Rapid characterization of chemical constituents of Gansuibanxia decoction by UHPLC-FT-ICR-MS analysis

Journal Pre-proof Rapid characterization of chemical constituents of Gansuibanxia decoction by UHPLC-FT-ICR-MS analysis Yue Cui, Huanhuan Yang, Jixue Jing, Ting Liu, Roujia Wang, Fuyu Di, Fei Han, Yunli Zhao, Zhiguo Yu

PII:

S0731-7085(19)31223-3

DOI:

https://doi.org/10.1016/j.jpba.2019.113029

Reference:

PBA 113029

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

17 May 2019

Revised Date:

24 October 2019

Accepted Date:

3 December 2019

Please cite this article as: Cui Y, Yang H, Jing J, Liu T, Wang R, Di F, Han F, Zhao Y, Yu Z, Rapid characterization of chemical constituents of Gansuibanxia decoction by UHPLC-FT-ICR-MS analysis, Journal of Pharmaceutical and Biomedical Analysis (2019), doi: https://doi.org/10.1016/j.jpba.2019.113029

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Rapid characterization of chemical constituents of Gansuibanxia decoction by UHPLC-FT-ICR-MS analysis

Yue Cuia, Huanhuan Yanga, Jixue Jinga, Ting Liub, Roujia Wanga, Fuyu Dia, Fei Hana, Yunli Zhaoa, *, Zhiguo Yu a, * School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,

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a

Shenhe District, Shenyang 110016, China b

The Precise Medicine Center, Key Laboratory of Environmental Pollution and

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Microecology, Liaoning Province; College of Basic Medical Sciences, Shenyang

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Medical College, No. 146, North Huanghe Street, Huanggu District, Shenyang

Co-corresponding authors: E-mail address: [email protected]; [email protected];

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*

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110034, China

Highlights



An UHPLC-FT-ICR-MS method was developed for chemical constituents characterization of GSBXD.



A total of 62 chemical constituents were detected in GSBXD.



Chemical constituents in GSBXD were first chemically defined or tentatively

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identified.

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Abstract

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Gansuibanxia decoction (GSBXD) is one of the most famous traditional Chinese medicine (TCM). It is a herbal formula used for treating hydrops, such as cancerous

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ascites, pleural effusion, pericardial effusion, etc. However, the chemical constituents of GSBXD were still unclear. In this study, an UHPLC-FT-ICR-MS method was

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established and applied to the separation and characterization of the chemical

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constituents of GSBXD. A total of 62 components were chemically defined or tentatively identified, including diterpenoids, triterpenoids, flavonoids, monoterpene glycosides and alkaloids. The results is meaningful for a better understanding of the material basis of GSBXD and can be the basis for its further in vitro and in vivo studies.

Keywords: Gansuibanxia decoction; UHPLC-FT-ICR-MS; Chemical constituents; Traditional Chinese Medicine

1. Introduction

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Gansuibanxia decoction (GSBXD) is a classic traditional Chinese medicine (TCM). This herbal formula with about 2000-year clinical application history was first recorded in Han dynasty. According to Synopsis of Golden Chamber written by

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Zhongjing Zhang, an outstanding physician in Chinese history, GSBXD is composed

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of four herbs including kansui (Kansui radix), pinellia ternata (Pinelliae rhizoma),

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liquorice (Glycyrrhizae radix et rhizoma) and peony (Paeoniae radix). It is used for the treatment of prolonged elema which is called Liuyin in Chinese since the ancient

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time. The common symptoms are thirsty, arthralgia of extremities, coldness of back, shortness of breath and pulse condition sinking according to TCM theories. Nowadays,

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GSBXD is applied to treating serious effusions: pericardial effusion, pleural effusion, hydronephrosis, and even cancerous effusion [1]. It is also have effects on chronic

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diarrhea, asthma and autonomic nervous dysfunction [2]. Although GSBXD is widely used in treating effusion, its chemical constituents are

still unclear which makes the further investigation of GSBXD difficulty. Usually, the TCM herbal formulas are composed of not less than two kinds of herbs, and each herb has large amounts of chemical constituents which is quite different from chemical

drugs. Therefore, the chemical constituents of herbal formula must be quite complex and not easy to detect and characterize not only for the abundant kinds but also for the amounts of compounds. Besides, the different properties of these chemical constituents also make characterization difficulty. Chemical constituents is the basis to know what exit in GSBXD. Without clear chemical components, the effective components can’t be fully understood, needless to say deep studies in vivo. Therefore,

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it’s urgent to investigate the chemical constituents of GSBXD and this could be meaningful for its quality control and pharmacological studies.

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Up to date, many modern analysis techniques have been used for the exploration of

the chemical constituents of TCM. Wang et al [3] investigated the chemical

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constituents in SiJunZiTang using UPLC-Q-TOF-MS/MS. Su et al [4] identified 33

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chemical constituents in Chaihu-Shu-Gan-San by LC-LTQ-Orbitrap-MS. Ultrahigh performance liquid chromatography (UHPLC) with the best separation ability in

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chromatography fields coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) which has the highest resolution in mass spectrum fields

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can be the most powerful analytical techniques for the separation and tentative

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characterization of complex chemical constituents in TCM herbal formulas. Liu et al [5] detected and characterized 134 compounds in Gegenqinlian decoction, including triterpenoids, flavonoids and alkaloids within 25 min. Guan [6] rapidly characterized 120 compounds in Sijunzi decoction, including ginsenosides, sesquiterpenes, etc. In this study, an UHPLC-FT-ICR-MS method with rapid and sensitive advantages was developed and applied to the detection and characterization of chemical

constituents in GSBXD. To the best of our knowledge, it is the first time to profile the chemical components in GSBXD. And the results can be useful for the further investigations of GSBXD both in vitro and in vivo.

2. Materials and methods 2.1. Chemicals and reagents

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All the raw herbs including kansui, pinellia ternata, liquorice and peony were bought from GuoDa Pharmacy (Shenyang, China). References (purity > 98%) of liquiritin, liquiritigenin, isoliquiritigenin, glycyrrhetinic acid and glyrrhizic acid were

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purchased from Chengdu Must Bio-Technology Co., Ltd (Chengdu, China). Formic

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acid and methanol of chromatographic grade were purchased from Concord Technology (Tianjin, China). Acetonitrile of LC-MS grade was bought from

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Sigma-Aldrich (St. Louis, MO, USA). Purified water was provided from Hangzhou

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Wahaha Corporation (Hangzhou, China). 2.2. Preparation of GSBXD samples

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Kansui (5 g), pinellia ternata (12 g), liquorice (5 g) and peony (15g) were weighted

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and under reflux together for three times with 370 mL, 300 mL and 220 mL 95% methanol, respectively. The filtrates were evaporated and dried. Appropriate dried powder was dissolved in methanol to obtain 0.34 g/mL GSBXD solution (calculated as raw herbs) before analysis. The solution was centrifuged at 12100 g for 5 min and filtered through a 0.22 μm membrane. An aliquot of 5 μL of GSBXD solution sample was injected for analysis.

2.3. Instruments and analytical conditions Agilent 1260 UHPLC system (Agilent, USA) was used for chromatographic analysis. A HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters Corporation, Milford, UK) with column temperature 35 °C was used for chromatographic separation. The gradient elution with mobile phase acetonitrile (A) - 0.1% formic acid in water (B)

(A) from 25 to 28 min. The flow rate was 0.3 mL/min.

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was set as follows: 5-50% (A) from 0 to 6 min; 50-95% (A) from 6 to 25 min; 95%

Bruker Solarix 7.0T FT-ICR-MS system (Bruker, Germany) was used for mass

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spectrum analysis. ESI positive mode was carried out. The capillary voltage was 4.5 kV; nebulizer gas pressure was 4 bar; dry gas flow was 8 L/min; dry gas temperature

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was 200 °C; nebulizing gas was N2; collision gas was Ar. The scan range was m/z

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100-1500. The collision energy was from 10-30 eV to acquire fragments.

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3. Results and discussion

3.1. Optimization of LC and MS conditions

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LC conditions were optimized, including mobile phase, flow rate, injection volume,

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etc. to obtain a good separation and short analysis time. Finally, acetonitrile - 0.1% formic acid in water which showed good peak shapes, more amounts of peaks and better intensity was selected as mobile phase. The flow rate and injection volume were 0.3 mL/min and 5 μL, respectively. For MS conditions optimization, both the positive and negative mode were tested. However, the intensity of compounds in GSBXD in positive mode is quite higher than

negative mode. Besides, more compounds can be detected in positive mode according to the base peak intensity (BPI) chromatograms. Finally, a more sensitive positive mode was selected which can show more information of the chemical constituents in GSBXD. Other parameters of MS were also optimized, such as capillary voltage, nebulizer gas pressure, dry gas flow, etc. The results indicated that the developed

constituents in GSBXD. 3.2. Identification of the chemical constituents in GSBXD

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UHPLC-FT-ICR-MS method in this study is appropriate for the detection of chemical

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First, an in-house database was built by the results searched from Chemspider, PubMed, Science Direct of Elsevier and CNKI (Chinese National Knowledge

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Infrastructure). The compounds in each herb in GSBXD were summarized by their

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chemical names, molecular formulas, accurate molecular mass, chemical structures and relevant fragments. The in-house database was used for the comparison and

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characterization of the compounds in GSBXD. DataAnalysis (Bruker, Germany) is a software supporting Bruker Solarix 7.0T FT-ICR-MS system (Bruker,

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Germany). It can predict molecular formula based on the chromatography and

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mass information. We match the in-house database with the predicted molecular formula to determine what can be chemically defined (with standard references) and tentatively characterized (without standard references). Also, there may be multiple formula for certain mass peak. These formulas were searched in our in-house database to confirm if they have been reported in the four herbs in GSBXD in literatures. If they were reported, they would be considered; if not,

they would be ignored. A total of 62 chemical constituents in GSBXD were chemically identified or tentatively characterized, including 16 diterpenoids, 14 triterpenoids, 21 flavonoids, 10 monoterpenoids and 1 alkaloids. And the majority of them are bioactive components in each herb. Fig. 1 shows the BPI chromatogram of GSBXD. The details of the compounds are shown in Table 1. The chemical structures of the compounds are shown in Fig. 2.

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3.2.1. Characterization of compounds from kansui in GSBXD

There were 19 compounds tentatively identified from kansui, including 3

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triterpenoids and 16 diterpenoids. The triterpenoids from kansui, including kansenonol, 11-oxo-kansenonol and kansenone are also the bioactive components in

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kansui [7]. Diterpenoids are the main bioactive components in kansui with

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anti-leukemia, anti-allergic, anti-virus and anti-tumor activities [8-10]. There are two types of diterpenoids in kansui, one is ingenol type and another is jatrophone type.

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Ingenol type diterpenoids is a kind of tetracyclic diterpenoids which is combined by a 5-membered ring, two 7-membered rings and a 3-membered ring. In parent nucleus,

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ketone group bridges C-8 and C-10, one double bond connects C-1 and C-2, and

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another one connects C-6 and C-7. Jatrophone type diterpenoids belong to macrocyclic diterpenoids, combined by a 5-membered ring and a 12-membered ring. Usually, their substituent groups can be hydroxy and acyloxy, and fragments generally produced by the loss of corresponding carboxylic acids of these acyloxys from the precursor ions. Taking compound K7 as an example to explain the identification process. First, precursor ion [M+Na]+ at m/z 753.27588 was found at 16.44 min. The

molecular formula was predicted as C37H46O15 using DataAnalysis (Bruker, Germany) within 5 ppm. Then its fragments were obtained in MS/MS experiments, including 693.25471, 633.23369 and 513.19142 which were the daughter ions [M+Na-HOAc]+, [M+Na-2HOAc]+

and

[M+Na-4HOAc]+,

respectively.

The

fragments

were

compromised with our in-house database and literatures [11], and the compound was tentatively identified as kansuinin A. The MS/MS mass spectrum of kansuinin A is

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shown in Fig. 3(A).

3.2.2 Characterization of compounds from liquorice in GSBXD

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There were 32 compounds detected from liquorice in GSBXD. The main

compounds chemically defined or tentatively identified in liquorice including 1

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triterpenoid, 10 triterpene saponins and 21 flavonoids and their glycosides. One

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triterpenoid from liquorice is glycyrrhetinic acid which has anti-inflammatory activities [12]. Studies reported that triterpenes saponins from liquorice have

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anti-inflammatory [13], anticancer [14] and hepatoprotective [15] activities. Glycyrrhizic acid was used to illustrate the identification process. Precursor ion

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[M+H]+ m/z 823.41352 was found at 11.20 min. The major fragments were found at

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m/z 647.38058, 471.33275 and 453.33715. MS/MS fragments at m/z 647.38058 indicated the loss of glucuronic acid of the precursor ion, m/z 471.33275 indicated another loss of glucuronic acid from the product ion at m/z 647.38058, and m/z 453.33715 indicated loss of H2O from the product ion at m/z 471.33275. According to the literatures [5] and compared with the reference standard, the compound was chemically defined as glycyrrhizic acid. Fig. 3(B) shows the MS/MS mass spectrum

of glycyrrhizic acid. Studies showed that flavonoids and their glycosides which we chemically defined or tentatively identified are the bioactive constituents in liquorice [16]. Liquiritin apioside is a representative flavonoid glycosides in liquorice. The identification of liquiritin apioside was taking as an example for illustration. The precursor ion [M+H]+ was at m/z 551.17722 observed at 7.37 min. The molecular formula was predicted as

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C26H30O13 using DataAnalysis (Bruker, Germany) within 5 ppm. Daughter ions at m/z

419.13438 and 257.08105 were [M+H-api]+ and [M+H-api-glc]+, respectively.

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According to the in-house database and literatures [5, 6], the compound was

tentatively identified as liquiritin apioside. And the MS/MS mass spectrum is shown

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in Fig. 3(C).

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3.2.3. Characterization of compounds from peony in GSBXD Totally, 10 monoterpenoids and monoterpene glycosides were tentatively

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identified in GSBXD and they were from peony. It has been reported that

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monoterpene glycosides are the major bioactive components of peony with anti-inflammatory, neuroprotective and hepatoprotective activities [17]. Paeoniflorin

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and albiflorin are isomers and they were used as examples to illustrate the characterization process. The precursor ion at m/z 503.15274 and 503.15321 were observed at 8.16 and 8.19 min, respectively. And their molecular formulas were speculated as C23H28O11 with 5ppm. Both of the two compounds showed the same fragments at m/z 381, 341 and 219. Daughter ion at 381 indicated a loss of

benzoyloxy, daughter ions at m/z 341 and 219 were [M+Na-glc]+ and [M+Na-benzoyloxy-glc]+, respectively. According to the in-house database and literatures [4], the two compounds were paeoniflorin and albiflorin. Based on their different skeletons of aglycones, the dehydration ability of C4 hydroxyl was different. Albiflorin is more polar than paeoniflorin and can be more quickly eluted than paeoniflorin on reserved phase column [4, 18]. Therefore, the retention time of

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albiflorin is shorter in this separation conditions. And the isomers can be distinguished

with each other. Compound Pa4 and Pa6 were tentatively identified as albiflorin and

paeoniflorin, respectively. The MS/MS mass spectrum of paeoniflorin is shown in Fig.

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3(D).

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3.2.4. Characterization of compounds from Pinellia ternatain in GSBXD

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There were two compounds tentatively identified from Pinellia ternata. One was violanthin which is isoflavone glycoside, and another was adenosine which is alkaloid.

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It has been reported that alkaloids were one kind of major active components in Pinellia ternata with anti-tumor, antiemetic, anticonvulsive, sedative and hypnotic

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activities [19]. However, only one alkaloid was tentatively identified in GSBXD and

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it was from Pinellia ternata. Adenosine was used to illustrate the fragmentation pathway. Precursor ion [M+H]+ at m/z 268.10403 was observed at 1.51 min. In the MS/MS experiments, fragments were at m/z 136.06178 which meant the loss of ribose, and the compound was tentatively identified as adenosine. Fig. 3(E) shows the MS/MS mass spectrum of adenosine.

4. Conclusion In this study, the chemical constituents in GSBXD were profiled using UHPLC-FT-ICR-MS technology. It is the first time to detect the chemical components in GSBXD. A total of 62 constituents including diterpenoids, triterpenoids, monoterpenoids, alkaloids, flavonoids and their glycosides were identified or tentatively characterized. These compounds were the major bioactive components of

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kansui, liquorice, peony and pinellia ternata according to the previous reports. The

established UHPLC-FT-ICR-MS method with the advantages of good separation, high

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resolution, high sensitive and high accuracy for the detection of chemical components

in GSBXD could be the basis for the further study of this classic TCM formula, and

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also help us to have a better understanding of the pharmacodynamic material basis of

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Declaration of interests

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TCM.

The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

Acknowledgement This study was supported by the Project for National Basic Science Personnel Training Fund (No. J1103606) and the National Natural Science Foundation of China (NO. 81573629).

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Figure captions

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Fig. 1. Representative base peak intensity (BPI) chromatogram of GSBXD.

Fig. 2. Chemical structures of the compounds detected in GSBXD. glu A: glucuronic

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-p

acid; glu: glucose; gal: galactose; rha: rhamnose; api: apiose; rib: ribose.

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Fig. 3. MS/MS spectra of typical compounds. A: kansuinin A; B: glycyrrhizic acid; C:

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liquiritin apioside; D: paeoniflorin; E: adenosine.

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Table

Compound

Rt (min)

K1

12.99

2

K2

3

K3

4

K4

5

K5

6

K6

7

K7

Molecular formula

Selected ion

Measured mass (m/z)

Accuracy mass (m/z)

Error (ppm)

Product ion

References

(m/z)

Kansuinin I

C38H42O13

M+Na

729.25217

729.25173

0.61

607, 579

[11]

14.32

Kansuinin B

C38H42O14

M+Na

745.24712

745.24667

0.61

623

[11]

15.28

Kansuinin G

C34H43O12N

M+Na

680.26763

680.26773

-0.15

-

[11]

15.48

Kansuinin M

C35H42O13

M+Na

693.25269

693.25173

1.39

633, 573, 175

[11]

15.93

Kansuinin H

C37H46O16

M+Na

769.28004

769.27779

2.92

375, 297

[11]

15.98

Kansuinin K

C40H44O15

M+Na

787.25716

787.25723

-0.09

605

[11]

16.44

Kansuinin A

C37H46O15

M+Na

753.27588

753.27291

3.94

693, 633, 513

[11, 20]

Jo ur

1

Identification

na l

No.

Pr

Table 1. Chemical constituents identified in GSBXD by UHPLC-FT-ICR-MS

16.53

Kansuinin D

C41H47NO15

M+Na

9

K9

18.07

3-O-benzoyl-20-deoxyingenol

C27H32O5

M+Na

10

K10

18.31

Kansuinin E

C41H47NO14

M+H

11

K11

18.85

11-oxo-kansenonol

C30H46O4

12

K12

19.16

Kansuinin L

C41H47NO13

13

K13

20.96

Kansuiphorin C

C29H34O6

14

K14

21.19

Kansenonol

15

K15

22.17

3-O-(2’E, 4’E/Z-decadienoyl) ingenol

16

K16

22.59

Kansuinin F

17

K17

24.71

3-O-(2’E,4’E/Z-decadienoyl)20-O-acetylingenol

18

K18

19

K19

816.28349

816.28378

-0.36

756, 369

[11, 20]

459.21426

459.21420

0.14

441, 337

[11]

778.30684

778.30696

-0.16

718, 658

[11]

M+H

471.34680

471.34688

-0.16

453, 359

[11]

M+Na

784.29278

784.29392

-1.45

724

[11]

M+Na

501.22506

501.22476

0.60

297

[11]

C30H48O3

M+H

457.36789

457.36763

0.56

439

[11]

C30H42O6

M+Na

521.28779

521.28735

0.85

353, 335

[11, 20]

C42H48O14

M+Na

799.29485

799.29360

1.57

739, 679

[11]

C32H44O7

M+Na

563.29926

563.29794

2.35

503, 395, 335

[11, 20]

e-

Pr

na l

Jo ur

f

oo

K8

pr

8

25.33

Kansuinone

C30H50O3

M+H

459.21426

459.38329

1.12

319

[11]

27.25

3-O-(2,3-dimethylbutanoyl)-

C36H56O8

M+Na

639.38782

639.38674

0.90

523, 467, 351

[11]

C18H24O12

M+H

433.13465

433.13405

1.38

371, 127

[6]

13-O-dodecanoyl ingenol

20

G1

6.14

Licoagroside B

6.40

Vicenin-2

C27H30O15

M+H

22

G3

6.80

Schaftoside

C26H28O14

M+H

23

G4

7.17

Violanthin

C27H30O14

M+H

f

oo

G2

595.16664

595.16573

1.53

551, 505

[21]

565.15540

565.15517

0.40

547, 445, 385

[21]

579.17113

579.17085

0.48

-

[6]

M+H

551.17722

551.17592

2.40

419, 257

[5, 6]

M+H

419.13428

419.13366

1.49

257, 137

[6, 21]

C26H30O13

M+H

551.17649

551.17592

1.03

419, 257

[6]

C22H22O9

M+H

431.13429

431.13366

1.47

269, 253, 237, 197

[21]

C35H36O15

M+H

697.21356

697.21272

1.20

257

[6]

(Pi2) G5

7.37

Liquiritin Apioside

C26H30O13

25*

G6

8.25

Liquiritin

C21H22O9

26

G7

8.34

Isoliquiritin Apioside

27

G8

8.61

Ononin

28

G9

8.63

Licorice glycoside B

29*

G10

8.66

30

G11

31

G12

32

G13

33

G14

34

G15

35

G16

na l

Pr

e-

24

pr

21

C21H22O9

M+H

419.13426

419.13366

1.43

257, 137

[5]

8.95

Uralsaponin P

C42H64O16

M+H

825.42700

825.42671

0.35

455

[6]

9.04

Licorice glycoside E

C35H35O14N

M+H

694.21413

694.21303

1.58

257

[6]

9.15

Liquiritigenin

C15H12O4

M+H

257.08083

257.08083

0.01

239, 137

[6]

9.17

Uralsaponin T

C48H74O19

M+H

955.49151

955.48970

1.89

-

[6]

9.26

Uralsaponin F

C44H64O19

M+H

897.41349

897.41146

2.26

721, 545, 375

[6]

9.30

Licorice saponin A3

C48H72O21

M+H

985.46640

985.46388

2.56

807, 471

[5]

Jo ur

Isoliquiritin

9.64

22β-acetoxyl-glycyrrhizin

C44H64O18

M+H

37

G18

9.74

Licorice saponin G2

C42H62O17

M+H

38*

G19

10.66

Isoliquiritigenin

C15H12O4

M+H

39

G20

10.90

Formononetin

C16H12O4

40

G21

11.03

Yunganoside B1

C48H76O19

41*

G22

11.20

Glycyrrhizic acid

C42H62O16

42

G23

11.47

Licorice saponin B2

43

G24

11.79

Echinatin

44

G25

12.18

Uralsaponin W

45

G26

12.39

Glycycoumarin

46

G27

12.95

47

G28

48

G29

49*

G30

50

G31

51

G32

f

oo

G17

881.41695

881.41652

0.49

705, 511

[6]

839.40784

839.40596

2.24

487, 469

[6]

257.08088

257.08083

0.21

137

[21]

M+H

269.08108

269.08083

0.92

254, 237

[6]

M+Na

979.48866

979.48733

1.36

-

[6]

M+H

823.41352

823.41109

2.95

647, 471, 453

[5]

C42H64O15

M+Na

831.41414

831.41372

0.51

-

[21]

C16H14O4

M+H

271.09666

271.09648

0.65

239

[6]

C42H62O15

M+H

807.41760

807.41616

1.78

-

[6]

C21H20O6

M+H

369.13435

369.13326

2.94

313, 285

[21]

Licoflavonol

C20H18O6

M+H

355.11820

355.11763

1.60

-

[6]

14.21

Licopyranocoumarin

C21H20O7

M+H

385.12844

385.12819

0.66

-

[6]

15.12

Licoricone

C22H22O6

M+H

383.14946

383.14891

1.44

368

[21]

18.85

Glycyrrhetinic acid

C30H46O4

M+H

471.34680

471.34688

-0.16

453, 407, 137

[5]

20.30

Kanzonol M

C23H26O6

M+H

399.17995

399.18022

-0.67

-

[6]

23.54

Kanzonol F

C26H28O5

M+H

421.20055

421.20095

-0.94

-

[6]

e-

Pr

na l

Jo ur

pr

36

5.91

Oxypaeoniflorin

C23H28O12

M+Na

53

Pa2

6.12

Mudanpioside E

C24H30O13

M+Na

54

Pa3

6.60

Isomaltopaeoniflorin

C29H38O16

M+Na

55

Pa4

8.16

Albiflorin

C23H28O11

56

Pa5

8.18

Paeonilactone C

C17H18O6

57

Pa6

8.19

Paeoniflorin

C23H28O11

58

Pa7

8.20

Galloy paeoniflorin

59

Pa8

8.24

Paeoniflorigenone

60

Pa9

8.26

Paeonilactone B

61

Pa10

9.75

Benzoyl paeoniflorin

62

Pi1

1.51

Adenosine

f

oo

Pa1

519.14807

519.14730

1.48

357, 219

[22]

549.15861

549.15786

1.37

381, 219

[22]

665.20598

665.20523

1.13

-

[23]

M+Na

503.15274

503.15237

0.74

381, 341, 219

[4, 18]

M+H

319.11748

319.11763

-0.48

-

[24]

M+Na

503.15321

503.15237

1.67

381, 341, 219

[4, 18]

C30H32O15

M+Na

655.16307

655.16336

-0.45

-

[18]

C17H18O6

M+H

319.11735

319.11763

-0.87

-

[24]

C10H12O4

M+H

197.08141

197.08084

2.88

105

[24]

C30H32O12

M+Na

607.17986

607.17861

2.06

341, 289

[4]

C10H13N5O4

M+H

268.10403

268.10402

0.04

136

[25]

na l

Pr

e-

pr

52

Jo ur

K: Kansui radix; G: Glycyrrhizae radix et rhizoma; Pa: Paeoniae radix; Pi: Pinelliae rhizoma; -: not detected.

*: chemically defined by standard references.