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Rapid Identification of Chemical Constituents in Artemisia argyi Lévi. et Vant by UPLC-Q-Exactive-MS/MS
Artemisia argyi Lévi. et Vant is a traditional Chinese medicine with a long history, and its buds and seedlings can be used as vegetables. However, the investigations on the chemical constituents of A. argyi are not sufficient. In this paper, ultra-high performance liquid chromatography tandem hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive-MS/MS) was used to identify the chemical constituents of A. argyi. The Q Exactive mass spectrometer was used to collect MS and MS2 data. Finally, 125 compounds were preliminarily identified in A. argyi by comparing the retention time and accurate molecular weight with standard databases such as MZVault, MZCloud, and BGI Library (self-built standard Library by BGI Co., Ltd), including flavonoids, phenylpropanoids, terpenoids, and organic acids.
Artemisia argyi Lévi. et Vant belongs to Asteraceae family, widely distributed in China. A. argyi is a traditional Chinese medicine with a long history, and its buds and seedlings can be used as vegetables. According to flora of China, A. argyi has functions of warming menstrual cycle, removing dampness, dispersing cold, hemostasis, anti-inflammation, relieving asthma, relieving cough, calming fetus and anti-allergy with whole grass as medicine [1, 2]. According to Chinese Pharmacopoeia (2020), Artemisiae argyi folium (A. argyi leaves) has minor poison, its nature and flavour are warm, pungent, and bitter, and its channel tropism is in liver, spleen, and kidney. A. argyi leaves have warm meridian to stop bleeding, disperse cold, and relieve pain and external clearing damp antipruritic effect .
At present, research about chemical constituents of A. argyi was focused on A. argyi leaves. Research showed that the chemical constituents of A. argyi leaves include volatile oil, flavonoids, terpenoids, phenylpropanoids, organic acids, and steroids. Among them, more than 200 volatile oils from A. argyi leaves have been identified, and 182 nonvolatile components have been isolated and identified [2, 4]. Pharmacological studies have shown that A. argyi leaves have many pharmacological effects, such as antibacterial, antiviral, hemostatic, anti-tumor, protecting liver and gallbladder, and anti-oxidation, relieving cough and asthma, analgesia, anti-inflammatory, and immune regulation. Moxibustion has the effect of warming and dispersing cold and treating all diseases [2, 4–6]. With the development of the health industry, more and more attention has been paid to the study of A. argyi leaves and moxa . In recent years, systematic studies on the chemical constituents of A. argyi leaves or A. argyi have gradually increased [7, 8]. With the mature application of LC-MS/MS technology in the rapid identification of plant and food stuff ingredients [9–11], research studies on the rapid analysis of nonvolatile components of A. argyi leaves or A. argyi are gradually increasing [12–15], but there are few identified compounds. Ultra-high performance liquid chromatography tandem hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive-MS/MS) is a new type of liquid chromatography-mass spectrometry developed in recent years, which has the characteristics of high resolution, good quality and precision, and strong qualitative and quantitative ability [16, 17]. Samples can be separated quickly by ultra-high performance liquid chromatography, and accurate molecular weight can be determined by high-resolution mass spectrometry to obtain molecular formula of compounds. Zhumadian region is abundant with A. argyi; however, research about chemical components of A. argyi from Zhumadian region was less. Therefore, in this study, UPLC-Q-Exactive-MS/MS combined with standard substance database was used to rapidly identify the nonvolatile chemical components of A. argyi from Zhumadian region.
2. Materials and Methods
Rotary evaporator (N-1300) was purchased from EYELA. Ultra-performance liquid chromatograph (Waters 2D UPLC) was purchased from Waters, USA. High-resolution mass spectrometer (Q Exactive) was purchased from Thermo Fisher Scientific, USA. Hypersil GOLD aQ column (100 mm × 2.1 mm, 1.9 μm) was purchased from Thermo Fisher Scientific, USA. Low temperature high speed centrifuge (Centrifuge 5430) was purchased from Eppendorf. Vortex finder (QL-901) was purchased from Qilinbeier Instrument Manufacturing Co., Ltd. Pure water meter (Milli-Q) was purchased from Integral Millipore Corporation, USA.
d3-Leucine, 13C9-Phenylalanine, d5-Tryptophan, and 13C3-Progesterone were used as internal standard. Methanol (A454-4) and acetonitrile (A996-4) were both in mass grade, which were purchased from Thermo Fisher Scientific, USA. Ammonium formate (17843–250G) was obtained from Honeywell Fluka, USA. Formic acid (50144–50 mL) was obtained from DIMKA, USA. 95% ethanol (20190320) was obtained from Tianjin Fuyu Fine Chemicals Co., Ltd. Water was supplied by a pure water meter.
A. argyi was collected in July 2020 in Wanhei Village, Shangcai County, Zhumadian City, and identified as the aerial part of A. argyi by professor Li Changqin of Henan University. The specimens (No. 2020-08-10) were saved in National Research and Development Center of Edible Fungi Processing Technology, Henan University.
2.4. Preparation of Sample
10 g of A. argyi powder was accurately weighed and impregnated with 25 times 70% ethanol for 2 times at room temperature, each time for 2 days. The extraction was filtered, and filtrate was combined and concentrated. The extract was added with 70% ethanol to 1 g/mL of the original materials and then reserved. 200 μL was sent to BGI Co., Ltd., for chemical composition identification.
2.5. Chromatographic Conditions
Hypersil GOLD aQ column (100 mm × 2.1 mm, 1.9 μm) was used to do LC-MS experiment. The mobile phase was 0.1% formic acid-water (liquid A) and 0.1% formic acid-acetonitrile (liquid B). The following gradients were used for elution: 0–2 min 5% B; 2–22 min 5%–95% B; 22–27 min 95% B; 27.1–30 min 5% B. The flow rate was 0.3 mL/min, the column temperature was 40°C, and the injection volume was 5 μL.
2.6. Mass Spectrometry Conditions
The mass range was 150–1500, the MS resolution was 70000, the AGC was 1e6, and the maximum injection time was 100 ms. According to the strength of the MS ions, TOP3 was selected for fragmentation. The MS2 resolution was 35000, AGC is 2e5, the maximum injection time was 50 ms, and the fragmentation energy were set as 20, 40, and 60 eV. Ion source (ESI) parameter settings: sheath gas flow rate was 40, aux gas flow rate was 10, spray voltage (|KV|) of positive ion mode was 3.80, spray voltage (|KV|) of negative ion mode was 3.20, ion capillary temp was 320°C, and aux gas heater temp was 350°C.
2.7. Data Analysis
UPLC-MS/MS technology was used to systematically analyze the chemical constituents of A. argyi. High-resolution mass spectrometer Q Exactive (Thermo Fisher Scientific, USA) was used to collect data in positive and negative ion modes, respectively, to improve the chemical constituent coverage. Raw mass spectrum data collected by UPLC-MS/MS were imported into Compound Discoverer 3.1 (Thermo Fisher Scientific, USA) for data processing. It mainly includes peak extraction, retention time correction within and between groups, additive ion merging, missing value filling, background peak labeling, and metabolite identification. Finally, the molecular weight, retention time, peak area, and identification results of the compound were derived. The compounds were identified by comparing the retention time, accurate molecular weight, and MS2 data with standard databases such as MZVault, MZCloud, and BGI Library (self-built standard Library by BGI Co., Ltd).
3.1. Total Ion Chromatogram
3.2. Identification Results of Chemical Composition
In this study, UPLC-Q-Exactive-MS/MS technology was used to rapidly identify the chemical constituents in A. argyi. The identification of compounds was based on the retention time, MS data, and MS2 data compared with the standard database (MZVault, MZCloud and BGI Library (self-built standard Library by BGI Co., Ltd)). Most flavonoids, phenylpropanoids, and organic acids were easily deprotonated and responded in negative ion mode. Most steroids and terpenoids were easily protonated and responded in positive ion mode. The identification results are shown in Table 1. A total of 125 chemical constituents were identified in A. argyi, including 49 flavonoids, 30 organic acids, 13 phenylpropanoids, 9 terpenoids, 5 amino acids, 2 steroids, 1 phenolic acid, 1 alkaloid, and 15 other compounds.
Eupatilin and jaceosidin as flavonoids and chlorogenic acid as phenylpropanoids were the main components of A. argyi, which were often used as the index components for content determination and quality evaluation [18–22], and the content of total flavonoids in A. argyi leaves was as high as 4.48%–11.46% . Flavonoids and phenylpropanoids of A. argyi were also the active ingredients. The flavonoids in A. argyi leaves had anti-tumor, anti-oxidation, anti-platelet aggregation, gastrointestinal smooth muscle protection, and other pharmacological effects. And the phenylpropanoids in A. argyi leaves had pharmacological effects such as antibacterial, anti-inflammatory, antiviral, free radical scavenging, liver protection and gallbladder protection, and lowering blood pressure and blood lipid .
Previous studies showed that the chemical components isolated from the A. argyi leaves include flavonoids, terpenoids, phenylpropanoids, organic acids, steroids, etc. , and the above chemical components were all contained in A. argyi from Zhumadian, indicating that the chemical components in A. argyi from Zhumadian were relatively diverse with good quality. In this study, in addition to the reported flavonoids, eupafolin, eupatilin, jaceosidin, apigenin, kaempferol, luteolin, hispidulin, isoschaftoside, eriodictyol, naringenin, acacetin, and artemetin isolated from A. argyi leaves, A. argyi from Zhumadian contained 37 other flavonoids such as schaftoside, rutin, isovitexin, and taxifolin. These results revealed that there were abundant flavonoids in A. argyi from Zhumadian. A. argyi from Zhumadian contained most of the reported phenylpropanoids identified from A. argyi , such as neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid C, scopoletin, and isofraxidin; besides, A. argyi from Zhumadian also contained other phenylpropanoid compounds, such as 2-hydroxycinnamic acid, esculetin, fraxetin, and 3-coumaric acid. In addition, A. argyi from Zhumadian was rich in terpenoids and organic acids. There were many kinds of active ingredients in A. argyi from Zhumadian, and it was worthy to study systematically to find more natural active ingredients.
Ultra-high performance liquid chromatography (UPLC) has the advantages of high analytical speed, high sensitivity, and solvent saving compared with high-performance liquid chromatography (HPLC) . UPLC is often combined with mass spectrometry for rapid detection of chemical constituents of traditional Chinese medicine. Ultra-high performance liquid chromatography tandem quadruple-time of flight mass spectrometry (UPLC-Q-TOF-MS/MS) and UPLC-Q-Exactive-MS/MS are currently commonly used. Compared to traditional LC-MS/MS, UPLC-Q-Exactive-MS/MS has higher resolution, which can eliminate the interference of sample matrix . Wang et al.  investigated the chemical constituents of A. argyi produced in Nanyang by UPLC-Q-TOF-MS/MS technology, and 23 chemical constituents were identified, including 12 flavonoids and flavonoid glycosides, 9 phenylpropionic acids, and 1 coumarin. Li et al.  used RRLC-TOFMS technology to rapidly identify the chemical components in A. argyi leaves and identified 31 chemical components. In this study, 125 chemical constituents of A. argyi from Zhumadian were preliminarily identified by UPLC-MS/MS. All of them contain isochlorogenic acid C and eupatilin. The chemical constituents identified in this study were relatively comprehensive, which provides a certain reference for the subsequent studies of A. argyi.
In this study, a total of 125 chemical constituents of A. argyi were identified by UPLC-Q-Exactive-MS/MS technology. The UPLC-Q-Exactive-MS/MS technology could be used to quickly and preliminarily identify the chemical constituents of A. argyi, providing a basis for further study on the pharmacological substance basis and resource utilization of A. argyi.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that there are no conflicts of interest regarding this study.
This study was funded by the Basic Project in Science and Technology Agency of Kaifeng City (1908007).
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