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. 2020 Feb 18;25(4):898. doi: 10.3390/molecules25040898

Rapid Characterizaiton of Chemical Constituents of the Tubers of Gymnadenia conopsea by UPLC–Orbitrap–MS/MS Analysis

Xin Wang 1, Xiang-Jian Zhong 1, Na Zhou 1, Ning Cai 1, Jia-Hui Xu 1, Qing-Bo Wang 1, Jin-Jie Li 1, Qian Liu 1, Peng-Cheng Lin 2, Xiao-Ya Shang 1,*
PMCID: PMC7070944  PMID: 32085417

Abstract

Gymnadenia conopsea R. Br. is a traditional Tibetan medicinal plant that grows at altitudes above 3000 m, which is used to treat neurasthenia, asthma, coughs, and chronic hepatitis. However, a comprehensive configuration of the chemical profile of this plant has not been reported because of the complexity of its chemical constituents. In this study, a rapid and precise method based on ultra-high performance liquid chromatography (UPLC) combined with an Orbitrap mass spectrometer (UPLC–Orbitrap–MS/MS) was established in both positive- and negative-ion modes to rapidly identify various chemical components in the tubers of G. conopsea for the first time. Finally, a total of 91 compounds, including 17 succinic acid ester glycosides, 9 stilbenes, 6 phenanthrenes, 19 alkaloids, 11 terpenoids and steroids, 20 phenolic acid derivatives, and 9 others, were identified in the tubers of G. conopsea based on the accurate mass within 3 ppm error. Furthermore, many alkaloids, phenolic acid derivates, and terpenes were reported from G. conopsea for the first time. This rapid method provides an important scientific basis for further study on the cultivation, clinical application, and functional food of G. conopsea.

Keywords: Gymnadenia conopsea, UPLC–Orbitrap–MS/MS, chemical constituents, rapid characterization

1. Introduction

Gymnadenia conopsea R. Br. is a perennial herb belonging to the Orcidaceae family and is widely distributed in Tibet, Xinjiang, Qinghai, Gansu, and Sichuan in China [1]. The tubers of this plant are similar to the palm of the human hand, so was given the Chinese name “shou zhang shen”. G. conopsea has widely been used as a traditional Tibetan remedy and traditional health food for the treatment of neurasthenia, asthma, coughs, and chronic hepatitis [2,3,4]. In recent years, modern pharmacological experiments have demonstrated that the ethanol extract or fractions obtained from the tubers of G. conopsea have effects on Alzheimer’s disease and are anti-viral [5,6,7]. A number of previous studies have reported the isolation and structural determination of different categories in this plant, including glucosyloxybenzyl-2-isobutylmalates, phenanthrenes, and stilbenes [8]. however, traditional separation and identification methods require a large amount of materials and take a long time, and only the main components can be obtained, which do not fully explain the chemical profile of this plant. At the same time, the resources of this plant are rare and blind separation is a waste of resources. A comprehensive configuration of the chemical profile of G. conopsea could be used as guidance for further study of active components, and also could save resources. Therefore, a rapid and sensitive method to figure out the chemical components in the tubers of G. conopsea was urgently needed.

A rapid, efficient, and precise method focused on identification of chemical components is very important for complex herb medicines. Recently, based on the highly efficient separation performance of ultra-high performance liquid chromatography (UPLC) and high sensitivity of mass spectrometry (MS), UPLC coupled with high-resolution mass spectrometry (HRMS) has become an important tool for characterization of chemical components in natural products [9]. Furthermore, a combination of UPLC separation with an Orbitrap MS system (UPLC–Orbitrap–MS/MS) has been widely used for screening and identification of chemical components in herbal medicines because of the advantages in terms of the peak capacity, resolution, separation time, and detection sensitivity [10,11,12].

In this study, a method based on UPLC–Orbitrap–MS/MS was established for rapid and sensitive characterization of various chemical components in the tubers of G. conopsea for the first time. A total of 91 components belonging to seven categories in the tubers of G. conopsea were identified in a short time, which will provide a basis for further study of the relationship between the constituents and pharmacology.

2. Results and Discussion

2.1. Optimization of Ultra-High Performance Liquid Chromatography (UPLC) and Mass Spectrometry (MS) Conditions

In order to obtain the optimal elution conditions for the separation and analytical sensitivity of constituents, a series of parameters (mobile phase, flow rate, and column temperature) were investigated. According to the previous reports [13], there are many glycoside compounds in the tubers of G. conopsea. A comparative study based on the chromatographic separation and detection sensitivity revealed that the best performance was achieved with methanol as the organic part of the mobile phase. Due to the compounds containing carboxyl and phenolic hydroxyl, the moiety was tailed on the C18 column, which could be improved by adding a small amount of organic acid. The alkaloid compounds generally showed better mass spectrometric responses in positive ionization mode. Therefore, it was finally decided that methanol/0.1% formic acid aqueous solution was used as the mobile phase. Finally, a column temperature of 40 °C and a flow rate of 0.3 mL/min were set to reduce the pressure and obtain better separation.

Some parameters of heated electrospray ionization (HESI) sources (spray voltage, source heater temperature, capillary temperature, sheath gas flow, auxiliary gas flow, capillary voltage, and S-lens voltage) were also optimized to obtain high sensitivity for most compounds. The optimal conditions were set as follows: spray voltage, 4 kV/3.5 kV (positive/negative); source heater temperature, 350 °C; capillary temperature, 350 °C; sheath gas flow, 50 arb; auxiliary gas flow, 10 arb; and S-lens RF level, 50. The mass scan range was set at m/z 150–2250 Da in the full scan mode, and the resolution was set at 70,000. To acquire the more abundant MS/MS2 spectrum, the MS/MS energy was set at 20, 40, and 60 V as stepped normalized collision energy (NCE) and the resolution was set at 17,500.

2.2. Identification of Main Constituents in G. conopsea Extract

The total ion chromatogram (TIC) of G. conopsea extract in positive- and negative-ion modes are shown in Figure 1. A total of 91 chemical constituents were identified, including 17 succinic acid ester glycosides, 9 stilbenes, 6 phenanthrenes, 19 alkaloids, 11 terpenoids and steroids, 20 phenolic acid derivatives, and 9 others (the chemical structures and MS2 spectra of some constituents see Figure S1–S41). The compounds identification process contained many steps. Firstly, the analysis data were imported into the Compound Discoverer 2.1 software (The workflow tree see Figure S42), which includes the OTCML database and the free chemical structure database, including Massbank, NIST, ChemSpider, and mzCloud. The chemical elemental composition for each target peak was accurately assigned within a mass error of 3 ppm. Then, the formulas that were obtained from Compound Discovery were searched in the self-built chemical database of gymnadenia to match the known structures in this genus. For those formulas not included in this genus, we referred to the database search results for confirmation. Then, the fragment ions were used to further confirm the chemical structures. The retention time, compound name, formula, m/z values of adduct ions and MS/MS fragment ions in positive/negative ESI modes, mass error, and accurate molecular mass are shown in Table 1.

Figure 1.

Figure 1

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The total ion chromatograms of the tubers of G. conopsea, extracted by ultra-high performance liquid chromatography combined with an Orbitrap mass spectrometer (UPLC–Orbitrap–MS/MS) in positive- and negative-ion modes.

Table 1.

All the identified components from G. conopsea extract and their ultra-high performance liquid chromatography mass spectrometer (UPLC–MS/MS) data.

No R.T. (min) Compound Name Formula Exact Mass Error (ppm) Adduct Ion (m/z) MS2 Fragment (m/z) Ref.
Succinic Acid Ester Glycosides
9 4.605 coelovirins E C14H24O11 368.13181 −0.14 367.12473 [M − H]− 293.12454, 187.06120, 143.07137 a, 99.08157 [14]
16 8.430 dactylorhin C C14H23O10 352.13690 0.09 351.12982 [M − H]− 179.05595, 171.06635,127.07648 a [15]
28 10.072 coelovirins D C27H40O17 636.22664 0.15 635.21948 [M − H]− 349.11404 a, 293.12415, 277.12915,143.07129 [14]
29 10.308 grammatophylloside C C24H28O12 508.28186 2.09 507.14993 [M − H]− 221.04546,203.03497 a, 177.05568, 149.06070, 107.05019 [16]
31 10.748 Coelovirin B C21H30O12 474.17371 0.63 473.16614 [M − H]− 367.12451, 293.10284, 187.06094, 159.06616,143.0729, 115.07640, 99.08151 a [14]
32 11.08 (−)-(2R,3S)-1-(4-β-d-glucopyranosyloxybenzyl)-2-O-β-d-glucopyranosyl-4-{4-[α-d-glucopyranosyl-(1-4)-β-d-glucopyranosyloxy]-benzyl}-2-isobutyltartrate C46H66O28 1066.37406 −0.06 1065.37610 [M − H]− 797.27228 a, 635.21936, 455.17773, 293.12411 [4]
33 11.291 dactylorhin B C40H56O23 904.32147 1.42 903.31238 [M + H] + 739.40845, 635.21973 a, 473.16724, 349.11383, 293.12393 [15]
35 11.678 loroglossin C34H46O18 742.26858 0.04 741.26056 [M − H]− 455.15555, 285.09799, 349.11484, 277.12958 a, 187.09761, 123.04520 [17]
36 11.756 dactylorhin E C27 H40 O16 620.23185 −0.34 619.22369 [M − H]− 439.16074, 285.09821, 179.05609,153.05569 a [15]
44 13.063 coelovirins A C21H30O11 458.17903 0.49 457.17169 [M − H]− 285.09793, 189.07683, 171.06650,153.05566, 127.07648 a 123.04527 [14]
46 13.420 (−)-(2R,3S)-1-(4-β-d-glucopyranosyloxybenzyl)-4-methyl 2-isobutyltartrate C22H32O12 488.18950 0.25 487.18188 [M − H]− 189.07649, 171.06628, 153.05579, 129.09218 a, 99.08157 [4]
47 13.420 dactylorhin A C40H56O22 888.32675 1.49 887.32123 [M − H]− 619.22485,439.16113, 323.09833, 153.05572 a, 171.06639, 127.07654 [15]
48 13.425 gymnoside II C21H30O11 458.17897 0.35 457.17175 [M − H]− 285.09827,171.06633, 153.05576, 127.07654,123.04524, 99.08158 [15]
52 14.412 gymnoside III C42H58O23 930.33937 −1.11 929.33154 [M − H]− 661.23553, 619.22565 481.17163, 439.16144, 153.05579 a [5]
53 14.431 gymnosides VII C50H62O24 1046.36365 1.21 1045.35632 [M − H]− 741.26141, 635.21967, 455.15485, 349.11420, 293.12424 a [5]
54 14.436 gymnoside I C21H30O11 458.17897 0.35 457.17169 [M − H]− 351.12991 171.06636, 127.07649 a, 123.04526, 99.08160 [15]
55 14.440 militarine C34H46O17 726.27387 0.51 725.26599 [M − H]− 457.17157 a, 285.09799, 153.05573, 127.07654, 123.04519 [17]
Stilbenes
38 11.995 isorhapontigenin C15H14O4 258.08932 −0.42 259.09647 [M + H]+ 227.07019,199.07533 a, 135.04410, 107.04953 [18]
39 12.018 rhaponticin C21H24O9 420.14210 −0.16 419.13513 [M − H]− 256.07437, 241.05089 a, 213.05588 [19]
40 12.116 piceatannol C14H12O4 244.07371 −0.57 243.06630 [M − H]− 149.02441 a, 121.02955, 93.03458 [20]
57 14.568 dihydro-resveratroll C14H14O3 230.09433 −0.05 229.14445 [M − H]− 123.04518, 121.02949 a 107.05019, 93.03454 [21]
64 17.405 batatasin III C15 H16O3 244.11001 0.23 245.11731 [M − H]− 227.10683, 151.07535, 137.05969, 121.06501 a [22]
69 19.445 3,3′-dihydroxy-4-(4-hydroxybenzyl)-5-methoxybibenzyl C22H22O4 350.15206 0.71 349.14474 [M − H]− 255.10283, 243.10271 a, 227.07153, 106.04240, 93.03458 [23]
72 19.998 bulbocodin C C29H28O5 456.19405 0.83 455.18674 [M − H]− 361.14493 a, 331.09796, 304.11102, 255.10280, 93.03461 [24]
73 20.542 bulbocodin D C29H28O5 456.19372 0.88 455.18680 [M − H]− 440.09048, 361.1088 a, 349.10840, 255.06645, 93.03416 [24]
76 22.298 3,3′-dihydroxy-2,6-bis(4-hydroxybenzyl)-5-methoxybibenzyl C29H28O4 440.19894 0.42 439.19168 [M − H]− 424.16870, 345.14984 a, 333.11353, 93.03459 [25]
Phenanthrenes
71 19.863 1-((4-hydroxyphenyl)methyl)-4-methoxy-2,7-phenanthrenediol C22H18O4 346.12087 1.03 347.12778 [M + H]+ 253.08589 a, 235.07544, 207.08047, 107.04955, [26]
74 21.160 gymconopin A C22H20O4 348.13616 0.02 347.12888 [M − H]− 332.10544 a, 239.07147, 226.06348, 93.03457 [26]
75 21.191 9,10-dihydro-2-methoxy-4,5-phenanthrenediol C15H14O3 242.09439 0.25 243.10161 [M + H]+ 228.07809, 225.09105 a, 211.07533 197.09607 [26]
82 26.152 blestriarene A C30H26O6 482.17309 0.03 481.16586 [M − H]− 466.14246, 241.05086 a, 210.06853 [26]
83 26.438 gymconopin C30H26O6 482.17308 0.27 481.16583 [M − H]− 241.05081,225.09227, 210.06870 a [26]
84 27.870 blestriarene B C30H24O6 480.15759 0.63 481.16461 [M + H]+ 257.08075 a, 225.05467, 211.07530, 207.04405 [26]
Phenolic Acid Derivatives
7 4.203 (−)-4-[β-d-glucopyranosyl-(1-4)-β-d-glucopyranosyloxy]benzyl alcohol] C19H28O12 448.15814 0.15 447.15176 [M − H]− 341.10901 a,179.05614, 161.04562, 119.03497, 89.02443 [5]
11 4.877 (+)-4-[α-d-glucopyranosyl-(1-4)-β-d-glucopyranosyloxy]benzyl alcohol C19H28O12 448.15811 0.12 447.15079 [M − H]− 341.10901 a,179.05614, 161.04575, 89.02444, 71.01380 [5]
13 7.711 4-methoxyphenyl
β-d-glucopyranoside		 C13H18O7 286.10521 −0.16 285.09793 [M − H]− 179.11877, 161.04642, 123.04515 a [27]
17 8.943 dactylose B C12H16O6 256.09481 0.49 255.08772 [M − H]− 237.11345,237.07713, 165.05467, 123.04523 a [28]
18 9.049 phenyl-3-deoxyheopyranoside C12H16O5 240.09993 −0.63 239.09271 [M − H]− 179.07149 a, 162.06873, 121.02957 [29]
21 9.267 isoferulic acid C10H10O4 194.05803 0.64 195.06535 [M + H]+ 177.05464 a, 149.05975, 145.02840, 117.03376 [30]
22 9.549 ferulic acid C10H10O4 194.05808 −0.88 195.06541 [M − H]− 177.05453, 149.05968, 145.02832 a, 117.03370 [31]
23 9.562 p-doumaric acid C9H8O3 164.04738 −0.23 163.04010 [M − H]− 119.05019 a, 93.03452 [30]
25 9.621 (E)-4-methoxycinnamic acid C10H10O3 178.06311 −0.69 179.07040 [M + H]+ 147.04402 a, 137.05974, 119.04941, 91.05477 [31]
34 11.595 tremuloidin C20H22O8 390.13185 −0.97 389.12460 [M + H]+ 341.10324, 193.05069 a, 150.03229, 134.03743 [32]
43 12.631 chlorogenic acid C16H18O9 354.09569 1.67 353.08841 [M − H]− 179.03511 a,135.04527, 177.01929, 109.02952 [33]
45 13.353 quercetin-3β-D-glucoside C21H20O12 464.09555 −0.15 463.08832 [M − H]− 300.02747 a, 271.02481, 255.02997 [34]
49 13.665 cirsimarin C23H24O11 476.13197 −0.22 475.12469 [M − H]− 307.08240 a, 167.03502, 152.01154 [35]
50 14.041 astragalin C21H20O11 448.10073 −0.39 447.09341 [M − H]− 284.03262, 255.03510 a, 227.03510 [36]
56 14.470 kaempferol-7-O-glucoside C21H20O11 448.10072 −0.36 449.10794 [M + H]+ 287.05487 a, 258.05228, 145.04948 [37]
59 14.609 desmethylxanthohumol C18H22O5 340.13105 0.07 341.13831 [M + H]+ 323.12762, 217.08611, 153.05446, 137.05969 a, 187.07526 [38]
61 14.917 isorhamnetin C16H12O7 316.05854 −0.74 317.06573 [M + H]+ 302.04196 a, 274.04684, 273.03922, 153.01820 [39]
63 16.015 naringenin chalcone C15H12O5 272.06856 −0.33 271.06131 [M − H]− 177.01930, 151.00363 a, 145.02951, 119.05019 [40]
65 17.450 equol C15H14O3 242.09429 −0.72 243.10172 [M − H]− 228.07822, 211.07527, 149.05972, 135.04405, 123.04429,107.04951 a [41]
82 24.670 galangin C15H10O5 270.05291 −0.31 269.04562 [M − H]− 241.05077, 225.05580 a [42]
Alkaloids
1 1.112 dl-arginine C6H14N4O2 174.11176 −0.48 175.11899 [M + H]+ 158.09248,130.09763,116.07089, 112.08723, 70.06586 a [43]
3 1.946 Adenosine C10H13N5O4 267.09653 0.84 268.10388 [M + H]+ 136.06180a, 119.03542, [43]
4 1.961 6-quinolinecarboxylic acid C10 H7NO2 173.04785 0.03 174.05510 [M + H]+ 156.04442, 146.06017 a, 130.06531,128.04971 [44]
5 2.479 l-Phenylalanine C9H11NO2 165.07921 −1.40 166.08640 [M + H]+ 149.05977, 131.04926, 120.08099 a,103.05462 [45]
6 3.100 N-(4-methyoxyphenyl)-1H-pyrazolo [3,4-d]pyrimidin C12H11N5O 241.09636 −0.14 242.10341 [M + H]+ 136.06171, 107.04944 a [46]
8 4.329 trans-indole-3-acrylic acid C11H9NO2 187.06348 −0.29 188.07060 [M + H]+ 170.06012, 146.06004 a, 144.08080, 118.06541 [47]
10 4.856 Guanine C5H5N5O 151.04946 −0.34 152.05661 [M + H]+ 135.03011 a, 110.03517 [48]
12 5.444 5′-S-Methyl-5′-thioadenosine C11H15N5O3S 297.08965 −0.29 298.09668 [M + H]+ 136.06178 a, 163.04239, 145.03169 [49]
14 8.361 conopsamide A C14H21N3O4 295.15315 1.05 294.14621 [M − H]− 188.10416, 131.08266 a, [50]
15 8.420 befunolol C16H21NO4 291.14681 0.90 292.25405 [M + H]+ 277.13074, 151.03897, 124.11227 a, [51]
19 9.067 cyclo(tyrosy-tyrosyl) C18H18N2O4 326.12667 −0.05 327.13342 [M + H]+ 221.09201, 203.08133, 175.08655,158.06003, 107.04946 a [6]
24 9.596 cyclo(leucylprolyl) C11H18N2O2 210.13695 0.58 211.14403 [M + H]+ 193.08359, 183.14925, 138.12781, 127.08688, 114.09170, 70.06586 a [52]
26 9.758 N-(4-hydroxybenzy) adenine riboside C17H19N5O5 373.13861 −0.05 374.14581 [M + H]+ 242.10358, 148.06180, 136.06180 a, 107.04951 [53]
27 9.827 dibenzylamine C14H15N 197.12062 −0.89 198.12784 [M + H]+ 181.10126, 106.06558,91.05482 a [54]
30 10.699 (+)-chelidonine C20H19NO5 353.12643 −0.30 354.13321 [M + H]+ 336.12274,293.08057, 188.07043 a, 206.08098, 149.05965 [55]
37 11.822 (2E)-3-(4-hydroxy-phenyl)-N-[2-(4-hydroxy-phenyl)-ethyl]-acrylamide C17H17NO3 283.12083 0.06 284.12769 [M + H]+ 147.04390 a, 164.07062, 121.06493, 119.04931 [56]
42 12.834 2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylic acid C12H12N2O2 216.09012 −1.13 217.09723 [M + H]+ 144.08080 a, 156.08093, 118.06545 [57]
58 14.582 dl-tryptophan C11H12N2O2 204.08987 0.03 203.08272 [M − H]− 159.09279, 142.06619, 116.05058 a, 74.24770 [48]
78 23.937 N-phenyl-2-naphthylamine C16H13N 219.10478 0.08 220.11194 [M + H]+ 143.07289 a, 128.06215 [58]
Terpenoids and Steroids
41 12.664 mascaroside C26H36O11 524.22615 −0.73 523.21875 [M − H]− 361.6602 a, 179.07140, 165.05576, 101.02450 [59]
51 14.349 (±)-abscisic acid C15H20O4 264.13613 0.12 263.12869 [M − H]− 219.13905 a,204.11546, 201.12842, 151.07640 [60]
77 23.323 (3β,5α,9α)-3,6,19-trihydroxyurs-12-en-28-oic acid C30H48O5 488.35032 −0.29 489.35718 [M + H]+ 471.34665 a,453.33636, 435.32520, 265.21689 [61]
80 24.638 (3β,17β)-estr-5(10)-ene-3,17-diol C18H28O2 276.20882 0.12 277.21600 [M + H]+ 259.20557, 235.16937, 221.15327, 149.13251, 121.10139, 107.08587, 93.07037 a, [62]
85 28.595 17α-methyl-5α-androstane-3β,11β,17β-triol C20H34O3 322.25091 0.37 323.25797 [M + H]+ 305.24716, 277.21613 a, 259.20554, 179.14297, 151.11176, 135.11687, 107.08589 [63]
86 32.654 lup-20(29)-en-28-al C30H48O2 440.36543 −0.04 441.37292 [M + H]+ 423.36244 a, 405.35190, 191.14313, 151.11177, 109.10156, 123.08073 [64]
87 33.514 lupenone C30H48O 424.37052 −0.02 425.37735 [M + H]+ 407.36710 a, 231.21080, 191.17928, 177.16399, 109.10153 [65]
88 34.104 poriferasterol C29H48O 412.37052 −0.07 413.37762 [M + H]+ 395.36703 a,353.33051, 255.21051, 213.16359, 159.11682, 105.07026 [66]
89 35.684 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol C29H46O 410.35496 −0.12 411.36194 [M + H]+ 393.35141, 353.32016, 253.19467, 175.11179 a, 147.11678 [67]
90 40.568 lupeol C30H50O 426.38611 0.13 427.39322 [M + H]+ 409.38208, 191.17934, 121.10136, 109.10149, 95.08600 a [68]
91 41.305 (22E)-stigmasta-3,5,22-triene C29H46 394.35992 0.06 395.36719 [M + H]+ 297.25775, 241.19502, 173.13257, 159.11693, 145.10123 a [69]
Others
2 1.354 citric acid C6H8O7 192.02699 0.05 191.01979 [M − H]− 173.00919, 129.01920, 111.00877 a, 87.00876, [70]
20 9.247 butanedioic acid C8H14O5 190.08414 0.15 189.07680 [M − H]− 171.06630, 129.05573 a, 143.07171, 127.07654, 99.08161 [71]
60 14.911 pinoresinol C20H22O6 358.1417 0.75 359.14969 [M − H]− 163.03735, 137.05968 a, 131.04922 [72]
62 15.501 benzyl-[(6-oxo-7,8,9,10-tetrahydro-6H-benzo[c]chromen-3yl)oxy]-acetate C22H20O5 364.13133 −0.72 365.13849 [M + H]+ 271.09637, 239.07021, 147.04408, 107.04951 a [72]
66 18.242 aloeresin A C28H28O11 540.16377 −1.15 539.15643 [M − H]− 377.10330 a, 283.06125, 163.00378 [73]
67 19.175 frangulin B C20H18O9 402.09545 −0.9 401.08740 [M − H]− 357.06149, 313.07181, 121.02949 a [74]
68 19.422 cleomiscosin A C20H18O8 386.10051 −0.91 387.10724 [M + H]+ 357.06030 a, 329.06540, 301.07065, 245.04463, 149.05989 [75]
70 19.772 bis-(methylbenzylidene)-sorbitol C22H26O6 386.17321 −0.69 387.18051 [M + H]+ 105.07003 a, 119.04945, 103.05464 [75]
80 24.129 umbelliferone C9H6O3 162.03168 0.09 163.03894 [M + H]+ 135.04408 a,133.02847, 107.04951, 105.04509 [33]
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a Basepeak.

2.2.1. Succinic Acid Ester Glycosides

Succinic acid ester glycosides were the main components in G. conopsea, which consisted of succinic acid, glycosyl, and a benzyl moiety. A total of 17 succinic acid ester glycosides were identified in the tubers of G. conopsea extract, and the deprotonated molecules [M − H]− were found in the ESI–MS spectra for all compounds. All the esters glycosides could be classified into glycosyloxybenzyl 2-isobutylmalate and glycosyloxybenzyl 2-isobutyltartrate. In tandem mass spectra of succinic acid ester glycosides, the losses of H2O, COOH and C6H10O5 (glycose moiety), and C13H17O7 (glycosyloxybenzyl moiety) are commonly observed.

Compounds 16, 29, 31, 36, 44, 47, 48, and 52–55 were glycosyloxybenzyl 2-isobutylmalate. Among them, compound 16 showed a [M-H]− ion at m/z 351.12982, and gave fragment ions at 351.12982 179.05595, 171.06635, and 127.07648 corresponding to [M-H]−, [M-H-C6H10O5]−, [M-H-C6H10O5-H2O]−, and [M-H-C6H10O5-H2O-COOH]−, respectively; this compound was tentatively identified as dactylorhic C [15]. Except for 16, all other compounds had the glycosyloxybenzyl moiety and had similar fragmentation patterns. Taking compound 47 as an example, it had a [M-H]− ion at m/z 887.32123. The fragment ion m/z 619.22485 [M-H-C13H16O6]− was easily produced, which indicated that the glucopyranosyloxy-benzyl moiety was easily lost. Then, the fragment ion m/z 439.16113 [M − H − C13H16O6 − C6H10O5]−, with its high relative abundance, was easily produced from m/z 619.22485 by neural loss of the glycose moiety at C2–OH. Fragment ions m/z 323.09833, 171.06639, 153.05572, and 127.07654 were derived from the malate moiety by the loss of H2O and COOH. Compared with the literature data, compound 47 was identified as dactylorhin A [15]. The possible fragmentation mechanism of dactylorhin A is depicted in Figure 2. In a similar way, the other nine compounds were identified according to their molecular mass, formula, MS/MS fragments, and related literature studies, including grammatophylloside C (29) [16], coelovirin B (31) [14], dactylorhin E (36) [15], coelovirins A (44) [14], gymnoside II (48) [15], gymnoside III (52) [5], gymnosides VII (53) [5], gymnoside I (54) [14], and militarine (55) [17].

Figure 2.

Figure 2

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The possible fragmentation mechanism of dactylorhin A.

Compounds 9, 28, 32, 33, 35, and 46 were glycosyloxybenzyl 2-isobutyltartrates. The [M − H]− ion of compound 9 was shown at m/z 367.12473. Its MS2 fragment ions at m/z 293.12454 [M − H − C2H2O3]−, 187.06120 [M − H − C6H12O6]−, 143.07137 [M − H − C6H12O6 − CO2]−, and 99.08157 [M − H − C6H12O6 − CO2 − CO2]− were characteristic fragments of the tartrate moiety. All except compounds 9 have the same fragment of the glucopyranosyloxy-benzyl moiety (285 Da). Compounds 28, 32, 33, 35, and 46 showed a [M-H]− ion at m/z 635.21948, 1065.37610, 903.31238, 741.26056, and 487.18188. They have similar fragmentation patterns, including ions at m/z 349.11383, 293.12393, and 277.12915, which were identified as coelovirins D [14], (−)-(2R,3S)-1-(4-β-d-glucopyranosyloxybenzyl)-2-O-β-d-glucopyranosyl-4-{4-[α-d-glucopyranosyl-(1-4)-β-d-glucopyranosyloxy]benzyl}-2-isobutyltartrate [4], dactylorhin B [4], loroglossin [17], and (−)-(2R,3S)-1-(4-β-d-glucopyranosyloxybenzyl)-4-methyl-2-isobutyltartrate [4], respectively. The possible fragmentation mechanism of dactylorhin B (33) is depicted in Figure 3.

Figure 3.

Figure 3

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The possible fragmentation mechanism of dactylorhin B.

2.2.2. Stilbenes

Stilbenes were structures containing one or more C6-C2-C6 units, which were widely distributed in medicinal plants. A total of eight stilbenes in the tubers of G. conopsea extract were identified in positive and negative ion modes. According to their molecular mass, formula, MS/MS fragments, and related literature studies, compounds 38, 39, 40, 57, 64, 69, 72, 73, and 76 were considered to be isorhapontigenin [18], rhaponticin [19], piceatannol [20], dihydro-resveratrol [21], batatasin III [22], 3,3′-dihydroxy-4-(4-hydroxybenzyl)-5-methoxybibenzyl [23], bulbocodin C [24], bulbocodin D [24], and 3,3′-dihydroxy-2,6-bis(4-hydroxybenzyl)-5-methoxybibenzyl [25], respectively.

Taking compound 57 as an example, it had a [M − H]− ion at m/z 229.14445, and the highest relative abundance ion m/z 121. 02949 [M − H − C6H4O2]− was easily yielded by the breakage of the C2-chain. The fragments ions at m/z 123.04515, 107.05019, and 93.03454 were formed in the same fragmentation pattern. Its fragmentation process was the same as in the literature and was identified as dihydro-resveratroll [21]. The possible fragmentation mechanism of compound 57 is depicted in Figure 4.

Figure 4.

Figure 4

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The possible fragmentation mechanism of dihydro-resveratrol.

2.2.3. Phenanthrenes

Six phenanthrenes were identified from the extract of the G. conopsea extract, including 1-((4-hydroxyphenyl)methyl)-4-methoxy-2,7-phenanthrenediol (71) [26], gymconopin A (74) [26], 9,10-dihydro-2-methoxy-4,5-phenanthrenediol (75) [26], blestriarene A (82) [26], gymconopin (83) [26], and blestriarene B (84) [26].

A typical phenanthrene, 9,10-dihydro-2-methoxy-4,5-phenanthrenediol (75), was taken as an example to investigate the MS/MS fragmentation pattern of this type of compound in G. conopsea. The protonated molecular ion of compound 75 was m/z 243.10161 [M + H]+ in positive ESI mode, and its dehydration of C11–OH yielded the fragment ion m/z 225.09105 [M + H − H2O]. The fragment ion m/z 211.07533 [M + H − OCH3]+ was produced by the loss of methoxy at C-13. Then, the continuous dehydration and breakage of the C-ring formed the fragment ion m/z 197.09607 (Figure 5).

Figure 5.

Figure 5

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The possible fragmentation mechanism of 9,10-dihydro-2-methoxy-4,5-phenanthrenediol.

2.2.4. Phenolic Acid derivatives

Phenolic acids were structures containing one or more phenolic hydroxyl moieties, which were widely distributed in medicinal plants. A total of 20 phenolic acid derivates in the tubers of G. conopsea extract were identified in negative and positive ion modes. Among them, compounds 7, 11, 13, 17, and 18 were aromatic glycosides. The loss of hexose residues (glycose 162 Da, rhamnose 146Da) was often seen in these compounds. Taking compound 7 as an example, the deprotonated molecular ion m/z 447.15176 was detected in the spectrum. Fragment ion m/z 341.10901 [M − H − 106]− with the highest relative abundance was easily produced from m/z 447.15176 [M − H]− by cleavage of the glycoside band. The fragment ions m/z 179.05614 and 161.04562 were glycose moieties. Compounds 21–23 and 25 were phenylpropanoids, which were considered to be isoferulic acid, ferulic acid, p-coumaric acid, and (E)-4-Methoxycinnamic acid [30,31]. There were four flavonoid glycosides and five flavonoids, which were identified as quercetin-3β-d-glucoside (45) [34], cirsimarin (49) [35], astragalin (50) [36], kaempferol-7-O-glucoside (56) [37], desmethylxanthohumol (59) [38], isorhamnetin (61) [39], naringenin chalcone (63) [40], equol (65) [41], and galangin (82) [42], respectively.

2.2.5. Alkaloid

A total of 19 alkaloids were identified from the extract of G. conopsea, including amino acids, adenosine, indoles, cyclic peptides, and amides. As depicted in Table 1, in positive ion mode, compounds 3, 6, 12, and 26 were considered as adenosine [43], N-(4-methyoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin [46], 5′-S-Methyl-5′-thioadenosine [49], and N-(4-hydroxybenzy)-adenine-riboside [53], respectively. Taking compound 6 as an example, it had a [M + H]+ ion at m/z 242.10341 in the spectrum. Two main fragment ions at m/z 136.06171 and 107.04944 were obviously observed. Among them, the most abundant fragment ion m/z 136.06171 was suggested by the loss of the phenol residue [M + H − 107]+. The fragment ion at m/z 107.04944 was identified as purine. Compared to the MS spectra data and references, compound 6 was tentatively identified as N-(4-methyoxyphenyl)-1H-pyrazolo[3,4-d] pyrimidin [46].

Compounds 19 and 24 had similar fragmentation behavior and showed [M + H]+ ions at m/z 327.13342 and 211.14403, respectively. According to reference mass spectra and fragmentation spectra reported in the literature studies, two cyclic peptides were identified as cyclo (tyrosy-tyrosyl) [6] and cyclo (leucylprolyl) [52] in the tubers of G. conopsea. The other 13 alkaloids were identified according to their molecular mass, formula, MS/MS fragments, and related literature studies, which are shown in Table 1.

2.2.6. Terpenoid and Steroid

Terpenoids and steroids were derived from methylglutaric acid (MWA). Eleven terpenoids and steroids were identified in this study, including one sesquiterpenoid, one diterpenoid, four triterpenoids, and five steroids. Compound 51 had [M − H]− ion at m/z 263. 12869, and its fragments were at m/z 219.13905 [M-H-COO]−, 204.11546 [M-H-COO-CH2]−, 201.12842 [M − H − COO − H2O]−, and 151.07640 [M − H − C6H8O2]−. Its fragmentation process was the same as the literature and identified as abscisic acid [60].

In tandem mass spectra of terpenoids and steroids in this plant, the neutral losses of H2O (18 Da) and CO (28 Da) are commonly observed. Compounds 77, 87, 89, and 90 were triterpenoids, which gave [M + H]+ ions at m/z 489.35718, 425.37735, 411.36194, and 427.39322, respectively. Thus, they were (3β,5α,9α)-3,6,19-trihydroxyurs-12-en-28-oic acid [60], lupenone [65], 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol [67], and lupeol [68]. Compound 88 was taken as an example to investigate the MS/MS fragmentation pattern of this type of compound in G. conopsea. The protonated molecular ion of compound 88 was m/z 413.37762 [M + H]+ in positive ESI mode, and its dehydration of C3-OH with the adjacent hydrogen easily yielded the fragment ion m/z 395.36703 [M + H − 18]+. The following fragmentation pattern of fragment m/z 395.36703 was the breakage of the side chain to produce the fragment m/z 255.21051 [M + H − 158]+. This was consistent with the literature, and the fragment was identified as poriferasterol [66].

2.2.7. Others

Aside from those listed above, another 9 compounds, namely compounds 2, 20, 60, 66–68, and 80, were considered to be citric acid [70], succinic acid [71], pinoresinol [72], benzyl-[(6-oxo-7,8,9,10-tetrahydro-6H-benzo[c]chromen-3-yl)oxy]-acetate [72], aloeresin A [73], frangulin B [74], cleomiscosin A [75], bis(methylbenzylidene)sorbitol [75], and umbelliferone [33], respectively. As a typical representative, the MS/MS fragmentation of citric acid was firstly investigated. Its deprotonated molecular ion was m/z 191.01979 [M − H]− in negative ESI mode, and its main fragmentation pattern was 173.00919 [M − H − 18]−. The fragment m/z 129.01920 [M − H − 62]− was yielded through decarboxylation and dehydration. The most abundant fragment ion m/z 111.00877 [M − H − 80]− was produced from the fragment m/z 129.01920.

3. Materials and Methods

3.1. Chemicals and Reagents

Methanol, acetonitrile, and formic acid (all MS grade) were purchased from Fisher Scientific (Fisher Scientific, Pittsburgh, PA, USA). Dimethyl sulfoxide (DMSO, HPLC grade) was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). The ultra-pure water was purified by a Milli-Q ultrapure water system (Merck Millipore, Milford, MA, USA). All other regents used were of at least analytical grade.

3.2. Materials and Sample Preparation

The tubers of G. conopsea were collected in Xining City, Qinghai province, China, in August 2018. A botanical voucher specimen of this plant was preserved at the authors’ laboratory and was identified by Professor Pengcheng Lin of Qinghai University for Nationalities.

First, 1.0 g aliquots of the tuber powders were weighed and transferred into a 100 mL Erlenmeyer flask. Next, 50 mL of 95% aqueous methanol solution was added, and then extracted ultrasonically for 1 h. Then, the fluid was filtered and concentrated under reduced pressure in a rotary evaporator. Subsequently, the concentrated extract was dissolved in methanol. Then, the above herb extract solution was filtered through a 0.22 μm PTFE membrane as the sample.

3.3. UPLC–Orbitrap–MS/MS

The UPLC separation was carried out on a Thermo Vanquish Flex Binary RSLC platform (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a diode array detector (DAD). Chromatographic separation was conducted on a Thermo Accucore aQ C18 (150 × 2.1 mm, 2.6 μm; Thermo Fisher Scientific, Waltham, MA, USA) kept at 40 °C. The 0.1% formic acid aqueous solution (v/v, A) and methanol (B) were used as the mobile phase. The gradient elution with a flow rate of 0.3 mL/min was performed as follows: 6–20% B at 0–5 min, 20–21% B at 5–6 min, 21–30% B at 6–7 min, 30–34% B at 7–10 min, 34–40% B at 10–11 min, 40–57% B at 11–17 min, 57–65% B at 17–18 min, 65–90% B at 18–30 min, 90–97% B at 30–37 min, 97–100% B at 37–45 min. The injection volume was set at 2 μL.

The UPLC–Orbitrap–MS/MS detection was conducted on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The MS analysis was carried out by the ESI source in both positive- and negative-ion modes and the specific parameters were set as mentioned above. In the MS/MS experiments, the five most intensive ions from each full MS scan were selected for MS/MS fragmentation. The UPLC–MS/MS data were analyzed using Xcalibur 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA), Compound Discoverer 2.1 (Thermo Fisher Scientific, Waltham, MA, USA) loaded with OTCML database 1.0 (Thermo Fisher Scientific, Waltham, MA, USA) and Mass Frontier (Thermo Fisher Scientific, Waltham, MA, USA) were employed to process the UPLC–MS data.

4. Conclusions

In this study, an UPLC–Orbitrap–MS/MS approach was firstly developed and applied for rapid separation and identification of the main chemical constituents in the tubers of G. conopsea. Based on the high separation speed of UPLC, accurate MS data, and the fragment ion identification strategy, a total of 91 compounds, including 17 succinic acid ester glycosides, 9 stilbenes, 6 phenanthrenes, 19 alkaloids, 11 terpenoids and steroids, 20 phenolic acid derivatives, and 9 others, were identified by comparison of their accurate masses, fragment ions, retention times, and literature studies. Many compounds, such as alkaloids and terpenoids, were reported for G. conopsea for the first time. According to the types of compounds identified from this plant, several low polar compounds were identified, which are worthy of further study. This rapid method provides an important scientific basis for further study on the cultivation, clinical application, and functional food of G. conopsea.

Supplementary Materials

Click here for additional data file. (661.3KB, pdf)

The following Supplementary Materials are available online: The Figures S1–S42 showed the chemical structures and available raw MS2 spectra of some compounds identified from the tubers of G. conopsea.

Author Contributions

Conceptualization, X.W. and X.-Y.S.; formal analysis, N.C.; investigation, X.W., X.-J.Z., N.Z., and P.-C.L.; software, J.-H.X., and Q.-B.W.; supervision, J.-J.L., Q.L., and X.-Y.S.; writing—original draft, X.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the grants from the Scientific Research Common Program of Beijing Municipal Commission of Education (Grant No. KM201911417014 and KM201811417003), Premium Funding Project for Academic Human Resources Development in Beijing Union University (BPHR2019DZ02), and Key projects of the Beijing Natural Sciences Foundation and Beijing Municipal Education Committee (No. KZ201811417049).

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of the compounds are not available from the authors.

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