.39 - 06, 28-deglucosyl-

24 -H
6 O16 54 1.4 471.34 achyranthosideC+O
70
PT

17.0 C36H58 +HC 679.40 - 471.34

25 Chikusetsusaponin 1+O
1 O9 OO 43 2.1 70
631.38
E

43,
17.7 C42H64 823.40 - 569.38 Achyranthoside F-
CC

26 -H
5 O16 98 2.8 41, C6H10O5(cleavage)
455.35
26
A

631.38
46,
18.4 C41H64 763.42 569.38 Pseudoginsenoside RT1-
27 -H 0.5
2 O13 78 43, C6H10O5(cleavage)
455.35
25
28 18.9 C41H62 -H 793.40 0.9 631.38 Achyranthoside C-
39
 1 O15 23 40, C6H10O5(cleavage)
455.35
11
647.38
19.1 C40H60 779.38 - 12, 28-deglucosyl-
29 -H
7 O15 51 1.0 471.34 achyranthosideE+O
70
19.3 C36H58 +HC 663.41 455.35 Achyranthoside D-
30 1.2
4 O8 OO 21 11 C17H24O17(cleavage)
647.38

T
20.1 C41H60 807.37 - 02,
31 -H Achyranthoside iv+O

IP
1 O16 72 4.5 471.34
76

R
20.5 C36H56 631.38 - 455.35 Zingibroside R1-
32 -H
9 O9 46 0.8 25 C6H10O5(cleavage)

SC
631.38
49,
28-deglucosyl-
20.8 C38H58 705.38 569.
33 -H 0.3 achyranthosideE-
5 O12 58
U
3837,
455.35
C2H2O2(cleavage)
N
21
21.2 C30H46 453.33 - 453.33
A
34 -H Oleanolic acid -H2
6 O3 54 4.4 54
M

Hederagenin-28-O-β-D-
21.4 C30H48 471.34 471.34
35 -H 3.7 glucopyranosyl ester-
7 O4 97 97
C6H10O5(cleavage)
ED

617.40
21.6 C36H58 697.36 - 51, Sulfachyranthoside B-
36 -H
6 O11S 22 0.8 455.35 C11H12O12(cleavage)
PT

28
Chikusetsusaponin IVa
21.6 C38H60 659.41 455.35
37 -H 0.4 ethyl ester-
8 O9 68 23
E

C6H10O5(cleavage)
Chikusetsusaponin IVa
CC

22.2 C37H58 645.40 455.35

38 -H 1.7 methyl ester-
1 O9 19 24
C6H10O5(cleavage)
24.4 C30H48 455.35 455.35
39 -H 0.1 oleanolic acid
A

0 O3 31 31

40