The Analysis of Leontopodium leontopodioides (Willd.) Beauv. Chemical Composition by GC/MS and UPLC-Q-Orbitrap MS

Leontopodium leontopodioides (Willd.) Beauv. (L. leontopodioides.) has been used to treat lung diseases in traditional Chinese medicine (TCM). However, a systematic analysis of its chemical components has not been reported so far. In this study, UPLC-Q-Orbitrap MS and GC-MS were applied to investigate the chemical composition of the water extracts and essential oils of L. leontopodioides. UPLC-Q-Orbitrap MS adopts a heating electrospray ionization source, collecting primary and secondary mass spectrometry data in positive and negative ions, respectively, and uses Compound Discoverer 3.2 software to analyze the collected raw data. As a result, a total of 39 compounds were identified from their high-resolution mass spectra in both positive and negative ionization modes, including 13 flavonoids and their glycosides, 15 phenolic acids, 4 oligosaccharides and glycosides, 4 pentacyclic triterpenoids, and 3 other compounds. Among them, 18 chemical components have not been reported in L. leontopodioides. In the GC-MS section, two common organic solvents (n-hexane and diethyl ether) were used to extract essential oils, and the mass spectra were recorded at 70 eV (electron impact) and scanned in the range of 35∼450 m/z. Compounds were identified using NIST (version 2017), and the peak area normalization method was used to calculate their relative amounts. Finally, 17 components were identified in the volatile oil extracted with n-hexane, accounting for 80.38% of the total volatile oil, including monoterpenoids, phenylpropene, fatty acids, and aliphatic hydrocarbons. In the volatile oil extracted with diethyl ether, 16 components were identified, accounting for 73.50% of the total volatile oil, including phenylpropene, aliphatic hydrocarbons, monoterpenoids, fatty acids, and esters. This study was the first to conduct a comprehensive analysis of the chemical composition of the L. leontopodioides water extract and its essential oil, and a comprehensive chemical composition spectrum was constructed, to lay a foundation for its further pharmacodynamic material basis and quality evaluation.


Introduction
As a traditional Chinese medicine, Leontopodium leontopodioides (Willd.)Beauv.has the functions of clearing away the pulmonary heat, relieving cough, and expectorating phlegm and is generally used to treat lung diseases in TCM [1].L. leontopodioides belongs to the Asteraceae family and is a perennial herb with a height of 5∼45 cm, and it is widely distributed in northeast, north, and northwest China and grows in arid grasslands, loess slopes, gravel, and mountain grasslands at an altitude of 100∼3200 m [2], as shown in Figure 1.Previous studies of L. leontopodioides were isolated by chromatographic methods, such as silica gel, ODS, Sephadex LH-20, and HPLC, and identifed by chemical and physical methods, especially spectral analysis [3][4][5].Modern pharmacological studies have shown that L. leontopodioides has anti-infammatory, antibacterial, antioxidant, hypoglycemic, diuretic, and other efects [6][7][8][9].Chen [10] et al. used chemical and spectroscopic methods to study a 70% EtOH extract of the whole plants of Leontopodium leontopodioides (Wild.)Beauv obtained leontoaerialosides A (1), B (2), C (3), D (4), and E (5).Zhao et al. [11] found that chlorogenic acid and ferulic acid are components with obvious antioxidant efects in L. leontopodioides, and most of the chemical components related to antioxidant activity are phenolic acids.Wu et al. [12] obtained an abundant higher monomer compound by silica gel column chromatography and preparative thin-layer chromatography in ethyl acetate parts of the alcohol extract; through analysis of ultraviolet, infrared, hydrogen, and carbon spectrum, it was presumed as parahydroxyl-acetophenone. Gao et al. [13] reported the essential oil from the aerial parts of L. leontopodioides and found that it not only has low antioxidant activity but also possesses a potent antibacterial activity against S. aureus and B. subtilis.Although some chemical components, such as favonoids, phenylpropanoids, phenolics [14], and essential oils, have been isolated from L. leontopodioides, systematic analysis of its chemical components has not been investigated.More importantly, L. leontopodioides has not been recorded in the Chinese Pharmacopoeia.Terefore, it is necessary to carry out a systematic and comprehensive study of the chemical composition in order to elucidate its pharmacodynamic material basis.At present, there is no research on the chemical composition of the water extract and essential oil of L. leontopodioides at the same time.

Experimental
2.1.Sample Preparation.Te whole plant of L. leontopodioides was powdered.Pulverized samples of L. leontopodioides (50g) were accurately weighed, add 15 times of distilled water, decocting 3 times for 1 hour each time, combining the three fltrates, evaporating, drying and weighing to prepare the L. leontopodioides water extrac, and its yield was 27.63%.Fifty grams of crushed L. leontopodioides was precisely weighed, and the supercritical CO 2 extraction method was used for 5 h at a temperature of 45 °C and a pressure of 18 MPa to obtain 1.20 mL of dark green essential oil of L. leontopodioides.
An appropriate amount of L. leontopodioides water extract was weighed, and 1 mL of 80% methanol was added to prepare a solution with a concentration of 10 mg•mL −1 , vortexed, ultrasonicated for 10 min, and centrifuged at 14000 rpm for 10 min.Ten, 0.8 mL of the supernatant was placed in a centrifuge tube and centrifuged again, and the supernatant was placed into a sample bottle for analysis by UHPLC-MS.

Methods.
In this study, water extracts and essential oils of L. leontopodioides were analyzed using UPLC-Q-Orbitrap MS and GC-MS techniques, respectively.Featuring high resolution, high sensitivity, and high speed, ultra-performance liquid chromatography quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS), a cutting-edge molecular separation and determination technique, has been applied to the analysis of various complex samples [15,16].Based on UPLC-Q-Orbitrap MS technology, a rapid identifcation method was established for the chemical composition of the water extract of L. leontopodioides.According to the precise molecular mass and fragmentation information of the compounds, the main compounds were identifed by means of databases and references, and their cracking laws were discussed, which provided a data basis for further elucidating their pharmacodynamic material basis.GC-MS a highly effective and versatile analytical technique is widely used in pharmaceutical industries for analytical research and development, quality control, and quality assurance [17].Te essential oil was extracted by supercritical carbon dioxide extraction and then dissolved in n-hexane and diethyl ether.Te resulting fractions were analyzed by GC-MS, using NIST (version 2017) for similarity search, enabling identifcation of the components, while calculating their relative amounts using peak area normalization.Te instruments and materials used in the experiment are listed in Table 1.

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International Journal of Analytical Chemistry International Journal of Analytical Chemistry Te heating electrospray ionization source (HESI) was used as the ion source to detect the positive and negative ion modes, the positive spray voltage was 3.2 kV, the negative spray voltage was 3.0 kV, the detection method was full MS/ dd-MS2, the sheath gas fow was 40 arb, the auxiliary gas fow rate was 15 arb, the capillary temperature was 320 °C, and the auxiliary gas heater temperature was 350 °C.Te resolution of MS was 70000, the resolution of MS/MS was 17500, and the mass spectrum was recorded with a positive ion spectrum scan range of m/z 100∼500.Unknown compounds were identifed using Compound Discoverer 3.2 software, and mzCloud (https://www.mzcloud.org/)and mzVault (self-built database) were used to identify compounds.

GC-MS Conditions
. Inject 1.0 μL essential oils dissolved in n-hexane/diethyl ether each in splitless mode.An HP-5 ms fused silica capillary column (30 m, inner diameter 0.25 mm, and flm thickness 0.25 μm) was used with helium as the carrier gas, and the oven temperature was increased from 80 °C to 90 °C at a rate of 3 °C/min (maintained for 2 min), 95 °C to 140 °C at a rate of 3 °C/min, 155 °C to 185 °C at a rate of 2 °C/min, and fnally 195 °C at a rate of 5 °C/min (hold for 8 min).Te total run time was 54.5 min, and the ion source temperature was set at 250 °C.Te GC interface temperature was 270 °C.Te mass spectra were recorded at 70 eV (EI) and were scanned in the range 35∼450 m/z.Compounds were identifed using the NIST Chemistry WebBook (https://webbook.nist.gov).

Compounds Confrmation of L. leontopodioides Water
Extract by UPLC-Q-Orbitrap MS.First, samples were injected according to the chromatographic and mass spectrometry conditions, and Compound Discoverer 3.2 software was used to search for the target compound peaks on the collected raw data and screen for compounds with a score greater than 80, and after matching, compounds were obtained, and the secondary fragmentation fragment ion information was analyzed to further accurately identify chemical components.As a result, a total of 39 chemical components were identifed from the water extract of L. leontopodioides, mainly including favonoids, phenolic acids, pentacyclic triterpenes, oligosaccharides, and glycosides.Te total ion chromatogram is shown in Figure 2, and the chemical composition identifcation results are shown in Table 2.

Identifcation of Phenolic Acids.
A total of 15 phenolic acid compounds (peaks 2, 3, 14, 17, 18, 21, 22, 23, 24, 26, 27, 29, 30, 33, and 34) were detected in the extract of L. leontopodioides, and these compounds responded better in negative ion mode.Te excimer ion of peak 21 with a retention time of 19.948 min and a molecular formula of  Relative Abundance   International Journal of Analytical Chemistry

Compound Confrmation of L. leontopodioides Essential
Oil by GC-MS.Samples were injected according to the GC-MS conditions and obtained a total ion chromatogram of volatile components in L. leontopodioides, and the obtained data were searched and matched by the mass spectrometry database of the National Institute of Standards and Technology (NIST 2017), and compounds with similarity scores above 80% were taken into account.After comparing the chemical composition of essential oils extracted with nhexane and diethyl ether, a total of 33 volatile compounds (there were seven identical components) were identifed, as shown in Figure 11 and Tables 3 and 4. Te relative content of each component was estimated by the peak area normalization method.It can be seen from Table 3 that the main components of the volatiles of L. leontopodioides extracted with n-hexane mainly included phenylpropene (64.52%), monoterpenes (10.96%), fatty acids (10.03%), and contained some aliphatic hydrocarbons.Among them, the components with higher content were methylconiferylaldehyde (14.77%), (E)-2,6-dimethoxy-4-(prop-1-en-1-yl) phenol (12.04%), and eugenol (11.51%).As shown in Table 4, the main components included fatty acids (28.99%), phenylpropene (28.37%), aliphatic hydrocarbons, and some esters.Among them, the components with higher content were methylconiferylaldehyde (9.61%), pentadecanoic acid (9.25%), and 8-methylnonanoic acid (8.63%).Furthermore, terpinolene, terpinen-4-ol, c-terpinene, methyleugenol, methylconiferylaldehyde, tetradecanoic acid, and n-hexadecanoic acid were identical components.

Discussion
4.1.UPLC-Q-Orbitrap MS Section.Flavonoids mainly exist in natural plants in the form of free or combined with sugar to form glycosides or in the form of carbon sugars, and they have anti-infammatory, antioxidant, antibacterial, antidiabetic, antihypertensive, and other pharmacological activities [18].Te mass spectrometry fragmentation characteristics of favonoid aglycones were mainly the loss of CO, COO, and CH 3 groups, or the loss of neutral molecules such as H 2 O and the occurrence of reverse Diels-Alder reaction (RDA) fragmentation to form a series of characteristic ion peaks.Flavonoid glycosides frst lose the glycosyl group to form the corresponding aglycone and then further cleave [19,20].In this study, taking luteolin, cynaroside, and astragalin as examples, the cracking rules of favonoids and their   International Journal of Analytical Chemistry glycosides were described, and it was found that the cracking rules of the three were consistent with those reported in the literature [21][22][23].Among the 13 favonoids and their glycosides obtained from the analysis, isoquercitrin, kaempferol, kaempferol-7-O-β-D-glucopyranoside, cymaroside, hyperoside, apigenin-7-O-β-D-glucoside, luteolin, quercetin, and apigenin compounds with previous reports [24][25][26][27][28] on the chemical composition of L. leontopodioides, scutellarein, morin, diosmetin, and astragalin have not been reported.Phenolic acids mainly contain carbonyl, carboxyl, and hydroxyl groups, so neutral fragments of CO, H 2 O, and CO 2 were easily lost in mass spectrometry collisions.Te cracking rules of protocatechuic acid and shikimic acid obtained by database analysis are consistent with those reported in the literature [29,30].Chlorogenic acid, quinic acid, protocatechuic acid, protocatechualdehyde, cryptochlorogenic acid, cafeic acid, p-coumaric acid, isochlorogenic acid B, isochlorogenic acid C, and salicylic acid have been reported [31,32].Moreover, basic research on pharmacodynamics found that protocatechuic acid, protocatechuic aldehyde, chlorogenic acid, and cafeic acid in L. leontopodioides can resist acute infammation [33].Cinnamic acid, citric acid, fumaric acid, and shikimic acid have not been reported in L. leontopodioides in previous research.
Te mass spectrometry fragmentation of pentacyclic triterpenoids was mainly loss of neutral molecules, such as H 2 O and CO, and the occurrence of Diels-Alder reaction, and the oligosaccharide and glycoside mass spectrometry was relatively simple.Te cracking rules of ursolic acid and sucrose were consistent with literature reports [34,35].In previous research, pentacyclic triterpenoids, oligosaccharides, and glycosides have not been reported in L. leontopodioides.Newly discovered pentacyclic triterpenoids have a wide range of pharmacological efects and important biological activities, including anti-infammatory, antibacterial, antiviral, immunomodulatory, blood sugar regulation, blood pressure lowering, and antitumor activities [36].In particular, ursolic acid has the same inhibitory efect on glycosidase in vivo and in vitro and has an obvious hypoglycemic efect [37].Oligosaccharides possess various bio-activities, including immune regulation, antitumor, antioxidation, and anti-infection, and modulate the gut microfora [38].In addition, there has been no research on the chemical composition of L. leontopodioides using UPLC-Q-Orbitrap MS technology at present.UPLC-Q-Orbitrap MS technology adopts full MS/dd-MS2 mode, which greatly shortens the analysis time and can quickly detect multiple chemical components, with its advantages of high separation, high resolution, and high sensitivity, and it can provide accurate mass, elemental composition, mass spectrometry fragments, and other information required for the structural characterization of compounds without the need for reference substances.Ten, the possible structure of the compound can be speculated for rapid qualitative analysis.Tis study collects data in both positive and negative ion modes to obtain more complete mass spectrometry data.Terefore, this method was used to analyze the water extract of L. leontopodioides in this study.Compared with previous studies, not only favonoids and phenolic acids, but also pentacyclic triterpenes, oligosaccharides, and glycosides, which have never been reported before, were obtained using UPLC-Q-Orbitrap MS.Te above research results show that the anti-infammatory, antibacterial, antioxidant, and hypoglycemic efects of L. leontopodioides may be derived from the presence of chemical components, such as favonoids, phenolic acids, pentacyclic triterpenes, oligosaccharides, and glycosides.

GC-MS Section.
Te essential oil of L. leontopodioides was extracted by supercritical carbon dioxide (SC-CO 2 ) extraction technology.As a new advanced "green" separation technology, SC-CO 2 is easy to operate and can not only extract and separate the desired substances quickly and efciently but also the yield and purity of the obtained substances are higher than those of traditional methods [39].International Journal of Analytical Chemistry Te composition of L. leontopodioides essential oil was analyzed by GC-MS, and the chemical composition of essential oils extracted with diferent organic solvents was compared, and it was found that extracting essential oils with n-hexane can obtain a large amount of phenylpropene compounds, such as eugenol.Te pharmacological efects of eugenol include antibacterial, anticancer, antioxidant, and other efects [40]; using diethyl ether to extract essential oils can obtain a large amount of fatty acids.Aparna et al. [41] through research suggested that the n-hexadecanoic acid might function as an anti-infammatory agent.Analysis of L. leontopodioides essential oil by GC-MS found that the high content of fatty acid components and phenylpropene components may be an essential ingredient for its medicinal efect.

International Journal of Analytical Chemistry
Compared with this, the results of this study are quite diferent, but the composition types are roughly the same, which may be related to the origin of L. leontopodioides, the extraction methods of volatile oil, and the extraction of volatile oil with diferent solvents.

Conclusions
In this study, UPLC-Q-Orbitrap MS and GC-MS analytical methods were established to comprehensively characterize the chemical composition of L. leontopodioides and provide a good research basis for the formulation compatibility and pharmacological mechanism of L. leontopodioides.However, the analysis of this study mainly focused on the identifcation and analysis of chemical components and did not carry out basic research on blood components and pharmacodynamic substances.Terefore, in the future, this analytical technique should be used to further improve the pharmacodynamic material basis of L. leontopodioides.At the same time, the mechanism of action of L. leontopodioides should be further elucidated by combining serum medicinal chemistry, network pharmacology, metabolomics, and other technologies.

Data Availability
Te data used to support the fndings of this study are included within the article.3 and 4. * Components that have not been reported in L. leontopodioides.3 and 4. * Components that have not been reported in L. leontopodioides.
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were identifed from the extract of L. leontopodioides.Te excimer ion of peak 35 with a retention time of 32.827 min and a molecular formula of C 15 H 10 O 6 given in negative ion mode was m/z 285.0403 [M − H] − , and it loses a molecule of carbon dioxide forming m/z 241.0501 [M − H − CO 2 ] − .At the same time, 1, 3 cracking could occur to generate fragment ions m/z 151.0035 [M − H] − and m/z 133.0294 [M − H] − .Among them, m/z 133.0294 consisted of residues on the B ring and C ring, and its intensity was larger than that of m/z 151.0035.Combined with the databases, peak 35 was identifed as luteolin.Its MS/MS spectrum and the fragmentation pathway are shown in Figure 3. Peak 9, with a retention time of 29.949 min and a molecular formula of C 21 H 20 O 11 , combined with the databases was identifed as cynaroside.It responds well in positive ion mode, and the excimer ion given in positive ion mode was m/z 449.1077 [M + H] + .In secondary mass spectrometry, luteoloside lost a glucose to form aglycone ion m/z 287.0549 [M + H − Glc] + , and the aglycone was further cleaved by RDA to form fragment ions m/z 153.0183 [ 1,3 A] − and m/z 135.0441 [ 1,3 B] − .Its MS/MS spectrum and the fragmentation pathway are shown in Figure 4. Te excimer ion of peak 31 in negative ion mode was m/z 447.0922 [M − H] − .Te excimer ion peaks were cracked and lost the fragment groups of C 6 H 10 O 5 and C 6 H 11 O 5 , respectively, and fragment ions of m/z 285.0393 [M − H − C 6 H 10 O 5 ] − and m/z 284.0324 [M − H − C 6 H 11 O 5 ] − were obtained, respectively.Subsequently, the fragment ion of m/z 284.0324 continued to fragment, losing 1 neutral CO molecule, and producing a fragment ion of m/z 257.0424 [M − H − C 6 H 11 O 5 − CO] − .At the same time, the fragment ion of m/z 285.0393 can continue to be fragmented, and after losing one neutral CO molecule, it rearranges and removes 2 H atoms, and a fragment ion of m/z 255.0298 [M − H − C 6 H 11 O 5 − CO − 2H] − was produced.Finally, the fragment ion continues to fragment and loses the CO molecule, producing a fragment ion of m/z 227.0349 [M − H − C 6 H 11 O 5 − CO − 2H − CO] − .Compared with the databases, peak 31 was identifed as astragalin.Its MS/MS spectrum and the fragmentation pathway are shown in Figure 5.
C 7 H 6 O 4 in negative ion mode was m/z 153.0192 [M − H] − .Trough the loss of CO 2 , the secondary spectrum generates fragment ion peaks at m/z 109.0293[M − H − CO 2 ] − .Compared with the databases, it was identifed as protocatechuic acid.Its MS/MS spectrum and the fragmentation pathway are shown in Figure 6.Te excimer ion of peak 24 with a retention time of 25.764 min and a molecular formula of C 7 H 10 O 5 in negative ion mode was m/z 173.0452 [M − H] − .Te excimer ion peak lost one molecule of H 2 O to generate m/z 155.0348 [M − H − H 2 O] − and also lost one molecule of H 2 O to generate m/z 137.0240 [M − H − 2H 2 O] − .Finally, this fragment ion continues to fragment and loses the COOH molecule, producing a fragment ion of m/z 93.0344 [M − H − 2H 2 O − COOH] − .Compared with the databases, peak 24 was identifed as shikimic acid.Its MS/ MS spectrum and the fragmentation pathway are shown in Figure 7.
3.1.3.Identifcation of PentacyclicTriterpenoids.A total of four pentacyclic triterpenoids (peaks 10, 12, 13, and 39) were identifed in this study.Ursolic acid is a pentacyclic triterpenoid with a retention time of 48.962 min and a molecular formula of C 30 H 48 O 3 .Te excimer ion given in the positive ion mode was m/z 457.3670 [M + H] + .After it

Figure 3 :
Figure 3: MS/MS spectrum and the fragmentation pathway of luteolin.

Figure 4 :
Figure 4: MS/MS spectrum and the fragmentation pathway of cynaroside.

Figure 6 :
Figure 6: MS/MS spectrum and the fragmentation pathway of protocatechuic acid.

Figure 7 :
Figure 7: MS/MS spectrum and the fragmentation pathway of shikimic acid.

Figure 8 :
Figure 8: MS/MS spectrum and the fragmentation pathway of ursolic acid.

Figure 10 :
Figure 10: MS/MS spectrum and the fragmentation pathway of piperine.

Table 1 :
Instruments and materials.
22 O 11 in negative ion mode was m/z 341.1085 [M − H] − .Its negative ion mode of MS 2 spectra revealed 179.0560 [M − H − Glc] and 161.0454 [M − H − Glc − H 2 O].Based on a comprehensive database, it was tentatively identifed as sucrose, and its MS/ MS spectrum and the fragmentation pathway are shown in Figure 9. + and m/z 115.0542 [M + H − C 5 H 11 N − CH 2 O − CO − CO] + .Compared with the database, this compound was identifed as piperine.Its MS/MS spectrum and the fragmentation pathway are shown in Figure 10.