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Analysis of triterpenoids in Ganoderma resinaceum using liquid chromatography coupled with electrospray ionization quadrupole - time - of - flight mass spectrometry
Accepted Manuscript Title: Analysis of triterpenoids in Ganoderma resinaceum using liquid chromatography coupled with electrospray ionization quadrupole - time - of - flight mass spectrometry Authors: Lingxiao Chen, Xianqiang Chen, Shenfei Wang, Yao Bian, Jing Zhao, Shaoping Li PII: DOI: Reference:
S1387-3806(18)30311-7 https://doi.org/10.1016/j.ijms.2018.11.016 MASPEC 16090
To appear in:
International Journal of Mass Spectrometry
Received date: Revised date: Accepted date:
15 August 2018 4 November 2018 4 November 2018
Please cite this article as: Chen L, Chen X, Wang S, Bian Y, Zhao J, Li S, Analysis of triterpenoids in Ganoderma resinaceum using liquid chromatography coupled with electrospray ionization quadrupole - time - of - flight mass spectrometry, International Journal of Mass Spectrometry (2018), https://doi.org/10.1016/j.ijms.2018.11.016 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 proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Analysis of triterpenoids in Ganoderma resinaceum using liquid chromatography coupled with electrospray ionization quadrupole - time - of - flight mass spectrometry
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Lingxiao Chen1, Xianqiang Chen1, Shenfei Wang1, Yao Bian, Jing Zhao*,
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and Shaoping Li*
State Key Laboratory of Quality Research in Chinese Medicine, Institute
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of Chinese Medical Sciences, University of Macau, Macao SAR, China
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* Correspondence: E-mail: [email protected] (J. Zhao);
These authors contributed equally to this work.
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[email protected] (S. P. Li); Tel.: +853 88224692.
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Graphical abstract
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Highlights
The triterpenoids from Ganoderma resinaceum were analyzed by using UPLC-
A strategy for identification of triterpenoids has been developed.
Total 55 triterpenoids were identified from G. resinaceum.
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Abstract
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QTOF-MS.
The triterpenoids from Ganoderma resinaceum were investigated by using
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UPLC-QTOF-MS and their fragmentation behaviors are summarized in detail. Based
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on the fragmentation pathways, a strategy for identification of triterpenoids has been
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developed. The main triterpenoids in G. resinaceum can be classified into 6 types (type 1-1, 1-2, 2-1, 2-2, 3-1, 3-2) in three steps. Each step was controlled by the
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characteristic ions, which are summarized from fragmentation pathways of 34
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reference compounds. Total 55 triterpenoids were identified from G. resinaceum. Thirty-four of them were identified by using reference standards. The other unknown
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compounds were tentatively identified by using the developed identification strategy. This work is helpful for phytochemical investigation of G. resinaceum as well as
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quality control of materials containing triterpenoids.
Key words: Ganoderma resinaceum; QTOF; triterpenoids; fragmentation pathway; Identification 2
1. Introduction The genus Ganoderma, belongs to Ganodermataceae family, is a group of higher macrofungi widely growing in tropical and temperate regions [1, 2]. In China,
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Ganoderma is a well-known medicinal fungus and has been used for healthimprovement and longevity for over 2000 years [3, 4]. Also, it is involved in clinical
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treatment of various types of diseases, such as chronic bronchitis, neurasthenia,
antitumor, coronary heart disease, hypercholesterolemia, etc. [4-6]. Triterpenoids are
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one of the main components in many Ganoderma species. As reported, the majority of
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triterpenoids in Ganoderma species exhibit significant biological activities, including
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α-glucosidase inhibitory activities, anti-HIV, antitumor, antiviral, hepatoprotective,
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anti-hypertensive, cholesterol-reducing and anti-inflammatory [1, 5-8]. Therefore, the
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triterpenoids have been regarded as one important indicator for quality evaluation of G. lucidum and related species [4]. G. resinaceum Boud., a member of genus
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Genoderma, has long been used for enhancement of internal balance and functions of
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the immune system. Our previous work showed that triterpenoids are also one of the main ingredients in G. resinaceum. Additionally, many triterpenoids separated from G. resinaceum showed strong inhibitory effects against α-glucosidase [1, 2, 9-11].
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However, the chemical structures of triterpenoids in G. resinaceum has not been systematic studied. Therefore, the structural analysis of triterpenoids in G. resinaceum is very necessary, which is beneficial for understanding their structure-function relationships as well as quality evaluation of G. resinaceum. 3
Generally, the chemical compositions of Ganoderma species are very complex. The systematic chemical investigation needs isolation, purification and identification of compounds. This work is very tedious and time-consuming especially for compounds that are of trace amount. Liquid chromatography coupled with
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electrospray ionization mass spectrometry (LC-ESI-MS) is a very efficient tool that has been proved to be rapid, selective and sensitive for qualitative analysis of
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compounds in natural products [12-17].
The most common method for identification of compounds by using LC-ESI-MS
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is to compare the retention times and MS data with those of reference standards.
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However, many standards are very expensive, and it is also hard to acquire all
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reference compounds that required [13]. Moreover, this method is not suitable for
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identification of unknown compounds. It is no doubt that the fragmentation pathway
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of compound is depending on their structures. Additionally, the same type compounds such as triterpenoids usually have similar fragmentation pathway. Therefore,
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systematic study the fragmentation behavior is helpful for identification of
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compounds with similar structures. The fragmentation behaviors of triterpenoids isolated from G. lucidum. have
been reported for several papers [4, 13, 14, 18]. However, as far as we are aware, no
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systematic strategy or steps has been reported for triterpenoid resolution. In this work, the triterpenoids isolated from G. resinaceum have been studied using UPLCQTOFMS and their fragmentation behaviors were summarized in detail. Additionally, a systematic strategy for triterpenoid resolution has been developed. Based on this 4
strategy, triterpenoids in G. resinaceum have been comprehensively investigated. This work is helpful for phytochemical investigation of G. resinaceum as well as quality control of materials containing triterpenoids.
2. Materials and Methods
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2.1 Materials and chemicals The fruiting bodies of G. resinaceum was purchased from the market. The
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reference standards of triterpenoids used in this study (S1-S34) were isolated from the fruiting bodies of G. resinaceum in our previous work (Table 1). All these structures
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were identified by NMR and MS analysis [1, 2, 9-11]. Acetonitrile, methanol and
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acetic acid were of HPLC grade. HPLC grade water was prepared using a Milli‐Q
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2.2 Preparation of standard solutions
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water purification system (Millipore, MA, USA).
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In order to optimize the conditions for ESI-QTOF-MS analysis. Seven representative reference compounds including nortriterpenoids (S8, S33),
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tritepenoidic acids contain double bonds between C-16 and C-17 (S15, S25), typical
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tritepenoidic acids (S24, S6) and tritepenoidic acid contain an oxaspirolactone moiety consisting of a five-membered ether ring, a five-membered lactone ring, and a characteristic C-23 spiro carbon (S2). The stock standard solution of seven reference
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compounds was prepared in methanol at final concentrations of 0.1 mg/mL. Working standard solution was prepared by dilution with methanol to final concentrations of 1.0 μg/mL. All of the solutions were stored in the 4 ◦C fridge. 2.3 Sample preparation 5
Pressurized liquid extraction (PLE) was carried out on a Dionex ASE 350 system (Dionex Corp., Sunnyvale, CA, USA). Powdered G. resinaceum (1.0 g) was mixed with diatomaceous earth in a proportion (1:1) and placed in a 10 mL stainless steel extraction cell. And then extracted according to our previous study [19]. The
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extraction conditions of the PLE method were: solvent, ethanol; temperature,100℃; static extraction time, 5 min; pressure, 1500 psi; 60% of the flush volume for one
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cycle and one extraction time. The extract was centrifuged at 4000g for 10 min, and the supernatant was evaporated to dryness under vacuum. Subsequently, the residue
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was dissolved in 2 mL methanol and filtered through a 0.22 µm filter.
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Some triterpenoids in G. resinaceum are of trace amount and the material are
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very complex. Therefore, a solid-phase extraction method has been developed. An
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SPE cartridge was pretreated with 2 mL of water, 2 mL of methanol and 2 mL of
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water, successively. The SPE conditions were optimized using the G. resinaceum extract (data not shown) and the optimum conditions were: 400 μL sample diluted in
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3.6 mL water, and then loaded on the SPE column, after that the SPE column was
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washing by 30% methanol (2 mL) and then eluting by 80% methanol (2 mL). The eluting solution was evaporated to dry under N2 at room temperature. The residue was re-dissolved by 100 μL methanol followed by centrifugation at 10,000 rpm for 5 min.
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2.4 Chromatography A Dionex (Germering, Germany) Ultimate 3000 UHPLC system equipped with Ultimate 3000 degasser, pump, RS autosampler, and RS column compartment, coupled with diode‐array detector. Waters Acquity BEH C18 column (2.1×150 mm 6
i.d., 1.7 μm, Waters, Milford, MA, USA). was used for sample separation. The mobile phase was consisted of 0.2% acetic acid in water (A) and acetonitrile (B) with gradient elution: 0-8 min, 3% B-22% B; 8-25 min, 22% B-25% B; 25-40 min, 25% B35% B；40-65 min, 35%B - 100%B, 65-70 min, 100%B-100% B. The flow rate was
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0.3 mL/min, injection volume was 1 μL and the column temperature was 25 ℃, and the detection wavelength was set at 257 nm.
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2.5 ESI-QTOF-MS analysis
A high-resolution impact HD QTOF mass spectrometer (Bruker Daltonik GmbH,
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Bremen, Germany) equipped with an electrospray ionization (ESI) source was
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operated in the negative ion mode. The mass range was set at m/z 100–1500 in the full
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scan mode. The capillary voltage was set at 3500 V. The source temperature was set at
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250 ◦C. Nitrogen was used as the drying gas. The gas flow rate was set at 8 L/min.
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MS2 data analysis of the three highest intensive ion fragments was intelligently performed in real time.
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3. Results and discussion
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3.1 Optimization of ESI-QTOF-MS conditions The ionization efficiency of triterpenoids is very important, especially for the
analytes in trace amount. The ESI-QTOF-MS was carried out in the negative ion
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mode according to the literature report [13, 14]. The capillary voltage (V), flow rate of dry gas (L/min), dry temperature (℃) has been optimized in this study. The capillary voltage (V) at 2500, 3000, 3500 was investigated. The average abundance of ion [MH]- of each compound was listed in Table S1. The results showed that the average 7
abundance of ion [M-H]- of compound S2 and S8 increased significantly with the capillary voltage (2500-3500 V). However, the capillary voltage more than 3500 V was not investigated because the average abundance of ion [M-H]- of other compounds (S6, S15, S24, S25 and S33) decreased slightly in the test range.
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Additionally, the average abundance of ion [M-H]- of compound S2 increased slowly when the capillary voltage more than 3000V. Therefore, 3500 V was selected as the
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optimum capillary voltage. Subsequently, flow rate of dry gas (L/min) at 6, 8, 10 was
investigated. When the flow rate increased from 6 to 10 L/min, the average abundance
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of ion [M-H]- of compounds S2, S6, S8, S15, S33 increased correspondingly (the
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average abundance of ion [M-H]- increased slowly when the flow rate more than 8
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L/min). However, the average abundance of ion [M-H]- of compounds S24 and S25
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reduced because of the dehydration reaction (Table S1 and S2). Therefore, flow rate
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of dry gas at 8 L/min was selected as the optimum condition. Dry temperature (℃) at 200, 250, 300 have been investigated. It was found that the average abundance of ion
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[M-H]- of compounds S2, S6, S8, S15, S33 increased steadily with dry temperature.
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However, as shown in Table S1 and S2, the average abundance of ion [M-H]- of compounds S24 and S25 also reduced significantly because of the dehydration reaction (no quasi-molecular ion was detected at 300 ℃). Therefore, the dry
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temperature at 250 ℃ was selected as the optimum condition. 3.2 ESI-QTOF-MS analysis of triterpenoids reference compounds The thirty-four reference compounds (Table 1) could be classified into six groups (1-1, 1-2, 2-1, 2-2, 3-1, 3-2) according to their fragmentation pathways (Fig. 1). The 8
dominant fragmentation pathways of the compounds studied are cleavages of C-ring and D-ring. The specific fragmentation behavior of these six types of triterpenoids has been summarized below. 3.2.1 Fragmentation of type 1-1 triterpenoids
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The fragmentation characteristic of this type of compounds was cleavage of Dring. Take compound ganoderenic acid I as an example. As shown in Fig. 2, the [M-H]-
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ion at m/z 513.2850 produced an ion at m/z 301.1801 by direct cleavage of D-ring. The m/z 495.2735 was formed by loss of H2O. The ion at m/z 451.2839 could be
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obtained by the loss of CO2. The ion at m/z 381.2421 was produced by the loss of
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H2O at R3 position during the ring cleavage.
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3.2.2 Fragmentation of type 1-2 triterpenoids
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The fragmentation characteristic of this type of compounds was D-ring and C-
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ring cleavage. Take compound ganoderic acid AM1 as an example (Fig. 3). The [MH]- ion at m/z 513.2857 produced an ion at m/z 301.1842 by direct cleavage of ring D.
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The m/z 495.2764 formed by loss of H2O. The ion at m/z 451.2839 could be obtained
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by the loss of CO2. The difference between this type of triterpenoids and type 1-1 was that the triterpenoids belong to type 1-1 will lose one more H2O (R3 position) during the ring cleavage. Additionally, in some cases, the type 1-2 triterpenoids have the high
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abundance ion at m/z 249.1453 (C-ring cleavage). 3.2.3 Fragmentation of type 2-1 triterpenoids The fragmentation characteristic of the compounds (type 2-1) was D-ring and Cring cleavage. Take compound 3β,7β,15β-trihydroxy-11,23-dioxo-lanost-8,16-dien9
26-oic acid as an example (Fig. S1). The [M-H]- ion at m/z 515.3021 produced an ion at m/z 303.1958 by direct cleavage of D-ring. The m/z 497.2916 was formed by loss of H2O. The ion at m/z 453.3010 could be obtained by the loss of CO2. The ion at m/z 249.1489 was obtained by cleaving on ring C. Actually, this type of triterpenoids m/z
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303.1958 was easy loss 2 H to produce ion at m/z 301.1801. 3.2.4 Fragmentation of type 2-2 triterpenoids
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The fragmentation characteristic of compounds (type 2-2) was D-ring and C-ring cleavage. Take compound ganoderic acid B as an example (Fig. S2). The [M-H]- ion
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at m/z 515.3007 produced an ion at m/z 303.1957 by direct cleavage of D-ring. The
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m/z 497.2906 was formed by loss of H2O. The ion at m/z 479.3298 could be obtained
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by the process of loss of CO2. The ion at m/z 249.1492 was obtained by cleaving on
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ring C. The ion at m/z 285.1849 was obtained by the loss of H2O from the ion at m/z
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303.1957. The difference between type 2-1 and 2-2, was that type 2-2 has high abundance ions at m/z 249.1496.
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3.2.5 Fragmentation of type 3-1 triterpenoids
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The fragmentation characteristic of this type of compounds was D-ring cleavage. Take compound lucidone F as an example (Fig. S3). The [M-H]- ion at m/z 401.2348 produced an ion at m/z 301.1818 by directly cleaving on ring D. The m/z 283.1706
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formed by loss of H2O from the ion at m/z 301.1818. 3.2.6 Fragmentation of type 3-2 triterpenoids The fragmentation characteristic of this type of compounds was D-ring and Cring cleavage. Take compound lucidone B as an example (Fig. S4). The [M-H]- ion at 10
m/z 399.2198 produced an ion at m/z 301.1807 by directly cleaving on ring D. The m/z 283.1697 formed by loss of H2O from the ion at m/z 301.1807. The m/z 247.1331 was obtained by cleaving on ring C. 3.2.7 Fragmentation of R5 chain
identification of triterpenoids in G. resinaceum. The common side chain
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The fragmentation pathways of R5 side chains are very important for
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fragmentation behavior of triterpenoids has been reported in several papers [13, 14]. As the fragments data of reference compounds shown in Table 3, the dominant
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fragmentation pathways for triterpenic acid (the type of R5 side chain was 1, 4, 6, 7, 8,
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9, 10, 11, 16 in Fig. 1) are loss CO2 and H2O. Additionally, when the hydroxyl group
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at the position of C-20 (the type of R5 side chain was 1, 2, 6, 10, 16), the carbonyl
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group may be produced at this position by cleavage of the side chain. (the
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fragmentation pathways of representative compound S27, S13 was shown in Fig. S5 and S6, respectively). For type 2, 3, 12 side chains may firstly lose a molecule MeOH
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and subsequently lose CO2 (the fragmentation pathways of representative compound
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S13 was shown in Fig. S6). The triterpenoids contain a side chain such as type 5 and 15 is stable and the most abundant fragment ions are obtained from ring cleavage. As shown in Fig. S7, the side chain type 13 firstly loses a molecule of CO2, and
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subsequently loses the whole side chain (C5H8O4). 3.3 Strategy for identification of triterpenoids As shown in Fig. 4, the first step is extraction of the ion at m/z 301.1807 (d1 and d1’) and m/z 303.1966 (d2). The second step is checked R4 position (usually is the OH 11
group). If a compound has R4 group, the fragmentations has obvious ion at m/z (d1, d1’or d2 +△m R4). The third step is identification of the types of triterpenoids. The ion at m/z 283.1704 is used to distinguish d1 and d1’. The ion at m/z 247.1340 is used to distinguish type 3-1 and type 3-2. The ion at m/z 249.1496 is used to distinguish type
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1-1 and type 1-2. Additionally, in many cases, the loss of H2O in R3 position can be detected in type 1-1 triterpenoids. For compounds containing ion at m/z 303.1966.
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The ion at m/z 249.1496 is used to distinguish type 2-1 and type 2-2. Additionally,
when hydroxy group at position 15, the ion at m/z 303.1966 may loss 2 H to produce
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ion at m/z 301. Finally, based on fragmentation pathways of R5 side chain to identify
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compounds.
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3.4 Identification of triterpenoids in G. resinaceum
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The G. resinaceum extract solution was analyzed by the developed UPLC-
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QTOF-MS (Fig. 5). Total 55 triterpenoids were identified from G. resinaceum. Thirtyfour of them were identified by using reference standards (Table 1). The other
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unknown compounds were tentatively identified by using the developed identification
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strategy (Table 2). The MS data of triterpenoids identified from G. resinaceum were shown in Table 3.
After extraction of ion at m/z 301.1809, fifteen compounds belong to type 1 and
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3 were identified by using reference compounds (Table 1). The unknown compounds C2 showed an [M-H]- ion at m/z 547.2931. The high abundance fragments at m/z 301.1440 and m/z 317.1752, generated by the cleavage of ring D, suggest the presence of the (d1 or d1’ + △ 16). Therefore, R4 position might have a hydroxy group. The ion 12
at m/z 301.1440 might be generated by the ion at m/z 317.1752 loss a CH4 group. No obvious m/z 283.1547, suggests this compound was d1 type. The ion at m/z 249.1490 was not detected, suggest this compound might be type 1-1. For R5 side chain identification, the ions at m/z 529.2813, 503.3022, 485.2915, 467.2796, were
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generated by loss of H2O, CO2, H2O+CO2 and 2H2O + CO2. High abundance ions (loss H2O and CO2), suggest the basic chain of this compound was type 7. The loss of
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2 H2O suggested that this side chain has one additional hydroxy group. This side
chain fragmentation behavior was similar with reference standard S21.Therefore the
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R5 chain of this compound might be type 9. Similarly, other type 1-1 compounds C17
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were also identified (Table 2).
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Identification of compounds belong to type 1-2. For unknown Compound C18
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showed an [M-H]- ion at m/z 531.2962. The fragment at m/z 301.1802, generated by
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the cleavage of ring D, suggests the presence of the (d1 or d1’). No obvious m/z 283.1547 detected, suggested this compound was d1 type. The presentation of ion at
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319.1906 (d1 +△ 18), suggest this compound might have a hydroxy group at the R4
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position. The high abundance of m/z 249.1489, suggests this compound might be type 1-2. For R5 side chain identification, the m/z 513.2863 (loss of H2O) and 469.2961 (loss CO2), suggested the R5 chain might be type 7. Similarly, other type 1-2
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compound C6, C7, C13, C20 and C21 were also identified. After extraction of ion at m/z 303.1966, nineteen compounds belong to type 2 were identified by using reference compounds (Table 1). For unknown Compound C1 showed an [M-H]- ion at m/z 549.3071. The high abundance fragments at m/z 13
303.1955, generated by the cleavage of ring D, suggests the presence of the d2. The presentation of ion at 319.1906 (d1 +△ 16 Da), suggest this compound might have a hydroxy group at R4 position. No m/z 249.1489 was detected, suggested this compound might be type 2-1. For R5 side chain identification, the ions at m/z
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505.3165, 487.3061, were generated by loss of CO2 and H2O. This side chain fragmentation behavior was similar with reference standard S21 (type 9). Similarly,
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other type 2-1 compounds C12, C16 and C19 were also identified.
Identification of compounds belong to type 2-2. For unknown Compound C3
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showed an [M-H]- ion at m/z 547.2931. The fragment at m/z 303.1961, generated by
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the cleavage of ring D, suggests the presence of the d2. High abundance of m/z
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249.1494 was detected, suggested this compound might be type 2-2. For R5 side chain
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identification, the ions at m/z 401.2328, were generated by loss of the R5 chain. Given
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the mass and the high polarity of this compound, the R5 chain might have two hydroxy groups (type 16). Similarly, other type 2-2 compounds C4, C5, C8, C9-C11,
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C14 and C15 were also identified.
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Concluding remarks
In this work, the fragmentation pathways of the triterpenoids from G. resinaceum
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were thoroughly investigated by using UPLC-QTOF-MS in the negative ion mode. Based on the fragmentation pathways of 6 types (type 1-1, 1-2, 2-1, 2-2, 3-1, 3-2) of triterpenoids, a strategy for triterpenoids identification has been developed. About 55 triterpenoids were identified from the enrichment extract of G. resinaceum. The other unknown compounds were tentatively identified using the fragmentation rules. This 14
work is helpful for phytochemical investigation of G. resinaceum as well as quality control of materials containing triterpenoids.
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Acknowledgments The research was partially supported by grants from the National Natural
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Science Foundation of China (Nos. 81673389 and 81603069), the Science and Technology Development Fund of Macau (074/2016/A2、034/2017/A1 and
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040/2016/A) and the University of Macau (MYRG2015-00202 and MYRG2015-
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00122).
Conflict of Interest Statement
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The authors have declared no conflict of interest.
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walls with LC/MS/MS system, International Journal of Mass Spectrometry, 407
M
(2016) 77-85.
ED
[16] J. Kolniak-Ostek, J. Oszmiański, Characterization of phenolic compounds in different anatomical pear (Pyrus communis L.) parts by ultra-performance liquid
PT
chromatography photodiode detector-quadrupole/time of flight-mass spectrometry
CC E
(UPLC-PDA-Q/TOF-MS), International Journal of Mass Spectrometry, 392 (2015) 154-163.
[17] M. Yang, Z. Lu, K. Yu, Q. Wang, X. Chen, Y. Li, X. Liu, W. Wu, D.-a. Guo,
A
Studies on the fragmentation pathways of ingenol esters isolated from Euphorbia esula using IT-MSn and Q-TOF-MS/MS methods in electrospray ionization mode, International Journal of Mass Spectrometry, 323 (2012) 55-62. [18] X.-Y. Guo, J. Han, M. Ye, X.-C. Ma, X. Shen, B.-B. Xue, Q.-M. Che, 18
Identification of major compounds in rat bile after oral administration of total triterpenoids of Ganoderma lucidum by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry, Journal of pharmaceutical and biomedical analysis, 63 (2012) 29-39.
IP T
[19] J. Zhao, X.Q. Zhang, S.P. Li, F.Q. Yang, Y.T. Wang, W.C. Ye, Quality evaluation of Ganoderma through simultaneous determination of nine triterpenes and sterols
SC R
using pressurized liquid extraction and high performance liquid chromatography,
A
CC E
PT
ED
M
A
N
U
Journal of separation science, 29 (2006) 2609-2615.
19
Figure captions
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Fig. 1 The chemical structure of 6 type triterpenoids and the R5 side chain.
20
PT
ED
M
A
N
U
SC R
IP T
Fig. 2 Typical fragmentation pathways of type 1-1 triterpenoids.
A
CC E
Fig. 3 Typical fragmentation pathways of type 1-2 triterpenoids.
21
IP T SC R U N A M ED PT
A
CC E
Fig.4 Strategy for triterpenoids resolution.
22
IP T SC R U N A M
A
CC E
PT
ED
Fig.5 UPLC-QTOF-MS chromatogram of G. resinaceum extract.
23
Table 1. Chemical structures of the reference compounds isolated from G. resinaceum. No.
Name
Type
R1
R2
R3
R4
R5
DBa
S1
Lucidone E
1-1
OH
O
OH
H
5
-
S2
(17S,23S,25R)-17,23-epoxy-
1-1
OH
O
OH
H
13
-
3β,15α-dihydroxy-7,11-dioxo5α-lanosta-8-en-26,23-olide Ganoderenic acid I
1-1
OH
O
OH
H
4
-
S4
3β,15α-dihydroxy-7,11,23-
1-1
OH
O
OH
H
7
16, 17
O
H
6
-
trioxo-5α-lanosta-8,16-dien-26-
S5
20-hydroxy-ganoderic acid AM1
1-2
OH
O
S6
Ganoderic acid AM1
1-2
OH
O
S7
3β,24-dihydroxy-7,11, 15,23-
1-2
OH
O
SC R
oic acid
O
H
7
-
O
H
3
-
O
H
5
-
O
H
7
16, 17
O
O
H
4
-
O
O
OH
7
16, 17
tetraoxo-5α-lanosta-8-en-26-oic
S8
Lucidone D
1-2
OH
O
S9
3β-Hydroxy-7,11,15,23-
1-2
OH
O
N
tetraoxo-5α-lanosta-8,16-dien-
Ganoderenic acid H
1-2
OH
S11
3β,12β-dihydroxy-7,11,15,23-
1-2
OH
M
S10
A
26-oic acid
tetraoxo-5α-lanosta-8,16-dien26-oic acid 1-2
OH
O
O
OH
4
-
1-2
OH
O
O
H
2
-
2-1
OH
OH
OH
H
14
-
2-1
OH
OH
OH
H
7
16, 17
ED
3β,12β-dihydroxy-7, 11,15,23-
U
acid methyl ester
S12
IP T
S3
tetraoxo-5α-lanosta-8, 20E-dien26-oic acid
3β, 20-dihydroxy-7, 11, 15,23-
PT
S13
tetraoxo-5α-lanosta-8-en-26-oic acid methyl ester
3β,7β,15α,24-tetrahydroxy-
CC E
S14
11,23-dioxo-5α-27-norlanosta-8ene
S15
3β,7β,15β-Trihydroxy-11,23-
A
dioxo-lanost-8,16-dien-26-oic acid
S16
Lucidone C
2-1
OH
OH
OH
H
5
-
S17
Ganoderenic acid C
2-1
OH
OH
OH
H
4
-
S18
Ganoderic acid C2
2-1
OH
OH
OH
H
7
-
S19
Ganoderic acid L
2-1
OH
OH
OH
H
6
-
S20
3β,7β,15α,24-tetrahydroxy-
2-1
OH
OH
OH
H
11
-
11,23-dioxo-5α-lanosta-8,20E-
24
dien-26-oic acid S21
3β, 7β, 15α, 24-tetrahydroxy-11,
2-1
OH
OH
OH
H
9
-
23-dioxo-5α-lanosta-8-en-26-oic acid S22
ganoderic acid XL1
2-1
OH
OH
OH
H
10
-
S23
Lucidone A
2-2
OH
OH
O
H
5
-
S24
Ganoderic acid B
2-2
OH
OH
O
H
7
-
S25
3β,7β-dihydroxy-11,15,23-
2-2
OH
OH
O
H
7
16, 17
2-2
OH
OH
O
H
acid S26
3β,7β-dihydroxy-11,15,23trioxo-lanost -8,16-dien-2 6-oic
S27
Ganoderic acid I
2-2
OH
OH
S28
3β,7β,20α-trihydroxy-4, 4,14α-
2-2
OH
OH
H
6
-
O
H
15
-
2-2
OH
OH
O
H
13
-
OH
O
H
2
-
O
H
9
-
OH
O
H
11
-
U
pregn-8-ene 3β,7β, 24-trihydroxy-11,15, 23-
2-2
OH
S31
3β,7β,24-Trihydroxy-11,15,23-
2-2
OH
trioxo-5α-lanosta-8-en-26-oic acid S32
3β, 7β, 24-trihydroxy-11, 15, 23-
2-2
OH
M
Methyl ganoderate I
A
acid methyl ester
OH
N
trioxo-5α-lanosta-8-en-26-oic
S30
16, 17
O
trimethyl-11,15- dioxo-5α-
S29
12
SC R
acid methyl ester
IP T
trioxo-lanost -8,16-dien-26-oic
26-oic acid Lucidone F
S34
Lucidone B
3-1
O
OH
OH
H
5
-
3-2
O
OH
O
H
5
-
double bonds.
A
CC E
a DB,
PT
S33
ED
trioxo-5α-lanosta-8, 20E-dien-
25
I N U SC R
Table 2. Compounds identified by the developed strategy. R2
R3
R4
R5
DB
C1
2-1
OH
OH
OH
OH
9
-
C2
1-1
OH
O
OH
OH
9
-
C3
2-2
OH
OH
O
H
16
-
C4
2-2
OH
OH
O
H
9
-
C5
2-2
OH
OH
O
OH
7
-
C6
1-2
OH
O
O
H
16
-
C7
1-2
OH
O
O
H
8
-
C8
2-2
OH
OH
O
OH
1
-
C9
2-2
OH
OH
O
OH
6
-
C10
2-2
C11
2-2
C12
2-1
A
R1
M
Type
PT
ED
No.
OH
O
OH
7
16,17
OH
OH
O
H
11
-
OH
OH
OH
OH
9
16,17
CC E
OH
1-2
OH
O
O
H
11
-
C14
2-2
OH
OH
O
H
11
-
C15
2-2
OH
OH
O
OH
7
-
C16
2-1
OH
OH
OH
H
11
-
C17
1-1
OH
O
OH
OH
7
16,17
C18
1-2
OH
O
O
OH
7
-
C19
2-1
OH
OH
OH
H
11
-
C20
1-2
OH
O
O
H
8
-
C21
1-2
OH
O
O
OH
7
16,17
A
C13
a DB,
double bonds. 26
I N U SC R
Table 3. The MS data of the triterpenoids from G. resinaceum by UPLC-QTOF-MS analysis. [M-H]Peak
Compounda
RT (min)
Formula
Experimental
No.
Theoretical mass
4
5
C2
S28 C3
6.8
7
8
A
9
10
C4
C5
403.2492
7.6
9.1
9.1 9.8
CC E
6
S19
549.3061
10.3
10.8
549.3078
M
3
S16
5.3
533.3122
ED
2
C1
PT
1
A
mass
547.2914
403.2492
403.249 533.312
Fragments(m/z)
Error, ppm
549.3069 (8.09b), 505.3165 (10.94), 487.3047 (11.63), 350.1725 (21.95), 319.1911 1.5
C30H45O9-
0.6
-
403.2492 (6.35), 387.2181 (2.17), 369.2112 (2.83), 303.1975 (100), 285.1863 (9.58)
-
533.3133 (0.7), 515.3004 (2.36), 403.2524 (3.29), 303.1967 (100), 285.1884 (2.68)
(13.83), 317.1749 (18.57), 303.1959 (100), 249.1501 (2.55) C24H35O5
0.4
C30H45O8
547.2934 (12.51), 529.2803 (12.59), 503.3012 (58.12), 485.2905 (54.58), 467.2787 547.2913
-
0.3
C30H43O9
(7.75), 389.2331 (30.37), 371.2224 (93.60), 348.1576 (42.93), 317.1754 (70.18), 315.1603 (96.24), 301.1442 (100), 287.1651 (5.80) 403.2492 (32.05), 385.2384 (4.50), 359.2231 (27.77), 341.2137 (21.55), 329.1761
403.249
0.6
C24H35O5-
(16.29), 303.1968 (45.79), 287.1651 (17.42), 285.1856 (9.14), 249.1496 (100) 547.292
547.2913
1.4
C30H43O9
531.2972
531.2963
1.6
C30H43O8-
485.2915 (5.29), 401.2333 (100), 303.1964 (8.74), 249.1503 (11.34) 531.2974 (100), 513.2854 (10.60), 495.2749 (15.75), 469.2958 (81.10), 369.436 (20.39), 303.1965 (27.91), 249.1494 (7.99)
531.2973
531.2963
0.6
C30H43O8-
531.2966 (100), 513.2859 (67.02), 495.2749 (5.63), 451.2851 (7.45)，399.2541 (22.16), 319.1913 (9.18), 303.1964 (5.58), 193.0888 (3.28)
C6
11.1
545.2759
545.2756
0.5
C30H41O9
C7
12
527.2653
527.265
1
C30H39O8-
525.2593 (4.42), 399.2175 (55.84), 355.2273 (7.95), 301.1809 (100) 527.2653 (90.19), 509.2547 (98.96), 483.2754 (97.86), 465.2646 (58.01), 453.2280 (21.74), 317.1762 (100), 301.1804 (5.44) 547.2913 (2.08), 503.3011 (9.22), 485.2906 (100), 467.2796 (28.02), 441.2279 (9.7),
11
C8
12.6
547.292
547.2913
-
1.3
C30H43O9
401.2329 (16.52), 319.1916 (10.91), 305.1756 (50.77), 303.1602 (15.34), 303.1959 (22.51), 287.1645 (52.55), 265.1443 (17.08), 249.1500 (3.56)
27
15
S21
C9
531.2959
13.1
401.2321
13.3
533.3129
N U SC R
I 14
S23
12.9
531.2963
401.2333
13.6
547.2912
547.2913
0.1
(6.59), 303.1964 (100), 301.1809 (33.58), 287.1683 (10.10), 249.1531 (17.16) 401.2321 (29.55), 383.2215 (10.06), 371.1851 (5.64),357.2065 (4.62), 343.1895
C24H33O5-
3
1.7
533.312
487.3058 (7.56), 469.3006 (12.95), 425.2738 (6.05), 385.2395 (3.59), 383.2589
C30H43O8-
0.7
A
13
S20
M
12
(7.63), 303.1956 (100), 287.1642 (8.10), 285.1858 (14.51), 249.1486 (49.31) 489.323 (23.64), 471.3125 (31.17), 453.3013 (4.04), 334.1791 (14.36), 303.1971
C30H45O8(53.34), 301.1814 (100), 287.1654 (86), 249.1491 (3.59) 529.2794 (74.62), 511.2691 (100), 493.2588 (29.99), 467.2785 (25.69), 417.2276 C30H43O9-
(65.67), 399.2175 (54.57), 319.1911 (14.48), 303.1956 (20.37), 303.1619 (8.37),
17
S22
C11
13.9
14
14.8
CC E
18
C10
PT
16
ED
301.1803 (62.87), 265.1445 (6.18) 529.2797
517.3169
529.2801 (4.47), 511.2693 (100), 467.2787 (10.14), 401.2340 (1.2), 319.1905 (3.44), 529.2807
C30H41O8-
1.9
303.1952 (1.6), 303.1596 (0.8), 301.1797 (2.46), 265.1439 (5.36) 517.317 (100), 473.3267 (16.02), 455.3158 (5.38), 361.2382 (43.70), 343.2277 517.3171
C30H45O7-
0.3
251.1645 (7.76), 249.149 (5.89) 513.2869 (3.33), 487.3064 (24.70), 469.2953(55.81), 451.2851 (12.01), 383.2583 531.2956
531.2963
C30H43O8-
1.3
(31.58), 303.1963 (100), 249.1492 (95.32) -
19
S1
15
401.2339
401.2333
1.5
C24H33O5
20
S8
15.2
399.2158
399.2158
4.8
C24H31O5-
21
S15
15.31
515.3028
515.3014
2.7
C30H43O7-
401.2321 (2.83), 383.2225 (0.25), 301.1776 (100), 245.1517 (7.11) 399.2158 (19.54), 369.1686 (9.28), 313.1430 (15.43), 301.1803 (100), 249.1471 (0.5) 515.3028 (100), 497.2873 (58.71), 435.2873 (14.27), 303.1944 (11.13), 287.1634 (9.06), 249.1473 (9.07)
A 22
(22.74), 303.1963 (75.29), 301.1807 (38.95), 287.201 (23.74), 287.165 (32.52),
531.513.2846 (12.35), 495.2738 (25.36), 469.2946 (44.85), 451.2840 (33.37), S27
15.8
531.295
531.2963
-
2.5
C30H43O8
401.232 (100), 303.1958 (33.58), 287.1639 (3.42), 285.1861 (3.77), 275.1642 (9.13), 249.1496 (28.40)
-
23
S33
15.9
401.2333
401.2333
0.1
C24H33O5
24
S5
16.9
529.2821
529.2807
2.6
C30H41O8-
401.2333 (8.24), 385.2007 (3.3), 367.1901 (4.41), 301.1797 (100), 283.1688 (9.66) 511.2711 (4.42), 493.2612 (18.26), 467.2811 (67.29), 449.2698 (27.72), 419.2237 (15.50), 399.2203 (37.7), 301.1803 (100), 287.1661 (3.13), 249.1464 (6.31)
28
28
S31
C12
527.2716
18.6
529.2807
19.3
531.2969
N U SC R
I 27
S32
18.5
527.2709
529.2807
19.4
547.2915
547.2913
0.4
301.1817 (1.3), 303.1624 (0.8), 287.1684 (0.12) 485.291(19.09), 467.2804 (100), 449.2696 (16.48), 385.2388 (43.04), 303.1968
C30H41O8-
1
0.4
531.2963
509.2604 (78.79), 479.2134 (100), 467.2489(4.60), 435.2229 (8.37), 317.1811 (6.82),
C23H43O13-
1.8
A
26
S11
M
25
(25.38), 287.1654 (16.05), 285.1865 (11.06), 249.1499 (62.15) 487.3065 (4.5), 469.2968 (62.11), 425.2342 (100), 303.1963 (2.45), 289.1811
C30H43O8(79.78), 287.1653 (15.28), 249.1499 (27.56) 503.3020 (100), 473.2544 (11.38), 319.1913 (1.7), 303.1958 (2.2), 303.1601 (0.9), C30H43O9301.1807 (3.92)
C14
21.2
21.7
CC E
31
S17
20.2
PT
30
C13
32
33
A
34
S14
S18
S12
ED
483.2757 (9.29), 465.2643 (72.5), 453.2278 (22.46), 435.2177 (27.03), 397.2383 29
23.4
23.9
24.5
527.2653
515.3022
527.265
C30H39O8-
0.9
(22.87), 383.2224 (19.60), 301.1818 (100), 249.1490 (4.04) 515.3019 (100), 497.2911 (10.35), 453.3007 (6.24), 383.2599 (11.61), 303.1968 515.3014
C30H43O7-
1.1
(54.69), 287.1653 (43.14), 249.1501 (3.87), 193.0898(35.29) 511.2698 (11.78), 485.2912 (19.98), 467.2809 (59.30), 438.2408 (13.08), 385.2384
529.281
529.2807
C30H41O8-
0.6
(9.63), 303.1960 (100), 249.1499 (48.55) 489.3211 (15.30), 471.3067 (13.65), 303.1932 (50.49), 287.1986 (10.82), 249.1475 489.3210
489.3222
C29H45O6-
2.5
(8.4) 517.3172 (98.57), 499.3066 (100), 481.2954 (2.63), 455.3165 (14.84), 437.3058 517.318
517.3171
C30H45O7-
1.6
(6.15), 303.1961 (14.31), 301.1805 (25.38), 287.1645 (33.07), 249.1483 (1.6) 509.2551 (100), 479.2082 (61.35), 465.2653 (30.47), 435.2181 (9.81), 397.2338 527.2661
527.2650
-
2.0
C30H39O8
(8.58), 385.2386 (20.72), 362.1369 (20.97), 345.1703 (14.25), 317.1752 (14.01), 303.1603 (9.3), 301.1802 (9.14), 249.1489 (5.74) 531.2963 (100), 513.2852 (36.61), 469.2952 (15.38), 423.2896 (2.04), 391.1762
35
C15
25.5
531.2963
531.2963
-
1
C30H43O8
(3.86), 363.1807 (2.12), 319.1911 (17.2), 301.1807 (14.51), 287.165 (3.8), 265.1441 (6.17)
36
C16
26.6
531.2968
531.2963
-
0.8
C30H43O8
29
487.3060 (4.79), 303.1967 (100)
40
C18
C19
529.2807
29.4
511.2713
29.8
531.2952
N U SC R
I 39
S9
27.4
529.2807
511.2701
32.4
531.2957
531.2963
1.3
(3.82), 301.1813 (2.86) 493.2605 (16.1), 467.2811 (100), 449.2703 (21.2), 419.2233 (20.88),
C30H39O7-
2.1
2.2
531.2963
511.2707 (100), 481.2231 (18.32), 467.2807 (67.87), 437.2333 (14.57), 303.1601
C30H41O8-
0
A
38
C17
M
37
384.2307(18.20), 369.2074 (8.99), 301.1809 (3.2), 287.165 (1.0), 249.1498 (1.0) 513.2851 (100), 469.295 (35.71), 451.2841 (11.91), 425.2327 (4.17), 319.1925
C30H43O8(9.45), 301.1805 (26.44), 265.144 (40.25), 249.149 (7.92) 519.2948 (3.74), 487.3057 (36.49), 457.2589 (42.17), 439.2483 (3.05), 401.2684 C30H43O8(8.75), 303.1960 (100)
43
C20 C21
33.1
34.1 34.1
CC E
44
S34
33.1
PT
42
S24
45
A
46
47
S10
S6
S30
ED
497.2862 (100), 453.2970 (79.54), 303.1938 (55.90), 287.1639 (7.48), 285.1830 41
34.8
35
36.7
515.3009
399.2197
515.3014
C30H43O7-
1
(21.85), 249.1468 (43.54) 399.2178 (12.18), 369.1702 (4.60), 341.1753 (5.99), 301.1804 (100), 283.1694 399.2177
5
C24H31O5(5.99), 247.1328 (11.80) 493.2585(24.05), 467.2795 (100), 449.2688 (44.04), 419.2219 (29.37), 384.2293
511.2695
511.2701
C30H39O7-
1.2
(20.7), 369.2064 (9.36), 301.1804 (88.75), 249.1493 (26.93) 529.28
529.2807
1.4
C30H41O8-
513.2862
513.2858
1.2
C30H41O7-
511.2688 (100), 449.2687 (7.45), 384.2297 (3.99), 301.1812 (14.77), 265.1434 (4.65) 513.2855 (4.6), 451.2853 (26.29), 436.2619 (100), 301.1819 (27.42), 287.1650 (2.12), 249.15 (7.17) 513.2856 (5.41), 495.2762 (7.10), 451.2889 (31.33), 436.2663 (100), 301.1851
513.2867
513.2858
C30H41O7-
2.1
(32.00), 287.1673 (2.44), 249.1454 (8.78) 527.296 (2.56), 509.2514 (19.74), 491.2375 (5.53), 453.2244 (27.89), 435.2129, 545.3072
545.3061
-
2.3
C38H41O3
401.2298 (100), 303.1961 (18.35), 301.1792 (10.78), 285.1831 (2.42), 249.1481 (22.01) 527.2998 (3.96), 513.2842 (6.39), 495.2741 (3.21), 439.25 (29.97), 425.2328 (100),
48
S29
36.8
545.3113
545.312
C31H45O8-
1.1
303.1951 (1.31), 287.1641 (6.04), 249.1491 (3.45) 495.2712 (100), 480.2473 (7.67), 465.2239 (4.99), 451.2815 (12.28), 436.258 (7.74), 49
S25
37.1
513.2844
513.2858
C30H41O7-
2.6
409.2345 (4.18), 303.1967 (trace), 249.1471 (5.60)
30
I S3
38
543.2971
39
513.2851
543.2963
513.2858
S7
M
52
39.4
543.2956
543.2956
511.2701 (354604), 493.2598 (28.02), 399.2176 (8.26), 301.1811 (100), 287.1645
C31H43O8-
2.6
-
1.3
C30H41O7
A
51
S13
N U SC R
50
(0.4), 249.1493 (1.0) 513.2849 (91.23), 465.2289 (100), 451.2857 (80.26), 435.2561 (13.64), 421.2390 (40.16), 399.2541 (2.78), 381.2445 (9.69), 351.1983 (5.08), 326.1902 (8.22), 303.1957 (52.13), 301.1817 (80.47), 287.1640 (5.44), 193.0883 (14.93) 543.2958 (6.84), 511.2694 (70.58), 455.2442 (86.03), 437.2329 (100), 423.2186
C31H43O8-
1.4
(20.31), 385.2400 (9.84), 303.1964 (4.7), 301.1802 (6.4), 287.1650 (7.96), 249.1495 (11.77)
54
a
S2
48.2
50
513.2860
527.3066
513.2858
C30H41O7-
0.4
(5.25), 351.1965 (21.75), 303.1960 (1.2), 301.1803 (2.41), 287.1647 (0.37), 193.0886 (1.3) 527.3031 (100), 512.279(31.19), 495.2764 (73.01), 465.2291 (4.02), 383.2249
527.3073
C24H47O12-
1.2
(62.38), 303.1969 (1.5), 287.1653 (1.6), 249.1499 (11.22) 513.2846
513.2858
C30H41O7 -
2.4
513.2845 (90.48), 495.2733 (26.60), 469.2939 (6.06), 451.2833 (14.01), 381.2426 (91.75), 301.1800 (93.13), 289.1791 (5.7), 287.1638 (3.1), 193.0862 (8.14)
the compound codes are same with table 1 and table 2. abundance.
A
b
S26
41.4
CC E
55
S4
PT
53
ED
513.2860 (3.85), 483.2391 (29.72), 421.2386 (100), 381.2360 (4.81), 369.2071
31
S1387-3806(18)30311-7 https://doi.org/10.1016/j.ijms.2018.11.016 MASPEC 16090
To appear in:
International Journal of Mass Spectrometry
Received date: Revised date: Accepted date:
15 August 2018 4 November 2018 4 November 2018
Please cite this article as: Chen L, Chen X, Wang S, Bian Y, Zhao J, Li S, Analysis of triterpenoids in Ganoderma resinaceum using liquid chromatography coupled with electrospray ionization quadrupole - time - of - flight mass spectrometry, International Journal of Mass Spectrometry (2018), https://doi.org/10.1016/j.ijms.2018.11.016 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 proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Analysis of triterpenoids in Ganoderma resinaceum using liquid chromatography coupled with electrospray ionization quadrupole - time - of - flight mass spectrometry
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Lingxiao Chen1, Xianqiang Chen1, Shenfei Wang1, Yao Bian, Jing Zhao*,
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and Shaoping Li*
State Key Laboratory of Quality Research in Chinese Medicine, Institute
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of Chinese Medical Sciences, University of Macau, Macao SAR, China
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* Correspondence: E-mail: [email protected] (J. Zhao);
These authors contributed equally to this work.
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1
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[email protected] (S. P. Li); Tel.: +853 88224692.
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Graphical abstract
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Highlights
The triterpenoids from Ganoderma resinaceum were analyzed by using UPLC-
A strategy for identification of triterpenoids has been developed.
Total 55 triterpenoids were identified from G. resinaceum.
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Abstract
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QTOF-MS.
The triterpenoids from Ganoderma resinaceum were investigated by using
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UPLC-QTOF-MS and their fragmentation behaviors are summarized in detail. Based
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on the fragmentation pathways, a strategy for identification of triterpenoids has been
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developed. The main triterpenoids in G. resinaceum can be classified into 6 types (type 1-1, 1-2, 2-1, 2-2, 3-1, 3-2) in three steps. Each step was controlled by the
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characteristic ions, which are summarized from fragmentation pathways of 34
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reference compounds. Total 55 triterpenoids were identified from G. resinaceum. Thirty-four of them were identified by using reference standards. The other unknown
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compounds were tentatively identified by using the developed identification strategy. This work is helpful for phytochemical investigation of G. resinaceum as well as
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quality control of materials containing triterpenoids.
Key words: Ganoderma resinaceum; QTOF; triterpenoids; fragmentation pathway; Identification 2
1. Introduction The genus Ganoderma, belongs to Ganodermataceae family, is a group of higher macrofungi widely growing in tropical and temperate regions [1, 2]. In China,
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Ganoderma is a well-known medicinal fungus and has been used for healthimprovement and longevity for over 2000 years [3, 4]. Also, it is involved in clinical
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treatment of various types of diseases, such as chronic bronchitis, neurasthenia,
antitumor, coronary heart disease, hypercholesterolemia, etc. [4-6]. Triterpenoids are
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one of the main components in many Ganoderma species. As reported, the majority of
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triterpenoids in Ganoderma species exhibit significant biological activities, including
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α-glucosidase inhibitory activities, anti-HIV, antitumor, antiviral, hepatoprotective,
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anti-hypertensive, cholesterol-reducing and anti-inflammatory [1, 5-8]. Therefore, the
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triterpenoids have been regarded as one important indicator for quality evaluation of G. lucidum and related species [4]. G. resinaceum Boud., a member of genus
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Genoderma, has long been used for enhancement of internal balance and functions of
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the immune system. Our previous work showed that triterpenoids are also one of the main ingredients in G. resinaceum. Additionally, many triterpenoids separated from G. resinaceum showed strong inhibitory effects against α-glucosidase [1, 2, 9-11].
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However, the chemical structures of triterpenoids in G. resinaceum has not been systematic studied. Therefore, the structural analysis of triterpenoids in G. resinaceum is very necessary, which is beneficial for understanding their structure-function relationships as well as quality evaluation of G. resinaceum. 3
Generally, the chemical compositions of Ganoderma species are very complex. The systematic chemical investigation needs isolation, purification and identification of compounds. This work is very tedious and time-consuming especially for compounds that are of trace amount. Liquid chromatography coupled with
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electrospray ionization mass spectrometry (LC-ESI-MS) is a very efficient tool that has been proved to be rapid, selective and sensitive for qualitative analysis of
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compounds in natural products [12-17].
The most common method for identification of compounds by using LC-ESI-MS
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is to compare the retention times and MS data with those of reference standards.
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However, many standards are very expensive, and it is also hard to acquire all
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reference compounds that required [13]. Moreover, this method is not suitable for
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identification of unknown compounds. It is no doubt that the fragmentation pathway
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of compound is depending on their structures. Additionally, the same type compounds such as triterpenoids usually have similar fragmentation pathway. Therefore,
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systematic study the fragmentation behavior is helpful for identification of
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compounds with similar structures. The fragmentation behaviors of triterpenoids isolated from G. lucidum. have
been reported for several papers [4, 13, 14, 18]. However, as far as we are aware, no
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systematic strategy or steps has been reported for triterpenoid resolution. In this work, the triterpenoids isolated from G. resinaceum have been studied using UPLCQTOFMS and their fragmentation behaviors were summarized in detail. Additionally, a systematic strategy for triterpenoid resolution has been developed. Based on this 4
strategy, triterpenoids in G. resinaceum have been comprehensively investigated. This work is helpful for phytochemical investigation of G. resinaceum as well as quality control of materials containing triterpenoids.
2. Materials and Methods
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2.1 Materials and chemicals The fruiting bodies of G. resinaceum was purchased from the market. The
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reference standards of triterpenoids used in this study (S1-S34) were isolated from the fruiting bodies of G. resinaceum in our previous work (Table 1). All these structures
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were identified by NMR and MS analysis [1, 2, 9-11]. Acetonitrile, methanol and
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acetic acid were of HPLC grade. HPLC grade water was prepared using a Milli‐Q
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2.2 Preparation of standard solutions
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water purification system (Millipore, MA, USA).
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In order to optimize the conditions for ESI-QTOF-MS analysis. Seven representative reference compounds including nortriterpenoids (S8, S33),
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tritepenoidic acids contain double bonds between C-16 and C-17 (S15, S25), typical
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tritepenoidic acids (S24, S6) and tritepenoidic acid contain an oxaspirolactone moiety consisting of a five-membered ether ring, a five-membered lactone ring, and a characteristic C-23 spiro carbon (S2). The stock standard solution of seven reference
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compounds was prepared in methanol at final concentrations of 0.1 mg/mL. Working standard solution was prepared by dilution with methanol to final concentrations of 1.0 μg/mL. All of the solutions were stored in the 4 ◦C fridge. 2.3 Sample preparation 5
Pressurized liquid extraction (PLE) was carried out on a Dionex ASE 350 system (Dionex Corp., Sunnyvale, CA, USA). Powdered G. resinaceum (1.0 g) was mixed with diatomaceous earth in a proportion (1:1) and placed in a 10 mL stainless steel extraction cell. And then extracted according to our previous study [19]. The
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extraction conditions of the PLE method were: solvent, ethanol; temperature,100℃; static extraction time, 5 min; pressure, 1500 psi; 60% of the flush volume for one
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cycle and one extraction time. The extract was centrifuged at 4000g for 10 min, and the supernatant was evaporated to dryness under vacuum. Subsequently, the residue
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was dissolved in 2 mL methanol and filtered through a 0.22 µm filter.
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Some triterpenoids in G. resinaceum are of trace amount and the material are
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very complex. Therefore, a solid-phase extraction method has been developed. An
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SPE cartridge was pretreated with 2 mL of water, 2 mL of methanol and 2 mL of
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water, successively. The SPE conditions were optimized using the G. resinaceum extract (data not shown) and the optimum conditions were: 400 μL sample diluted in
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3.6 mL water, and then loaded on the SPE column, after that the SPE column was
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washing by 30% methanol (2 mL) and then eluting by 80% methanol (2 mL). The eluting solution was evaporated to dry under N2 at room temperature. The residue was re-dissolved by 100 μL methanol followed by centrifugation at 10,000 rpm for 5 min.
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2.4 Chromatography A Dionex (Germering, Germany) Ultimate 3000 UHPLC system equipped with Ultimate 3000 degasser, pump, RS autosampler, and RS column compartment, coupled with diode‐array detector. Waters Acquity BEH C18 column (2.1×150 mm 6
i.d., 1.7 μm, Waters, Milford, MA, USA). was used for sample separation. The mobile phase was consisted of 0.2% acetic acid in water (A) and acetonitrile (B) with gradient elution: 0-8 min, 3% B-22% B; 8-25 min, 22% B-25% B; 25-40 min, 25% B35% B；40-65 min, 35%B - 100%B, 65-70 min, 100%B-100% B. The flow rate was
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0.3 mL/min, injection volume was 1 μL and the column temperature was 25 ℃, and the detection wavelength was set at 257 nm.
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2.5 ESI-QTOF-MS analysis
A high-resolution impact HD QTOF mass spectrometer (Bruker Daltonik GmbH,
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Bremen, Germany) equipped with an electrospray ionization (ESI) source was
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operated in the negative ion mode. The mass range was set at m/z 100–1500 in the full
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scan mode. The capillary voltage was set at 3500 V. The source temperature was set at
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250 ◦C. Nitrogen was used as the drying gas. The gas flow rate was set at 8 L/min.
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MS2 data analysis of the three highest intensive ion fragments was intelligently performed in real time.
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3. Results and discussion
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3.1 Optimization of ESI-QTOF-MS conditions The ionization efficiency of triterpenoids is very important, especially for the
analytes in trace amount. The ESI-QTOF-MS was carried out in the negative ion
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mode according to the literature report [13, 14]. The capillary voltage (V), flow rate of dry gas (L/min), dry temperature (℃) has been optimized in this study. The capillary voltage (V) at 2500, 3000, 3500 was investigated. The average abundance of ion [MH]- of each compound was listed in Table S1. The results showed that the average 7
abundance of ion [M-H]- of compound S2 and S8 increased significantly with the capillary voltage (2500-3500 V). However, the capillary voltage more than 3500 V was not investigated because the average abundance of ion [M-H]- of other compounds (S6, S15, S24, S25 and S33) decreased slightly in the test range.
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Additionally, the average abundance of ion [M-H]- of compound S2 increased slowly when the capillary voltage more than 3000V. Therefore, 3500 V was selected as the
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optimum capillary voltage. Subsequently, flow rate of dry gas (L/min) at 6, 8, 10 was
investigated. When the flow rate increased from 6 to 10 L/min, the average abundance
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of ion [M-H]- of compounds S2, S6, S8, S15, S33 increased correspondingly (the
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average abundance of ion [M-H]- increased slowly when the flow rate more than 8
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L/min). However, the average abundance of ion [M-H]- of compounds S24 and S25
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reduced because of the dehydration reaction (Table S1 and S2). Therefore, flow rate
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of dry gas at 8 L/min was selected as the optimum condition. Dry temperature (℃) at 200, 250, 300 have been investigated. It was found that the average abundance of ion
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[M-H]- of compounds S2, S6, S8, S15, S33 increased steadily with dry temperature.
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However, as shown in Table S1 and S2, the average abundance of ion [M-H]- of compounds S24 and S25 also reduced significantly because of the dehydration reaction (no quasi-molecular ion was detected at 300 ℃). Therefore, the dry
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temperature at 250 ℃ was selected as the optimum condition. 3.2 ESI-QTOF-MS analysis of triterpenoids reference compounds The thirty-four reference compounds (Table 1) could be classified into six groups (1-1, 1-2, 2-1, 2-2, 3-1, 3-2) according to their fragmentation pathways (Fig. 1). The 8
dominant fragmentation pathways of the compounds studied are cleavages of C-ring and D-ring. The specific fragmentation behavior of these six types of triterpenoids has been summarized below. 3.2.1 Fragmentation of type 1-1 triterpenoids
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The fragmentation characteristic of this type of compounds was cleavage of Dring. Take compound ganoderenic acid I as an example. As shown in Fig. 2, the [M-H]-
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ion at m/z 513.2850 produced an ion at m/z 301.1801 by direct cleavage of D-ring. The m/z 495.2735 was formed by loss of H2O. The ion at m/z 451.2839 could be
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obtained by the loss of CO2. The ion at m/z 381.2421 was produced by the loss of
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H2O at R3 position during the ring cleavage.
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3.2.2 Fragmentation of type 1-2 triterpenoids
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The fragmentation characteristic of this type of compounds was D-ring and C-
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ring cleavage. Take compound ganoderic acid AM1 as an example (Fig. 3). The [MH]- ion at m/z 513.2857 produced an ion at m/z 301.1842 by direct cleavage of ring D.
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The m/z 495.2764 formed by loss of H2O. The ion at m/z 451.2839 could be obtained
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by the loss of CO2. The difference between this type of triterpenoids and type 1-1 was that the triterpenoids belong to type 1-1 will lose one more H2O (R3 position) during the ring cleavage. Additionally, in some cases, the type 1-2 triterpenoids have the high
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abundance ion at m/z 249.1453 (C-ring cleavage). 3.2.3 Fragmentation of type 2-1 triterpenoids The fragmentation characteristic of the compounds (type 2-1) was D-ring and Cring cleavage. Take compound 3β,7β,15β-trihydroxy-11,23-dioxo-lanost-8,16-dien9
26-oic acid as an example (Fig. S1). The [M-H]- ion at m/z 515.3021 produced an ion at m/z 303.1958 by direct cleavage of D-ring. The m/z 497.2916 was formed by loss of H2O. The ion at m/z 453.3010 could be obtained by the loss of CO2. The ion at m/z 249.1489 was obtained by cleaving on ring C. Actually, this type of triterpenoids m/z
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303.1958 was easy loss 2 H to produce ion at m/z 301.1801. 3.2.4 Fragmentation of type 2-2 triterpenoids
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The fragmentation characteristic of compounds (type 2-2) was D-ring and C-ring cleavage. Take compound ganoderic acid B as an example (Fig. S2). The [M-H]- ion
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at m/z 515.3007 produced an ion at m/z 303.1957 by direct cleavage of D-ring. The
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m/z 497.2906 was formed by loss of H2O. The ion at m/z 479.3298 could be obtained
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by the process of loss of CO2. The ion at m/z 249.1492 was obtained by cleaving on
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ring C. The ion at m/z 285.1849 was obtained by the loss of H2O from the ion at m/z
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303.1957. The difference between type 2-1 and 2-2, was that type 2-2 has high abundance ions at m/z 249.1496.
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3.2.5 Fragmentation of type 3-1 triterpenoids
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The fragmentation characteristic of this type of compounds was D-ring cleavage. Take compound lucidone F as an example (Fig. S3). The [M-H]- ion at m/z 401.2348 produced an ion at m/z 301.1818 by directly cleaving on ring D. The m/z 283.1706
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formed by loss of H2O from the ion at m/z 301.1818. 3.2.6 Fragmentation of type 3-2 triterpenoids The fragmentation characteristic of this type of compounds was D-ring and Cring cleavage. Take compound lucidone B as an example (Fig. S4). The [M-H]- ion at 10
m/z 399.2198 produced an ion at m/z 301.1807 by directly cleaving on ring D. The m/z 283.1697 formed by loss of H2O from the ion at m/z 301.1807. The m/z 247.1331 was obtained by cleaving on ring C. 3.2.7 Fragmentation of R5 chain
identification of triterpenoids in G. resinaceum. The common side chain
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The fragmentation pathways of R5 side chains are very important for
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fragmentation behavior of triterpenoids has been reported in several papers [13, 14]. As the fragments data of reference compounds shown in Table 3, the dominant
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fragmentation pathways for triterpenic acid (the type of R5 side chain was 1, 4, 6, 7, 8,
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9, 10, 11, 16 in Fig. 1) are loss CO2 and H2O. Additionally, when the hydroxyl group
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at the position of C-20 (the type of R5 side chain was 1, 2, 6, 10, 16), the carbonyl
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group may be produced at this position by cleavage of the side chain. (the
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fragmentation pathways of representative compound S27, S13 was shown in Fig. S5 and S6, respectively). For type 2, 3, 12 side chains may firstly lose a molecule MeOH
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and subsequently lose CO2 (the fragmentation pathways of representative compound
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S13 was shown in Fig. S6). The triterpenoids contain a side chain such as type 5 and 15 is stable and the most abundant fragment ions are obtained from ring cleavage. As shown in Fig. S7, the side chain type 13 firstly loses a molecule of CO2, and
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subsequently loses the whole side chain (C5H8O4). 3.3 Strategy for identification of triterpenoids As shown in Fig. 4, the first step is extraction of the ion at m/z 301.1807 (d1 and d1’) and m/z 303.1966 (d2). The second step is checked R4 position (usually is the OH 11
group). If a compound has R4 group, the fragmentations has obvious ion at m/z (d1, d1’or d2 +△m R4). The third step is identification of the types of triterpenoids. The ion at m/z 283.1704 is used to distinguish d1 and d1’. The ion at m/z 247.1340 is used to distinguish type 3-1 and type 3-2. The ion at m/z 249.1496 is used to distinguish type
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1-1 and type 1-2. Additionally, in many cases, the loss of H2O in R3 position can be detected in type 1-1 triterpenoids. For compounds containing ion at m/z 303.1966.
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The ion at m/z 249.1496 is used to distinguish type 2-1 and type 2-2. Additionally,
when hydroxy group at position 15, the ion at m/z 303.1966 may loss 2 H to produce
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ion at m/z 301. Finally, based on fragmentation pathways of R5 side chain to identify
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compounds.
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3.4 Identification of triterpenoids in G. resinaceum
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The G. resinaceum extract solution was analyzed by the developed UPLC-
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QTOF-MS (Fig. 5). Total 55 triterpenoids were identified from G. resinaceum. Thirtyfour of them were identified by using reference standards (Table 1). The other
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unknown compounds were tentatively identified by using the developed identification
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strategy (Table 2). The MS data of triterpenoids identified from G. resinaceum were shown in Table 3.
After extraction of ion at m/z 301.1809, fifteen compounds belong to type 1 and
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3 were identified by using reference compounds (Table 1). The unknown compounds C2 showed an [M-H]- ion at m/z 547.2931. The high abundance fragments at m/z 301.1440 and m/z 317.1752, generated by the cleavage of ring D, suggest the presence of the (d1 or d1’ + △ 16). Therefore, R4 position might have a hydroxy group. The ion 12
at m/z 301.1440 might be generated by the ion at m/z 317.1752 loss a CH4 group. No obvious m/z 283.1547, suggests this compound was d1 type. The ion at m/z 249.1490 was not detected, suggest this compound might be type 1-1. For R5 side chain identification, the ions at m/z 529.2813, 503.3022, 485.2915, 467.2796, were
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generated by loss of H2O, CO2, H2O+CO2 and 2H2O + CO2. High abundance ions (loss H2O and CO2), suggest the basic chain of this compound was type 7. The loss of
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2 H2O suggested that this side chain has one additional hydroxy group. This side
chain fragmentation behavior was similar with reference standard S21.Therefore the
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R5 chain of this compound might be type 9. Similarly, other type 1-1 compounds C17
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were also identified (Table 2).
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Identification of compounds belong to type 1-2. For unknown Compound C18
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showed an [M-H]- ion at m/z 531.2962. The fragment at m/z 301.1802, generated by
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the cleavage of ring D, suggests the presence of the (d1 or d1’). No obvious m/z 283.1547 detected, suggested this compound was d1 type. The presentation of ion at
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319.1906 (d1 +△ 18), suggest this compound might have a hydroxy group at the R4
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position. The high abundance of m/z 249.1489, suggests this compound might be type 1-2. For R5 side chain identification, the m/z 513.2863 (loss of H2O) and 469.2961 (loss CO2), suggested the R5 chain might be type 7. Similarly, other type 1-2
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compound C6, C7, C13, C20 and C21 were also identified. After extraction of ion at m/z 303.1966, nineteen compounds belong to type 2 were identified by using reference compounds (Table 1). For unknown Compound C1 showed an [M-H]- ion at m/z 549.3071. The high abundance fragments at m/z 13
303.1955, generated by the cleavage of ring D, suggests the presence of the d2. The presentation of ion at 319.1906 (d1 +△ 16 Da), suggest this compound might have a hydroxy group at R4 position. No m/z 249.1489 was detected, suggested this compound might be type 2-1. For R5 side chain identification, the ions at m/z
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505.3165, 487.3061, were generated by loss of CO2 and H2O. This side chain fragmentation behavior was similar with reference standard S21 (type 9). Similarly,
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other type 2-1 compounds C12, C16 and C19 were also identified.
Identification of compounds belong to type 2-2. For unknown Compound C3
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showed an [M-H]- ion at m/z 547.2931. The fragment at m/z 303.1961, generated by
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the cleavage of ring D, suggests the presence of the d2. High abundance of m/z
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249.1494 was detected, suggested this compound might be type 2-2. For R5 side chain
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identification, the ions at m/z 401.2328, were generated by loss of the R5 chain. Given
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the mass and the high polarity of this compound, the R5 chain might have two hydroxy groups (type 16). Similarly, other type 2-2 compounds C4, C5, C8, C9-C11,
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C14 and C15 were also identified.
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Concluding remarks
In this work, the fragmentation pathways of the triterpenoids from G. resinaceum
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were thoroughly investigated by using UPLC-QTOF-MS in the negative ion mode. Based on the fragmentation pathways of 6 types (type 1-1, 1-2, 2-1, 2-2, 3-1, 3-2) of triterpenoids, a strategy for triterpenoids identification has been developed. About 55 triterpenoids were identified from the enrichment extract of G. resinaceum. The other unknown compounds were tentatively identified using the fragmentation rules. This 14
work is helpful for phytochemical investigation of G. resinaceum as well as quality control of materials containing triterpenoids.
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Acknowledgments The research was partially supported by grants from the National Natural
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Science Foundation of China (Nos. 81673389 and 81603069), the Science and Technology Development Fund of Macau (074/2016/A2、034/2017/A1 and
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040/2016/A) and the University of Macau (MYRG2015-00202 and MYRG2015-
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00122).
Conflict of Interest Statement
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The authors have declared no conflict of interest.
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Q.-M. Che, Structural characterization of minor metabolites and pharmacokinetics of
ED
ganoderic acid C2 in rat plasma by HPLC coupled with electrospray ionization tandem mass spectrometry, Journal of pharmaceutical and biomedical analysis, 75
PT
(2013) 64-73.
CC E
[5] S. Choi, V.T. Nguyen, N. Tae, S. Lee, S. Ryoo, B.-S. Min, J.-H. Lee, Antiinflammatory and heme oxygenase-1 inducing activities of lanostane triterpenes isolated from mushroom Ganoderma lucidum in RAW264. 7 cells, Toxicology and
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applied pharmacology, 280 (2014) 434-442. [6] J.-G. Wu, Y.-J. Kan, Y.-B. Wu, J. Yi, T.-Q. Chen, J.-Z. Wu, Hepatoprotective effect of ganoderma triterpenoids against oxidative damage induced by tert-butyl hydroperoxide in human hepatic HepG2 cells, Pharmaceutical biology, 54 (2016) 91916
929. [7] R.S. El Dine, A.M. El Halawany, C.-M. Ma, M. Hattori, Anti-HIV-1 protease activity of lanostane triterpenes from the vietnamese mushroom Ganoderma colossum, Journal of natural products, 71 (2008) 1022-1026.
IP T
[8] I. Lee, J. Kim, I. Ryoo, Y. Kim, S. Choo, I. Yoo, B. Min, M. Na, M. Hattori, K. Bae, Lanostane triterpenes from Ganoderma lucidum suppress the adipogenesis in
SC R
3T3-L1 cells through down-regulation of SREBP-1c, Bioorganic & medicinal chemistry letters, 20 (2010) 5577-5581.
U
[9] X.-Q. Chen, L.-X. Chen, S.-P. Li, J. Zhao, A new nortriterpenoid and an ergostane-
N
type steroid from the fruiting bodies of the fungus Ganoderma resinaceum, Journal of
A
Asian natural products research, 19 (2017) 1239-1244.
M
[10] X.-Q. Chen, L.-X. Chen, J. Zhao, Y.-P. Tang, S.-P. Li, Nortriterpenoids from the
ED
Fruiting Bodies of the Mushroom Ganoderma resinaceum, Molecules, 22 (2017) 1073.
PT
[11] X.-Q. Chen, L.-G. Lin, J. Zhao, L.-X. Chen, Y.-P. Tang, D.-L. Luo, S.-P. Li,
CC E
Isolation, Structural Elucidation, and α-Glucosidase Inhibitory Activities of Triterpenoid Lactones and Their Relevant Biogenetic Constituents from Ganoderma resinaceum, Molecules, 23 (2018) 1391.
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[12] R. Schuhmacher, M. Sulyok, R. Krska, Recent developments in the application of liquid chromatography–tandem mass spectrometry for the determination of organic residues and contaminants, Analytical and bioanalytical chemistry, 390 (2008) 253256. 17
[13] C.R. Cheng, M. Yang, Z.Y. Wu, Y. Wang, F. Zeng, W.Y. Wu, S.H. Guan, Fragmentation pathways of oxygenated tetracyclic triterpenoids and their application in the qualitative analysis of Ganoderma lucidum by multistage tandem mass spectrometry, Rapid Communications in Mass Spectrometry, 25 (2011) 1323-1335.
IP T
[14] M. Yang, X. Wang, S. Guan, J. Xia, J. Sun, H. Guo, D.-a. Guo, Analysis of triterpenoids in Ganoderma lucidum using liquid chromatography coupled with
SC R
electrospray ionization mass spectrometry, Journal of the American Society for Mass Spectrometry, 18 (2007) 927-939.
U
[15] W. Ostrowski, B. Swarcewicz, M. Nolka, M. Stobiecki, Differentiation of
N
phenylpropanoid acids cyclobutane-and dehydrodimers isomers in barley leaf cell
A
walls with LC/MS/MS system, International Journal of Mass Spectrometry, 407
M
(2016) 77-85.
ED
[16] J. Kolniak-Ostek, J. Oszmiański, Characterization of phenolic compounds in different anatomical pear (Pyrus communis L.) parts by ultra-performance liquid
PT
chromatography photodiode detector-quadrupole/time of flight-mass spectrometry
CC E
(UPLC-PDA-Q/TOF-MS), International Journal of Mass Spectrometry, 392 (2015) 154-163.
[17] M. Yang, Z. Lu, K. Yu, Q. Wang, X. Chen, Y. Li, X. Liu, W. Wu, D.-a. Guo,
A
Studies on the fragmentation pathways of ingenol esters isolated from Euphorbia esula using IT-MSn and Q-TOF-MS/MS methods in electrospray ionization mode, International Journal of Mass Spectrometry, 323 (2012) 55-62. [18] X.-Y. Guo, J. Han, M. Ye, X.-C. Ma, X. Shen, B.-B. Xue, Q.-M. Che, 18
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IP T
[19] J. Zhao, X.Q. Zhang, S.P. Li, F.Q. Yang, Y.T. Wang, W.C. Ye, Quality evaluation of Ganoderma through simultaneous determination of nine triterpenes and sterols
SC R
using pressurized liquid extraction and high performance liquid chromatography,
A
CC E
PT
ED
M
A
N
U
Journal of separation science, 29 (2006) 2609-2615.
19
Figure captions
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Fig. 1 The chemical structure of 6 type triterpenoids and the R5 side chain.
20
PT
ED
M
A
N
U
SC R
IP T
Fig. 2 Typical fragmentation pathways of type 1-1 triterpenoids.
A
CC E
Fig. 3 Typical fragmentation pathways of type 1-2 triterpenoids.
21
IP T SC R U N A M ED PT
A
CC E
Fig.4 Strategy for triterpenoids resolution.
22
IP T SC R U N A M
A
CC E
PT
ED
Fig.5 UPLC-QTOF-MS chromatogram of G. resinaceum extract.
23
Table 1. Chemical structures of the reference compounds isolated from G. resinaceum. No.
Name
Type
R1
R2
R3
R4
R5
DBa
S1
Lucidone E
1-1
OH
O
OH
H
5
-
S2
(17S,23S,25R)-17,23-epoxy-
1-1
OH
O
OH
H
13
-
3β,15α-dihydroxy-7,11-dioxo5α-lanosta-8-en-26,23-olide Ganoderenic acid I
1-1
OH
O
OH
H
4
-
S4
3β,15α-dihydroxy-7,11,23-
1-1
OH
O
OH
H
7
16, 17
O
H
6
-
trioxo-5α-lanosta-8,16-dien-26-
S5
20-hydroxy-ganoderic acid AM1
1-2
OH
O
S6
Ganoderic acid AM1
1-2
OH
O
S7
3β,24-dihydroxy-7,11, 15,23-
1-2
OH
O
SC R
oic acid
O
H
7
-
O
H
3
-
O
H
5
-
O
H
7
16, 17
O
O
H
4
-
O
O
OH
7
16, 17
tetraoxo-5α-lanosta-8-en-26-oic
S8
Lucidone D
1-2
OH
O
S9
3β-Hydroxy-7,11,15,23-
1-2
OH
O
N
tetraoxo-5α-lanosta-8,16-dien-
Ganoderenic acid H
1-2
OH
S11
3β,12β-dihydroxy-7,11,15,23-
1-2
OH
M
S10
A
26-oic acid
tetraoxo-5α-lanosta-8,16-dien26-oic acid 1-2
OH
O
O
OH
4
-
1-2
OH
O
O
H
2
-
2-1
OH
OH
OH
H
14
-
2-1
OH
OH
OH
H
7
16, 17
ED
3β,12β-dihydroxy-7, 11,15,23-
U
acid methyl ester
S12
IP T
S3
tetraoxo-5α-lanosta-8, 20E-dien26-oic acid
3β, 20-dihydroxy-7, 11, 15,23-
PT
S13
tetraoxo-5α-lanosta-8-en-26-oic acid methyl ester
3β,7β,15α,24-tetrahydroxy-
CC E
S14
11,23-dioxo-5α-27-norlanosta-8ene
S15
3β,7β,15β-Trihydroxy-11,23-
A
dioxo-lanost-8,16-dien-26-oic acid
S16
Lucidone C
2-1
OH
OH
OH
H
5
-
S17
Ganoderenic acid C
2-1
OH
OH
OH
H
4
-
S18
Ganoderic acid C2
2-1
OH
OH
OH
H
7
-
S19
Ganoderic acid L
2-1
OH
OH
OH
H
6
-
S20
3β,7β,15α,24-tetrahydroxy-
2-1
OH
OH
OH
H
11
-
11,23-dioxo-5α-lanosta-8,20E-
24
dien-26-oic acid S21
3β, 7β, 15α, 24-tetrahydroxy-11,
2-1
OH
OH
OH
H
9
-
23-dioxo-5α-lanosta-8-en-26-oic acid S22
ganoderic acid XL1
2-1
OH
OH
OH
H
10
-
S23
Lucidone A
2-2
OH
OH
O
H
5
-
S24
Ganoderic acid B
2-2
OH
OH
O
H
7
-
S25
3β,7β-dihydroxy-11,15,23-
2-2
OH
OH
O
H
7
16, 17
2-2
OH
OH
O
H
acid S26
3β,7β-dihydroxy-11,15,23trioxo-lanost -8,16-dien-2 6-oic
S27
Ganoderic acid I
2-2
OH
OH
S28
3β,7β,20α-trihydroxy-4, 4,14α-
2-2
OH
OH
H
6
-
O
H
15
-
2-2
OH
OH
O
H
13
-
OH
O
H
2
-
O
H
9
-
OH
O
H
11
-
U
pregn-8-ene 3β,7β, 24-trihydroxy-11,15, 23-
2-2
OH
S31
3β,7β,24-Trihydroxy-11,15,23-
2-2
OH
trioxo-5α-lanosta-8-en-26-oic acid S32
3β, 7β, 24-trihydroxy-11, 15, 23-
2-2
OH
M
Methyl ganoderate I
A
acid methyl ester
OH
N
trioxo-5α-lanosta-8-en-26-oic
S30
16, 17
O
trimethyl-11,15- dioxo-5α-
S29
12
SC R
acid methyl ester
IP T
trioxo-lanost -8,16-dien-26-oic
26-oic acid Lucidone F
S34
Lucidone B
3-1
O
OH
OH
H
5
-
3-2
O
OH
O
H
5
-
double bonds.
A
CC E
a DB,
PT
S33
ED
trioxo-5α-lanosta-8, 20E-dien-
25
I N U SC R
Table 2. Compounds identified by the developed strategy. R2
R3
R4
R5
DB
C1
2-1
OH
OH
OH
OH
9
-
C2
1-1
OH
O
OH
OH
9
-
C3
2-2
OH
OH
O
H
16
-
C4
2-2
OH
OH
O
H
9
-
C5
2-2
OH
OH
O
OH
7
-
C6
1-2
OH
O
O
H
16
-
C7
1-2
OH
O
O
H
8
-
C8
2-2
OH
OH
O
OH
1
-
C9
2-2
OH
OH
O
OH
6
-
C10
2-2
C11
2-2
C12
2-1
A
R1
M
Type
PT
ED
No.
OH
O
OH
7
16,17
OH
OH
O
H
11
-
OH
OH
OH
OH
9
16,17
CC E
OH
1-2
OH
O
O
H
11
-
C14
2-2
OH
OH
O
H
11
-
C15
2-2
OH
OH
O
OH
7
-
C16
2-1
OH
OH
OH
H
11
-
C17
1-1
OH
O
OH
OH
7
16,17
C18
1-2
OH
O
O
OH
7
-
C19
2-1
OH
OH
OH
H
11
-
C20
1-2
OH
O
O
H
8
-
C21
1-2
OH
O
O
OH
7
16,17
A
C13
a DB,
double bonds. 26
I N U SC R
Table 3. The MS data of the triterpenoids from G. resinaceum by UPLC-QTOF-MS analysis. [M-H]Peak
Compounda
RT (min)
Formula
Experimental
No.
Theoretical mass
4
5
C2
S28 C3
6.8
7
8
A
9
10
C4
C5
403.2492
7.6
9.1
9.1 9.8
CC E
6
S19
549.3061
10.3
10.8
549.3078
M
3
S16
5.3
533.3122
ED
2
C1
PT
1
A
mass
547.2914
403.2492
403.249 533.312
Fragments(m/z)
Error, ppm
549.3069 (8.09b), 505.3165 (10.94), 487.3047 (11.63), 350.1725 (21.95), 319.1911 1.5
C30H45O9-
0.6
-
403.2492 (6.35), 387.2181 (2.17), 369.2112 (2.83), 303.1975 (100), 285.1863 (9.58)
-
533.3133 (0.7), 515.3004 (2.36), 403.2524 (3.29), 303.1967 (100), 285.1884 (2.68)
(13.83), 317.1749 (18.57), 303.1959 (100), 249.1501 (2.55) C24H35O5
0.4
C30H45O8
547.2934 (12.51), 529.2803 (12.59), 503.3012 (58.12), 485.2905 (54.58), 467.2787 547.2913
-
0.3
C30H43O9
(7.75), 389.2331 (30.37), 371.2224 (93.60), 348.1576 (42.93), 317.1754 (70.18), 315.1603 (96.24), 301.1442 (100), 287.1651 (5.80) 403.2492 (32.05), 385.2384 (4.50), 359.2231 (27.77), 341.2137 (21.55), 329.1761
403.249
0.6
C24H35O5-
(16.29), 303.1968 (45.79), 287.1651 (17.42), 285.1856 (9.14), 249.1496 (100) 547.292
547.2913
1.4
C30H43O9
531.2972
531.2963
1.6
C30H43O8-
485.2915 (5.29), 401.2333 (100), 303.1964 (8.74), 249.1503 (11.34) 531.2974 (100), 513.2854 (10.60), 495.2749 (15.75), 469.2958 (81.10), 369.436 (20.39), 303.1965 (27.91), 249.1494 (7.99)
531.2973
531.2963
0.6
C30H43O8-
531.2966 (100), 513.2859 (67.02), 495.2749 (5.63), 451.2851 (7.45)，399.2541 (22.16), 319.1913 (9.18), 303.1964 (5.58), 193.0888 (3.28)
C6
11.1
545.2759
545.2756
0.5
C30H41O9
C7
12
527.2653
527.265
1
C30H39O8-
525.2593 (4.42), 399.2175 (55.84), 355.2273 (7.95), 301.1809 (100) 527.2653 (90.19), 509.2547 (98.96), 483.2754 (97.86), 465.2646 (58.01), 453.2280 (21.74), 317.1762 (100), 301.1804 (5.44) 547.2913 (2.08), 503.3011 (9.22), 485.2906 (100), 467.2796 (28.02), 441.2279 (9.7),
11
C8
12.6
547.292
547.2913
-
1.3
C30H43O9
401.2329 (16.52), 319.1916 (10.91), 305.1756 (50.77), 303.1602 (15.34), 303.1959 (22.51), 287.1645 (52.55), 265.1443 (17.08), 249.1500 (3.56)
27
15
S21
C9
531.2959
13.1
401.2321
13.3
533.3129
N U SC R
I 14
S23
12.9
531.2963
401.2333
13.6
547.2912
547.2913
0.1
(6.59), 303.1964 (100), 301.1809 (33.58), 287.1683 (10.10), 249.1531 (17.16) 401.2321 (29.55), 383.2215 (10.06), 371.1851 (5.64),357.2065 (4.62), 343.1895
C24H33O5-
3
1.7
533.312
487.3058 (7.56), 469.3006 (12.95), 425.2738 (6.05), 385.2395 (3.59), 383.2589
C30H43O8-
0.7
A
13
S20
M
12
(7.63), 303.1956 (100), 287.1642 (8.10), 285.1858 (14.51), 249.1486 (49.31) 489.323 (23.64), 471.3125 (31.17), 453.3013 (4.04), 334.1791 (14.36), 303.1971
C30H45O8(53.34), 301.1814 (100), 287.1654 (86), 249.1491 (3.59) 529.2794 (74.62), 511.2691 (100), 493.2588 (29.99), 467.2785 (25.69), 417.2276 C30H43O9-
(65.67), 399.2175 (54.57), 319.1911 (14.48), 303.1956 (20.37), 303.1619 (8.37),
17
S22
C11
13.9
14
14.8
CC E
18
C10
PT
16
ED
301.1803 (62.87), 265.1445 (6.18) 529.2797
517.3169
529.2801 (4.47), 511.2693 (100), 467.2787 (10.14), 401.2340 (1.2), 319.1905 (3.44), 529.2807
C30H41O8-
1.9
303.1952 (1.6), 303.1596 (0.8), 301.1797 (2.46), 265.1439 (5.36) 517.317 (100), 473.3267 (16.02), 455.3158 (5.38), 361.2382 (43.70), 343.2277 517.3171
C30H45O7-
0.3
251.1645 (7.76), 249.149 (5.89) 513.2869 (3.33), 487.3064 (24.70), 469.2953(55.81), 451.2851 (12.01), 383.2583 531.2956
531.2963
C30H43O8-
1.3
(31.58), 303.1963 (100), 249.1492 (95.32) -
19
S1
15
401.2339
401.2333
1.5
C24H33O5
20
S8
15.2
399.2158
399.2158
4.8
C24H31O5-
21
S15
15.31
515.3028
515.3014
2.7
C30H43O7-
401.2321 (2.83), 383.2225 (0.25), 301.1776 (100), 245.1517 (7.11) 399.2158 (19.54), 369.1686 (9.28), 313.1430 (15.43), 301.1803 (100), 249.1471 (0.5) 515.3028 (100), 497.2873 (58.71), 435.2873 (14.27), 303.1944 (11.13), 287.1634 (9.06), 249.1473 (9.07)
A 22
(22.74), 303.1963 (75.29), 301.1807 (38.95), 287.201 (23.74), 287.165 (32.52),
531.513.2846 (12.35), 495.2738 (25.36), 469.2946 (44.85), 451.2840 (33.37), S27
15.8
531.295
531.2963
-
2.5
C30H43O8
401.232 (100), 303.1958 (33.58), 287.1639 (3.42), 285.1861 (3.77), 275.1642 (9.13), 249.1496 (28.40)
-
23
S33
15.9
401.2333
401.2333
0.1
C24H33O5
24
S5
16.9
529.2821
529.2807
2.6
C30H41O8-
401.2333 (8.24), 385.2007 (3.3), 367.1901 (4.41), 301.1797 (100), 283.1688 (9.66) 511.2711 (4.42), 493.2612 (18.26), 467.2811 (67.29), 449.2698 (27.72), 419.2237 (15.50), 399.2203 (37.7), 301.1803 (100), 287.1661 (3.13), 249.1464 (6.31)
28
28
S31
C12
527.2716
18.6
529.2807
19.3
531.2969
N U SC R
I 27
S32
18.5
527.2709
529.2807
19.4
547.2915
547.2913
0.4
301.1817 (1.3), 303.1624 (0.8), 287.1684 (0.12) 485.291(19.09), 467.2804 (100), 449.2696 (16.48), 385.2388 (43.04), 303.1968
C30H41O8-
1
0.4
531.2963
509.2604 (78.79), 479.2134 (100), 467.2489(4.60), 435.2229 (8.37), 317.1811 (6.82),
C23H43O13-
1.8
A
26
S11
M
25
(25.38), 287.1654 (16.05), 285.1865 (11.06), 249.1499 (62.15) 487.3065 (4.5), 469.2968 (62.11), 425.2342 (100), 303.1963 (2.45), 289.1811
C30H43O8(79.78), 287.1653 (15.28), 249.1499 (27.56) 503.3020 (100), 473.2544 (11.38), 319.1913 (1.7), 303.1958 (2.2), 303.1601 (0.9), C30H43O9301.1807 (3.92)
C14
21.2
21.7
CC E
31
S17
20.2
PT
30
C13
32
33
A
34
S14
S18
S12
ED
483.2757 (9.29), 465.2643 (72.5), 453.2278 (22.46), 435.2177 (27.03), 397.2383 29
23.4
23.9
24.5
527.2653
515.3022
527.265
C30H39O8-
0.9
(22.87), 383.2224 (19.60), 301.1818 (100), 249.1490 (4.04) 515.3019 (100), 497.2911 (10.35), 453.3007 (6.24), 383.2599 (11.61), 303.1968 515.3014
C30H43O7-
1.1
(54.69), 287.1653 (43.14), 249.1501 (3.87), 193.0898(35.29) 511.2698 (11.78), 485.2912 (19.98), 467.2809 (59.30), 438.2408 (13.08), 385.2384
529.281
529.2807
C30H41O8-
0.6
(9.63), 303.1960 (100), 249.1499 (48.55) 489.3211 (15.30), 471.3067 (13.65), 303.1932 (50.49), 287.1986 (10.82), 249.1475 489.3210
489.3222
C29H45O6-
2.5
(8.4) 517.3172 (98.57), 499.3066 (100), 481.2954 (2.63), 455.3165 (14.84), 437.3058 517.318
517.3171
C30H45O7-
1.6
(6.15), 303.1961 (14.31), 301.1805 (25.38), 287.1645 (33.07), 249.1483 (1.6) 509.2551 (100), 479.2082 (61.35), 465.2653 (30.47), 435.2181 (9.81), 397.2338 527.2661
527.2650
-
2.0
C30H39O8
(8.58), 385.2386 (20.72), 362.1369 (20.97), 345.1703 (14.25), 317.1752 (14.01), 303.1603 (9.3), 301.1802 (9.14), 249.1489 (5.74) 531.2963 (100), 513.2852 (36.61), 469.2952 (15.38), 423.2896 (2.04), 391.1762
35
C15
25.5
531.2963
531.2963
-
1
C30H43O8
(3.86), 363.1807 (2.12), 319.1911 (17.2), 301.1807 (14.51), 287.165 (3.8), 265.1441 (6.17)
36
C16
26.6
531.2968
531.2963
-
0.8
C30H43O8
29
487.3060 (4.79), 303.1967 (100)
40
C18
C19
529.2807
29.4
511.2713
29.8
531.2952
N U SC R
I 39
S9
27.4
529.2807
511.2701
32.4
531.2957
531.2963
1.3
(3.82), 301.1813 (2.86) 493.2605 (16.1), 467.2811 (100), 449.2703 (21.2), 419.2233 (20.88),
C30H39O7-
2.1
2.2
531.2963
511.2707 (100), 481.2231 (18.32), 467.2807 (67.87), 437.2333 (14.57), 303.1601
C30H41O8-
0
A
38
C17
M
37
384.2307(18.20), 369.2074 (8.99), 301.1809 (3.2), 287.165 (1.0), 249.1498 (1.0) 513.2851 (100), 469.295 (35.71), 451.2841 (11.91), 425.2327 (4.17), 319.1925
C30H43O8(9.45), 301.1805 (26.44), 265.144 (40.25), 249.149 (7.92) 519.2948 (3.74), 487.3057 (36.49), 457.2589 (42.17), 439.2483 (3.05), 401.2684 C30H43O8(8.75), 303.1960 (100)
43
C20 C21
33.1
34.1 34.1
CC E
44
S34
33.1
PT
42
S24
45
A
46
47
S10
S6
S30
ED
497.2862 (100), 453.2970 (79.54), 303.1938 (55.90), 287.1639 (7.48), 285.1830 41
34.8
35
36.7
515.3009
399.2197
515.3014
C30H43O7-
1
(21.85), 249.1468 (43.54) 399.2178 (12.18), 369.1702 (4.60), 341.1753 (5.99), 301.1804 (100), 283.1694 399.2177
5
C24H31O5(5.99), 247.1328 (11.80) 493.2585(24.05), 467.2795 (100), 449.2688 (44.04), 419.2219 (29.37), 384.2293
511.2695
511.2701
C30H39O7-
1.2
(20.7), 369.2064 (9.36), 301.1804 (88.75), 249.1493 (26.93) 529.28
529.2807
1.4
C30H41O8-
513.2862
513.2858
1.2
C30H41O7-
511.2688 (100), 449.2687 (7.45), 384.2297 (3.99), 301.1812 (14.77), 265.1434 (4.65) 513.2855 (4.6), 451.2853 (26.29), 436.2619 (100), 301.1819 (27.42), 287.1650 (2.12), 249.15 (7.17) 513.2856 (5.41), 495.2762 (7.10), 451.2889 (31.33), 436.2663 (100), 301.1851
513.2867
513.2858
C30H41O7-
2.1
(32.00), 287.1673 (2.44), 249.1454 (8.78) 527.296 (2.56), 509.2514 (19.74), 491.2375 (5.53), 453.2244 (27.89), 435.2129, 545.3072
545.3061
-
2.3
C38H41O3
401.2298 (100), 303.1961 (18.35), 301.1792 (10.78), 285.1831 (2.42), 249.1481 (22.01) 527.2998 (3.96), 513.2842 (6.39), 495.2741 (3.21), 439.25 (29.97), 425.2328 (100),
48
S29
36.8
545.3113
545.312
C31H45O8-
1.1
303.1951 (1.31), 287.1641 (6.04), 249.1491 (3.45) 495.2712 (100), 480.2473 (7.67), 465.2239 (4.99), 451.2815 (12.28), 436.258 (7.74), 49
S25
37.1
513.2844
513.2858
C30H41O7-
2.6
409.2345 (4.18), 303.1967 (trace), 249.1471 (5.60)
30
I S3
38
543.2971
39
513.2851
543.2963
513.2858
S7
M
52
39.4
543.2956
543.2956
511.2701 (354604), 493.2598 (28.02), 399.2176 (8.26), 301.1811 (100), 287.1645
C31H43O8-
2.6
-
1.3
C30H41O7
A
51
S13
N U SC R
50
(0.4), 249.1493 (1.0) 513.2849 (91.23), 465.2289 (100), 451.2857 (80.26), 435.2561 (13.64), 421.2390 (40.16), 399.2541 (2.78), 381.2445 (9.69), 351.1983 (5.08), 326.1902 (8.22), 303.1957 (52.13), 301.1817 (80.47), 287.1640 (5.44), 193.0883 (14.93) 543.2958 (6.84), 511.2694 (70.58), 455.2442 (86.03), 437.2329 (100), 423.2186
C31H43O8-
1.4
(20.31), 385.2400 (9.84), 303.1964 (4.7), 301.1802 (6.4), 287.1650 (7.96), 249.1495 (11.77)
54
a
S2
48.2
50
513.2860
527.3066
513.2858
C30H41O7-
0.4
(5.25), 351.1965 (21.75), 303.1960 (1.2), 301.1803 (2.41), 287.1647 (0.37), 193.0886 (1.3) 527.3031 (100), 512.279(31.19), 495.2764 (73.01), 465.2291 (4.02), 383.2249
527.3073
C24H47O12-
1.2
(62.38), 303.1969 (1.5), 287.1653 (1.6), 249.1499 (11.22) 513.2846
513.2858
C30H41O7 -
2.4
513.2845 (90.48), 495.2733 (26.60), 469.2939 (6.06), 451.2833 (14.01), 381.2426 (91.75), 301.1800 (93.13), 289.1791 (5.7), 287.1638 (3.1), 193.0862 (8.14)
the compound codes are same with table 1 and table 2. abundance.
A
b
S26
41.4
CC E
55
S4
PT
53
ED
513.2860 (3.85), 483.2391 (29.72), 421.2386 (100), 381.2360 (4.81), 369.2071
31