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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 46  |  Page : 145-152  

Application of ultra-performance liquid chromatography with time-of-flight mass spectrometry for the rapid analysis of constituents and metabolites from the extracts of Acanthopanax senticosus Harms leaf


Department of Pharmaceutical Analysis, National TCM Key Laboratory of Serum Pharmacochemistry, Laboratory of Metabolomics and Chinmedomics, Heilongjiang University of Chinese Medicine, Harbin 150040, China

Date of Submission13-Mar-2015
Date of Decision02-Apr-2015
Date of Web Publication2-Mar-2016

Correspondence Address:
Xijun Wang
Department of Pharmaceutical Analysis, National TCM Key Laboratory of Serum Pharmacochemistry, Laboratory of Metabolomics and Chinmedomics, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1296.177902

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   Abstract 

Acanthopanax senticosus (Rupr and Maxim) Harms (AS), a member of Araliaceae family, is a typical folk medicinal herb, which is widely distributed in the Northeastern part of China. Due to lack of this resource caused by the extensive use of its root, this work studied the chemical constituents of leaves of this plant with the purpose of looking for an alternative resource. In this work, a fast and optimized ultra-performance liquid chromatography method with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) has been developed for the analysis of constituents in leaves extracts. A total of 131 compounds were identified or tentatively characterized including triterpenoid saponins, phenols, flavonoids, lignans, coumarins, polysaccharides, and other compounds based on their fragmentation behaviors. Besides, a total of 21 metabolites were identified in serum in rats after oral administration, among which 12 prototypes and 9 metabolites through the metabolic pathways of reduction, methylation, sulfate conjugation, sulfoxide to thioether and deglycosylation. The coupling of UPLC-QTOF-MS led to the in-depth characterization of the leaves extracts of AS both in vitro and in vivo on the basis of retention time, mass accuracy, and tandem MS/MS spectra. It concluded that this analytical tool was very valuable in the study of complex compounds in medicinal herb.

Keywords: Acanthopanax senticosus (Rupr. and Maxim) Harms, chemical constituents, extracts, metabolites, Traditional Chinese herbal medicine, ultra-performance liquid chromatography method with quadrupole time-of-flight mass spectrometry


How to cite this article:
Zhang Y, Zhang A, Zhang Y, Sun H, Meng X, Yan G, Wang X. Application of ultra-performance liquid chromatography with time-of-flight mass spectrometry for the rapid analysis of constituents and metabolites from the extracts of Acanthopanax senticosus Harms leaf. Phcog Mag 2016;12:145-52

How to cite this URL:
Zhang Y, Zhang A, Zhang Y, Sun H, Meng X, Yan G, Wang X. Application of ultra-performance liquid chromatography with time-of-flight mass spectrometry for the rapid analysis of constituents and metabolites from the extracts of Acanthopanax senticosus Harms leaf. Phcog Mag [serial online] 2016 [cited 2018 Sep 19];12:145-52. Available from: http://www.phcog.com/text.asp?2016/12/46/145/177902

Highlight of Paper

  • A fast UPLC-QTOF-MS has been developed for analysis of constituents in leaves extracts
  • A total of 131 compounds were identified in leaves extracts
  • A total of 21 metabolites including 12 prototypes and 9 metabolites were identified in vivo.


Summary

  • Constituent's analysis of Acanthopanax senticosus Harms leaf by ultra-performance liquid chromatography method with quadrupole time-of-flight mass spectrometry.





   Introduction Top


Acanthopanax senticosus (Rupr and Maxim.) Harms (AS) is a commonly used traditional Chinese herbal medicine (TCHM), which is widely distributed in the Northeastern part of China. The root and stem of AS are called “Ciwujia” in China, and it has long been widely used as tonifying and replenishing, heart-nourishing tranquilizing medicinal. In recent years, a great number of chemical, pharmacological, and clinical studies on AS has proved that it has the effect on immune regulation,[1] antistress,[2] antifatigue,[3] antitumors,[4] and treating cerebrovascular diseases.[5] However, because of the extensive use of root, the Acanthopanax plant resources are gradually exhausted and even cause the ecosystem crisis. Hopefully, the effect of leaves drew more and more attention over the years, not only because they may have the similar effects on the root but also the leaves have the ability of regeneration, which could be a substitutable resource.

Screening and identification of chemical constituents in TCHM are the first and indispensable steps of the development of TCHM.[6] However, for TCHM, the precise characterization of components can be considered as a challenge because of its complexity and variability. LC-mass spectrometry (MS)/MS has proved to be a very powerful tool in profiling of natural products with the advantages of its high resolution, high sensitivity, and accurate mass measurement.[7] Moreover, MS/MS offers unique structure identification capabilities that allow for the characterization of the components in mixtures directly and quickly without the reference standards. At the same time, this method also avoids the tedious and difficult ask of isolation, separation, and purification of substances whose structures are similar and even which are present in only trace amounts, which is obviously superior to other traditional methods.[8],[9]

It is a major blockage to understanding and revealing the mystery of herbal medicines due to the lack of awareness in effective material basic and biological disposition. Information on identification of metabolites and metabolic fate of natural compounds in vivo is a key part of the equation in elucidating the effective constituents and understanding their potential effects.[10] Fortunately, serum pharmacochemistry has played an important role in explicating the effective constituents as well as their metabolites in vivo.[11] It has been recognized as an indispensable part in evaluating drug safety, efficacy, and drug-drug interactions in the whole pipeline of drug discovery and development.[12] Besides principal component analysis (PCA), partial least squared discriminant analysis (PLS-DA), and orthogonal projection to latent structure (OPLS-DA) are also developed to identify potential marker compounds.

Although comprehensively analyzing the chemical constituents having the medicinal and therapeutic potential of the natural plant is very crucial, the leaves of AS has not been studied in considerable details in this respect. Hence, the aim of our work here was to undertake a comprehensive characterization of leaves extracts of AS in order to get an in-depth knowledge of the active ingredients and identify compounds circulating in the blood stream by ultra-performance liquid chromatography method with quadrupole time-of-flight MS (UPLC-QTOF-MS).


   Experimental Top


Chemicals and materials

Acetonitrile (ACN) (high-performance liquid chromatography [HPLC] grade) was purchased from Merck (Darmstadt, Germany). Distilled water was further purified by a Milli-Q system (Millipore, Bedford, MA, USA). Formic acid (HPLC grade) was purchased from Tianjin Kermel Reagent Company (Tianjin, China). The OASIS HLB SPE C18 columns (6cc, 200 mg) were purchased from Waters (Milford, MA, USA). Leucine enkephalin was purchased from Sigma-Aldrich (MO, USA). The dried leaves of A. senticosus (Rupr. and Maxim.) Harms were purchased from Qinghe Forestry Bureau (Heilongjiang, China), and authenticated by Prof. Xijun Wang, Department of Pharmacognosy of Heilongjiang University of Chinese Medicine.

Ultra-performance liquid chromatography-mass spectrometry conditions

Separation and detection of the components was performed on a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA) coupled with a Waters Synapt High Definition TOF Mass system (Waters Corp., Milford, USA) equipped with the electrospray ionization. Chromatographic separations were achieved on an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm i.d., 1.8 um, Waters Corp.) at 40°C and the flow rate of the mobile phase was 0.50 mL/min. Mobile phase A consisted of 0.1% formic acid in ACN while mobile phase B consisted of 0.1% formic acid in water. The column was eluted with a linear gradient of 1–9% A over initial to 2.0 min, 9–20% A over 2.0–11.0 min, 20–45% A over 11.0–19.0 min, 45–100% A over 19.0–22.0 min.

The mass spectrometric full-scan data were acquired in the negative ion by V mode from 50 to 1500 Da with a 0.3 s scan time. Other conditions were as follows: Capillary voltage of 2.4 kV, sample cone voltage of 35 V, extraction cone voltage of 3.5 V, desolvation temperature of 300°C, source temperature of 110°C, cone gas flow of 50 L/h and desolvation gas flow of 650 L/h for negative ion mode. Data were centroided and mass was corrected during the acquisition using an external reference (Lock-Spray ) consisting of a 200 pg/mL solution of leucine enkephalin infused at a flow rate of 0.1 mL·min − 1 via a lockspray interface, generating a reference ion for negative ion mode ([M-H] = 554.2615 Da) to ensure accuracy during the MS analysis.

Preparation of sample solutions

The dried leaves of AS were crushed and was immersed 10 times with water for 2 h and then extracted by heating reflux for 2 h 2 times. The extract was merged and evaporated by rotary evaporation under vacuum. The residue was then freeze-dried. The dried powder of the leaves extracts (0.2 g) was accurately weighed and dissolved with 10 mL of 30% v/v methanol. After extracting in an ultrasonic bath for 30 min at room temperature, the solution was centrifuged at 13,000 rpm for 15 min, and the supernatant filtered through a 0.22 μm filter membrane before injecting 3 μl for UPLC-QTOF-MS analysis.

Preparation of drug administration and serum samples

Six-week-old male Sprague-Dawley rats were obtained from the Laboratory of Animal Center of the Heilongjiang University of Chinese Medicine. The animals were kept in a room maintained at 23–25°C and 50–60% humidity under a 12-h light/12-h dark cycle of artificial lighting starting at 7:00 h; food and water were available ad libitum. After an acclimation period of 1-week, all the rats were randomly divided into two groups of five rats each group: A Control group and dosed group. Prior to drug administration, the experimental animals were deprived of food for 16 h and were free to access the water. The freeze-dried powder of AS was dissolved in 0.5% CMC-Na to get a concentration equivalent to 0.6 g/ml. The dosed group was orally administered with AS extracts (1 mL/100 g body weight) while the control group received the same volume of 0.5% CMC-Na. The blood samples were collected from a hepatic portal vein at 60 min after the oral administration. and then the rat blood was immediately centrifuged at 4000 rpm for 15 min at 4°C. Forty microliters of phosphoric acid was added to 2.0 mL of the above supernatant and ultrasonicated for 1 min, and then vortexed for the 30s, and the serum samples were prepared using SPE column as follows: OASIS HLB SPE C18 columns previously activated using 3 mL of methanol and equilibrated with 3 mL water, successively. Then, 100% methanol was eluted, and the eluate was collected and evaporated to dryness under a gentle stream of nitrogen at 40°C. The residue was reconstituted in 100% methanol and vortex-mixed for 30 s, centrifuged at 13,000 rpm for 15 min at 4°C. A 5 μL aliquot of the solution was injected into the UPLC-QTOF-MS for analysis.

Data processing

All LC/MS data including retention time, accurate mass, and MS/MS spectra were acquired in the centroid mode by MarkerLynx software MassLynx V 4.1 software with QuanLynx program (Waters Corp., Milford, MA, USA). All mass spectra were aligned with mass tolerance of 0.02 Da and retention time window of 0.20 min. The noise elimination level was 6. Ion identification was based on the tR, m/z, and MS/MS spectra. The three-dimensional were introduced into the EZinfo 2.0 software (Waters Corp, Milford, MA, USA) for PCA orthogonal partial least-squares-discriminate analysis (OPLS-DA) with the purpose of visualizing discrimination between the dosed and control groups. The S-plot showing the combined covarianceP (1) and correlationP (corr) from the PLS-DA model was used to visualize the metabolites contributing to the discrimination.


   Results and Discussion Top


To characterize the chemical constituents of leaves extracts, a UPLC-QTOF-MS method was established [Figure 1]. Structures of chemical compounds were characterized or tentatively characterized by comparing their chromatographic and spectrometric data with authentic standards or literature data. Ultimately, a total of 131 compounds [Table S1] were identified or tentatively characterized including triterpenoid saponins, phenols, flavonoids, lignans, coumarins, polysaccharides, and other compounds. Flavonoids are very important bioactive constituents widely found in leaves of AS. Among these, rutin (60), hyperin (63), and quercitrin (74) are the most cited in the literature.[13],[14],[15]
Figure 1: Liquid chromatography-mass spectrometry chromatograms for the leaves extracts of Acanthopanax senticosus

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Table S1: Compounds identified in leaves extracts by UPLC-QTOF-MS in the negative ESI mode

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Compound 60 was characterized as rutin. For it, the [M-H] precursor ion at m/z 609 gave one prominent fragment ion at m/z300 [M-H-Rha-Glu] and subsequent fragmentation patterns with ions at m/z 271, 255, 243, and 150, whose fragmentation pathway was supported by comparing with its pure standard. For compound 63, the MS/MS spectrum also displayed a main peak at m/z 300, obviously by the elimination of a galactose unit (180-H2O = 162 Da) to yield the [M-H] ion of the aglycone quercetin. The presence of hyperoside in leaves of AS has been reported before, together with its mass spectrum of pure standard, the compound 59 was identified as being hyperoside. Compound 74 corresponded to quercitrin, the structure and fragmentation pathway of which are in complete agreement with that of the quercitrin standard sample. Flavonoids found in leaves extracts were characterized with typical anions of basic parent structures at m/z 300, 284 and 314 represented aglycone quercetin, kaempferol, isorhamnetin, respectively.

Eleutheroside E has been widely reported to be the key constituent of AS. For compound 58, the MS/MS spectra displayed exactly the same fragmentation patterns as those of the pure standard of Eleutheroside E. It had [M-H] at m/z 741 which gave rise to an ion at m/z 417 by losing two galactose units (162 Da). Compound 80, giving the [M-H] at m/z 417, exhibited the similar MS/MS behavior as compound 58, while its retention time was later than that of compound 58. Above those, compound 80 was proposed as syringaresinol. Triterpenoid compound are another kind of main ingredients in AS, which are mainly pentacyclic triterpene compounds such as oleanane-type and lupane-type. In the structure of triterpenoid saponins, sugar moieties are generally linked at C-3 or C-28 of their parent structures.[16]

Compound 131 gave an [M-H] ion at m/z 733, which exhibited the fragment, typical of glycosyl derivatives (loss of terminal sugars) with ions at m/z 587 [M-H-Rha] and m/z 455 [M-H-Rha-Ara]; this latter ion corresponded to the [M-H] ion of the aglycone oleanane. Based on these, compound 130 was proposed as Eleutheroside I. Compound 109 showed an [M-H] ion peak at m/z 1245, in accordance with an empirical molecular formula of C60H94O27. The fragmentation patterns in the negative-ion MS/MS of it indicated loss of ester-linked sugar chain at C-28 (m/z 733 [M-C20H33O15]). Combined with the previous literature, compound 109 was inferred as Acanthopanaxoside A. Compound 105 had an [M-H] at m/z 1187, whose empirical molecular formula was C58H92O25. Upon MS/MS fragmentation, it yielded ions at m/z 717 by losing a fragment of mass 469, which was another ester-linked sugar chain at C-28 and suggested the presence of one rhamnopyranosyl moiety and two glucopyranosyl moieties.

AS contains a large number of phenolic compounds, most of which exists as isomers. Compound 19, 27, and 28 had the same precursor ion at m/z 353 and the product ions of them were similar, among which the most significant difference is a change in base peak m/z 191 and m/z 179. After compared with pure standards, the three compounds were identified as neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid.

Compared with other kinds of compounds, only a few coumarin compounds have been reported from this plant. Isofraxidin is not only one of the main bioactive constituents but also regarded as the ideal marker compound for the quality assurance of this plant. As compound 38, its [M-H] at m/z 221 produced MS/MS daughter ions at m/z 206, 191, which indicated loss two methyl moieties in sequence. Then, losing a 28 atomic mass unit, ion m/z 191 produced ion at m/z 163. From this information, we could conclude that compound 38 was isofraxidin and this assignment was supported by comparing its MS/MS spectra with those of a pure standard.

Global metabolite identification of complex compounds of herbal medicine in biological systems is a very challenging task. Using the optimal reversed-phase UPLC-MS conditions, all the data containing the retention time, peak intensity, and exact mass were imported in the Masslynx™ software for multiple statistical analyzes [Figure 2]. As an unsupervised pattern recognition method, PCA can effectively identify the differences between the control and dosed group, which indicated that these differences were caused by some exogenous constituents absorbed in serum after dosing [Figure 3]. Combining the results of the S-plot with VIP value from PLS-DA, significant difference points were selected [Figure 4]. However, these difference points were not all exogenous constituents, including some endogenous ones caused by the drug. For screening exogenous constituents absorbed in serum, the trend plot was used, displaying the ions only existed in the dosed group, taking the [M-H] ion at m/z 609 ((tR = 8.23) for example, shown in [Figure 4]c. By means of the above analysis, 12 prototype constituents were finally identified in rat serum after an oral administration leaves extracts.
Figure 2: Liquid chromatography-mass spectrometry chromatograms of (a) control rat serum. (b) Dosed rat serum

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Figure 3: (a) Principal component analysis score plot for the control and dosed group. (b) Three-dimensional-principal component analysis plot for the control and dosed group

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Figure 4: (a) S-plot of OPLS-discriminant analysis result for control and dosed group in negative mode. (b) VIP value result for control and dosed group in negative mode. (c) The trend plot of 8.23–609.1529

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The main chemical constituents of leaves extracts of AS are composed of glycosides, including flavonoid glycosides, triterpenoid glycosides, and so on. However, these glycosides with the ability of water solubility are not easily absorbed in the intestines, and their biological availabilities are lower. Therefore, the deglycosylation process of plant glycosides is crucial for its pharmacological expression. These glycosides are generally hydrolyzed into active aglycones and then absorbed by intestinal flora.[17] In this study, we found flavonoid aglycones and triterpenoid aglycones, which were produced by these compounds with the same basic parent structure [Table S2].
Table S2: Identification of serum from leaves extracts-administrated-rats analyzed using UPLC-QTOF-MS

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   Conclusion Top


A simple, reliable, and sensitive method was developed to separate and identify chemical compounds of leaves extracts of AS by UPLC-QTOF-MS. By using this method, 131 compounds have been characterized or tentatively characterized including triterpenoid saponins, phenols, flavonoids, lignans, coumarins, and polysaccharides. The experimental results, therefore, demonstrate that UPLC-QTOF-MS is a powerful analytical tool in the study of chemical compounds of herbal medicine. It also set a good example for the rapid identification of bioactive constituents in plant extracts and made it possible to fulfill the requirements for a modern drug with characters of safety, efficacy, and stability.

Acknowledgments

This work was supported by grants from the Key Program of Natural Science Foundation of State (Grant No. 81430093, 81173500, 81373930, 81302905, 81202639), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (Grant No. 2011BAI03B03, 2011BAI03B06, 2011BAI03B08), National Key Subject of Drug Innovation (Grant No. 2009ZX09502-005), Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20122327120006), Fund Project of Heilongjiang Provincial Department of Education (Grant No. 12521498), Natural Science Foundation of Heilongjiang Province of China (H2015038).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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   Authors Top


Professor Xijun Wang, is a vice president of the Heilongjiang University of Chinese Medicine, China. He has published 150 articles in peer-reviewed international journals. His research directions focus on Serum pharmacochemistry of TCM, Metabolomics, and Chinmedomics studies. As the project leader, he has put forward academic ideas and research design of Serum pharmacochemistry of TCM. Integrated metabolomics with Serum pharmacochemistry of TCM, he is particularly involved in the development and establishment of theory and research method of Chinmedomics and their integration in clinical, biomedicine, and TCM studies. His findings have scientific values of mining TCM prescriptions, innovative drug design based on clinical experience, enhancing the academic level and clinical efficacy of Chinese medicine. He presided over the completion of the "Establishment and Application of Serum pharmacochemistry of TCM" won the second prize of 2002 National Science and Technology Progress Award; the completion of the "Prevention and Control Technology of Artificial Cultivation of Medicinal Plants" reward by the second prize of 2009 National Technology Invention; "Chinmedomics study of Chinese medicine formulae" won the first prize of Science and Technology of 2012 China Society of Integrated Traditional Chinese and Western Medicine.


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table S1], [Table S2]



 

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