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. 2017 Aug 3;22(8):1280. doi: 10.3390/molecules22081280

Nontargeted Metabolomic Analysis of Four Different Parts of Platycodon grandiflorum Grown in Northeast China

Cuizhu Wang 1,2, Nanqi Zhang 1,2, Zhenzhou Wang 1,2, Zeng Qi 1,2, Hailin Zhu 1,2, Bingzhen Zheng 1,2, Pingya Li 1,2,*, Jinping Liu 1,2,*
PMCID: PMC6152411  PMID: 28769024

Abstract

Platycodonis radix is extensively used for treating cough, excessive phlegm, sore throat, bronchitis and asthma in the clinic. Meanwhile, the stems, leaves and seeds of Platycodon grandiflorum (PG) have some pharmaceutical activities such as anti-inflammation and anti-oxidation effects, etc. These effects must be caused by the different metabolites in various parts of herb. In order to profile the different parts of PG, the ultra-high performance liquid chromatography combined with quadrupole time-of- flight mass spectrometry (UPLC-QTOF-MSE) coupled with UNIFI platform and multivariate statistical analyses was used in this study. Consequently, for the constituent screening, 73, 42, 35, 44 compounds were characterized from the root, stem, leaf and seed, respectively. The stem, leaf and seed contain more flavonoids but few saponins that can be easily discriminated in the root. For the metabolomic analysis, 15, 5, 7, 11 robust biomarkers enabling the differentiation among root, stem, leaf and seed, were discovered. These biomarkers can be used for rapid identification of four different parts of PG grown in northeast China.

Keywords: Platycodon grandiflorum, nontargeted metabolomic analysis, different part, UPLC-QTOF-MSE

1. Introduction

It is well-known that there are both chemical and pharmacological differences in different parts of herbs. Taking Aristolochia mollissima Hance as an example, the fruits are used to treat cough and asthma, the roots have obvious antihypertensive effects, while the stems and leaves are rheumatoid medicines. This phenomenon also exists in other herbs, such as Lycium barbarum, Polygonum Multiflorum Thunb., Trichosanthes kirilowii Maxim, Ephedra sinice Stapf, etc. [1].

As both food and medicine, Platycodon grandiflorum (Jacq.) A. DC. (PG) is known as “Jiegeng” in China, “Huridunzhaga” in Mongolia, “Kikyo” in Japan and “Doraji” in North Korea [2]. In clinical, the root of PG which has various biological activities, such as apophlegmatic and antitussive [3], anti-inflammation [4], immunoregulation [5], anti-oxidant [6], etc., has been widely used for the treatment of cough, excessive phlegm, and sore throat. In addition, the stem and leaf of PG also have anti-inflammatory [7] and anti-oxidant [8,9] activities, while research on the pharmacological effects of PG seed is currently non-existent.

PG is a rich source of different natural products with various structural patterns. Around 100 compounds have been isolated from the roots of PG, including steroidal saponins, flavonoids, phenolic acids, polyacetylenes, sterols, etc. [2]. Triterpenoid saponins, mainly of the oleanane family pentacyclic type, are the active components of the root of PG [10]. Several flavonoids and phenolic acids were isolated from the aerial parts of PG [11]. Two glycosides and four flavonoids were isolated from the seeds of PG [12]. Recently, instead of traditional separation and identification method, a combination of ultra-high performance liquid chromatography (UHPLC) separation, quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) detection and automated data processing software UNIFI with scientific library was innovatively used for screening and identifying chemical components in herbal medicines [13,14] and traditional Chinese medicine formulas [15]. In 2015, Lee et al. reported the global profiling of various metabolites in PG by UPLC-QTOF/MS [16]. In that paper, a total of 20 metabolites were characterized from the roots, and 56 compounds from stems and leaves of PG grown in Korea. Herbs collected from different regions will show certain differences both in chemical constituents and in pharmacological activities [17]. For example, saponins in the root of PG from different sites in Gyeongnam Province, Korea showed different contents [18]. The 1H-NMR-based metabolomics with OPLS-DA statistical models was used to cluster the ginseng samples from Korea and China, and the result suggested that the chemical profiles from two countries are quite different due to their different geographical origins [19]. Hence, in order to illustrate different chemical constituents from the different regions and from the different parts of the plants, and to better clarify the pharmacological fundamental substances of PG, the root, stem, leaf and seed of PG produced in Jilin Province, China were taken as samples in this paper.

Metabolomics, including targeted and untargeted complementary approaches, is primarily concerned with identification and quantitation of small-molecule metabolites (<1500 Da) [20]. Recently, because of its ability to profile diverse classes of metabolites, untargeted metabolomics has been widely used to compare the overall metabolic composition of different samples [21]. An untargeted analysis approach is mainly applied in metabolite identification through mass-based search followed by manual verification [20] Being a sensitive, efficient, reliable, accurate and nondestructive method, UPLC-QTOF-MS has been widely used recently in this kind of analysis, such as exploring the early detection of mycotoxins in wheat [22], estimating compliance to a dietary pattern [23], exploring the bioavailability of the secoiridoids from a seed/fruit extract in human healthy volunteers [24], evaluating the enantioselective metabolic perturbations in MCF-7 cells after treatment with R-metalaxyl and S-metalaxyl [25].

In this study we focus on both the quickly chemical components’ screening and the non-targeted metabolomic analysis of the root, stem, leaf and seed of PG. UPLC-QTOF-MSE, UNIFI platform and multivariate statistical analyses, such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to profile the four different plant parts and to find the biomarkers among these four parts of PG grown in northeast China.

2. Results

2.1. Identification of Components from Different Parts of PG

As a result, a total of 159 compounds were identified or tentatively characterized in both positive and negative mode from the four parts of PG, the base peak intensity (BPI) chromatograms are shown in Figure 1, and their chemical structures are shown in Figure 2. More specifically, 73, 42, 35, 44 compounds were characterized from the root, stem, leaf and seed respectively (Table 1), including triterpenoid saponins, organic acids, steroids, phenols, flavonoids, alcohols, amino acids, coumarins, terpenoids, alkaloids and amides and so on.

Figure 1.

Figure 1

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The representative base peak intensity (BPI) chromatograms of root in positive (A) and negative (B) modes; of stem in positive (C) and negative (D) modes; of leaf in positive (E) and negative (F) modes; of seed in positive (G) and negative (H) modes.

Figure 2.

Figure 2

Figure 2

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Chemical structures of compounds identified in PG.

Table 1.

Compounds identified from different parts of PG by UPLC-QTOF-MSE.

No. tR (min) Formula Experimental (Da) Theoretical (Da) Mass Error (ppm) Adducts MSE Fragmentation Component Name Source
1 * 0.59 C12H22O11 342.1169 342.1162 2.04 −H 323.0984, 195.0510, 161.0465 Sucrose D
2 * 0.60 C6H14O6 182.0797 182.0790 3.04 +Na 205.0689, 152.0713 Mannitol R
3 0.67 C12H17NO5 255.1114 255.1107 2.91 +H 256.1114, 226.1074, 122.0375 Radicamine A R
4 0.68 C20H18O14 482.0682 482.0697 −2.95 −H 343.0676, 301.0007, 274.0119, 191.0554, 152.0124 2,3-(S)-Hexahydroxydiphenoyl-d-glucose a S, L
5 * 0.71 C6H8O7 192.0278 192.0270 3.93 −H 191.0205, 173.0077, 111.0089 Citric acid R
6 * 0.75 C10H13N5O4 267.0974 267.0968 2.23 +H 218.1020, 136.0634 Adenosine D
7 0.82 C20H20O14 484.0857 484.0853 0.78 −H 313.0568, 183.0308, 169.0156, 152.0123 2,6-Di-O-Galloyl-β-d-glucose a S, L
8 0.85 C20H20O4 324.1347 324.1362 −4.38 +H 203.0708, 175.0758, 164.0463, 149.0602, 103.0556 Isobavachin a D
9 * 0.86 C9H11NO2 165.0796 165.0790 3.98 −H 164.0724, 147.0456, 103.0549 Phenylalanine R
10 0.95 C34H24O22 784.0751 784.0759 −1.05 −H 421.0417, 337.0214, 249.0416, 182.0223, 168.0074, 149.9967 Casuariin a S
11 0.97 C21H24O11 452.1341 452.1319 4.86 −H 299.0771, 289.0737, 271.0611, 165.0206, 137.0257 Curculigoside B a D
12 1.02 C19H18O6 342.1089 342.1103 −4.14 −H 211.0628, 181.0506, 179.0349, 161.0240, 151.0404 5,6,7,4′-Tetramethoxyflavone a R
13 1.24 C20H24O5 344.1609 344.1624 −3.98 +Na 222.0916, 194.0973, 182.0611, 127.0394 Schininallylol a R
14 * 1.35 C11H12N2O2 204.0903 204.0899 2.29 +H 188.0706, 144.0808, 132.0813, 118.0661 Tryptophan R
15 1.36 C21H21ClO11 484.0775 484.0772 0.45 −H 309.0630, 287.0594, 124.0163, 109.0291 Cyanidin 3-glucoside a L
16 1.37 C27H28N2O4 444.2034 444.2049 −3.41 −H 235.1215, 175.0626, 173.0464, 131.0364, 105.0356 Aurantiamide acetate a D
17 * 1.73 C16H18O9 354.0950 354.0951 −0.22 +H 192.0663, 163.0396, 145.0294, 135.0452 Chlorogenic acid R
18 2.15 C27H32O16 612.1712 612.1690 3.51 −H 593.1511, 461.1313, 303.0532, 285.0428, 177.0209, 151.0052 (2R,3R)-Taxifolin7-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside D
19 2.30 C30H26O12 578.1430 578.1424 0.98 −H 449.0876, 425.0875, 407.0777, 289.0718, 125.0257 Procyanidin B1 a D
20 * 2.31 C15H14O7 306.0738 306.0740 −0.53 +HCOO 179.0349, 167.0343, 163.0406, 161.0241, 109.0315 Gallocatechin R
21 * 2.34 C7H12O6 192.0637 192.0634 1.53 −H 173.0480, 127.0406, 116.0514, 111.0456 Quinine acid R
22 * 2.35 C9H8O4 180.0425 180.0423 1.60 −H 161.0241, 135.0451, 133.0297, 109.0315, 108.0224 Caffeic acid R
23 2.36 C16H18O8 338.0993 338.1002 −2.62 −H 191.0567, 177.0195, 161.0243, 119.0505, 105.0351 3-O-trans-Coumaroylquinic acid R
24 2.70 C25H34O12 526.2045 526.2050 −1.04 −H 363.1452, 315.1244, 179.0713, 167.0711, 149.0612 LucidumosideA a R
25 2.78 C22H26O7 402.1670 402.1679 −1.88 +HCOO 327.0884, 303.0885, 297.0421, 209.0844, 137.0256 Neociwujiaphenol a D
26 2.81 C41H28O27 952.0809 952.0818 −0.97 −H 605.0777, 479.0469, 481.0642, 453.0677, 246.0169 Geraniin a L
27 2.98 C17H26O7 342.1678 342.1679 −0.01 +HCOO 281.0651, 163.1130, 121.0300 Citrusin C D
28 2.99 C27H22O18 634.0813 634.0806 1.16 −H 601.0460, 463.0518, 419.0617, 301.0007, 291.0156, 275.0208 Sanguiin H-4 a S
29 3.05 C14H12O4 244.0745 244.0736 3.14 +HCOO 203.0721, 187.0402, 161.0250, 123.0457, 109.0303 cis-Osthenone D
30 3.24 C15H18O8 326.1003 326.1002 0.33 +HCOO 162.0552, 129.0199, 121.0304 4-O-β-d-glucopyranosyl-trans-cinnamic acid a R, D
31 * 3.28 C26H32O11 520.1968 520.1945 4.46 +H 443.0984, 341.1392, 163.075 Brusatol R
32 3.42 C22H26O8 418.1631 418.1628 0.72 −H 359.1465, 179.0726, 164.0477, 149.0251, 125.0254 (+)-Syringaresinol D
33 3.55 C27H24O18 636.0969 636.0963 0.99 −H 483.0791, 465.0679, 331.0667, 313.0578, 169.0163 2,4,6-Tri-O-galloyl-β-d-glucose a S,L
34 * 3.65 C11H6O4 202.0260 202.0266 −2.32 +HCOO 163.0419, 149.0244, 134.0373, 133.0304, Xanthotoxol S, L
35 3.67 C45H38O18 866.2079 866.2058 2.37 −H 575.1207, 407.0781, 289.0730, 179.0356 Arecatannin A1 a D
36 3.76 C32H36O12 612.2223 612.2207 2.59 −H 562.1866, 518.1583, 210.0880, 135.0462 Filixic acid ABA a R
37 3.78 C21H22O12 466.1128 466.1111 3.59 −H 285.0428, 177.0208, 165.0568, 151.0053, 137.0257, 124.0178 Taxifolin-3-O-glucoside a D
38 3.80 C34H26O22 786.0915 786.0916 −0.08 −H 615.0646, 597.0511, 445.0416, 301.0021, 125.0258 Collinin a S
39 3.82 C24H28O9 460.1739 460.1733 1.24 −H 414.1699, 389.1244, 193.0528, 137.0261, 125.0258 Sanjidin A a R
40 4.30 C22H24O6 384.1560 384.1573 −2.96 +HCOO 325.1065, 313.1078, 310.0838, 150.0322 Sophoflavescenol a R
41 4.33 C9H6O5 194.0211 194.0215 −2.35 +H 177.0183, 153.0178, 138.0309, 127.0398 3,5,7-Trihydroxychromone D
42 4.38 C29H42O18 678.2395 678.2371 3.54 −H 497.1692, 453.1789, 323.0997, 291.1258, 161.0471 TangshenosideI R
43 4.48 C27H30O16 610.1554 610.1534 3.24 −H 463.0844, 313.0580, 265.0370, 190.9983, 151.0043 Quercetin-7-O-rutinoside L
44 4.50 C28H24O16 616.1084 616.1064 3.27 −H 313.0580, 190.9983, 177.0206, 169.0158, 151.0043 2′′-O-Galloylhyperoside a S, L
45 4.53 C11H12O3 192.0791 192.0786 2.21 +H 193.0863, 167.0703, 161.0603 Myristicin R
46 4.66 C34H46O18 742.2707 742.2684 2.90 +HCOO 579.2040, 417.1564, 181.0520, 149.0248 Syringaresinol-di-O-β-d-glucoside a D
47 4.72 C33H40O19 740.2178 740.2164 1.92 −H 593.1506, 575.1401, 429.0824, 335.0414, 284.0336 Grosvenorine a S, L
48 * 4.93 C27H30O16 610.1550 610.1534 2.59 −H 401.0912, 301.0365, 299.0205, 247.0609 Rutin S, L, D
49 4.94 C26H42O8 482.2874 482.2880 −1.13 +HCOO 261.1352,179.1074,149.0608, 125.0589 17-O-β-d-Glucopyra-nosyl-16β-H-ent-kauran-19-oicacid a R
50 * 4.96 C15H10O7 302.0427 302.0427 0.15 +H 161.0264, 123.0099, 109.0306, 107.0153 Delphinidin S, L
51 4.97 C15H10O8 318.0368 318.0376 −2.25 +HCOO 300.0266, 264.0562, 176.0132, 148.0176 Quercetagetin L
52 5.12 C27H30O15 594.1609 594.1585 4.02 −H 285.0403, 161.0459, 151.0038, 135.0452 Kaempferol-3-O-neohesperidoside R
53 5.14 C21H20O12 464.0945 464.0955 −2.16 −H 313.0549, 300.0266, 284.0330, 151.0041 Quercimeritrin S,L,D
54 5.17 C21H22O11 450.1178 450.1162 3.61 −H 193.0156, 179.0574, 175.0051, 151.0052, 135.0468 Dihydrokaempferol-5-O-β-d-glucopyranoside D
55 5.24 C15H10O6 286.0463 286.0477 −4.95 −H 256.0372, 177.0180, 164.0487, 150.0300, 123.0439, 107.0134 ω-Hydroxyemodin a D
56 5.25 C17H16O9 364.0780 364.0794 −3.53 +HCOO 337.0566, 278.0432, 202.0248, 185.0254, 149.0251 Bergaptol-O-β-d-glucopyranoside L
57 5.26 C15H12O7 304.0568 304.0583 −4.92 −H 285.0366, 243.0329, 152.0099, 150.0300, 125.0238 Dihydroquercetin D
58 5.27 C21H20O11 448.1005 448.1006 −0.19 −H 285.0406, 283.0256, 179.0569 Luteolin-7-O-glucopyranoside R,D
59 5.40 C15H10O6 286.0479 286.0477 0.70 +H 149.0216, 139.0371, 123.0433, 111.0439 7-Hydroxy-1-methoxy-2-methoxyxanthone a S, L
60 5.57 C41H32O26 940.1163 940.1182 −1.96 −H 769.0887, 617.0782, 313.0565, 291.0150, 169.0158 1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranoside a S, L
61 5.72 C20H18O11 434.0853 434.0849 0.80 −H 300.0301, 195.0321, 151.0050, 109.0305 Quercetin-3-O-arabinoside S
62 5.76 C30H36O8 524.2409 524.2410 −0.19 +HCOO 453.1908, 339.1256, 195.0667, 165.0570 Saucerneol C a R
63 5.79 C23H24O13 508.1224 508.1217 1.36 −H 315.0519, 207.0291, 193.0506, 151.0044, 137.0246 Limocitrin-3-O-β-d-glucopyranoside a L
64 5.83 C27H30O14 578.1637 578.1636 0.24 −H 269.0475, 227.0364, 177.0203, 151.0050, 119.0513 Apigenin-7-O-β-d-rutinoside D
65 5.84 C21H20O11 448.1016 448.1006 2.36 −H 295.0843, 284.0340, 179.0362, 151.0411, 123.0102 Quercetin-3-O-α-l-rhamnoside S
66 5.86 C14H18O3 234.1243 234.1256 −4.58 +H 175.0746, 163.0746, 133.0647, 119.0860, 111.0811 Lobetyol R
67 6.02 C26H38O13 558.2326 558.2312 2.37 +Na 217.1197, 199.1096, 145.0642, 128.0613, 115.0541 Lobetyolinin R
68 6.12 C21H20O10 432.1040 432.1056 −3.82 −H 268.0367, 227.0341, 177.0181, 151.0037, 124.0168 Cosmosiin D
69 6.17 C15H12O6 288.0643 288.0634 3.23 −H 271.0623, 177.0181, 151.0037, 133.0297, 125.0254, 107.0143 Dihydrokaempferol D
70 6.32 C9H10O4 182.0584 182.0579 2.65 −H 166.0263, 151.0040, 135.0452, 108.0226 2,6-Dimethoxy benzoic acid D
71 6.59 C21H24O7 388.1509 388.1522 −2.96 +HCOO 358.1066, 301.0369, 243.0306, 231.0308, 151.0047 β-Hydroxyisovalerylshikonin a D
72 6.61 C20H18O10 418.0892 418.0900 −1.74 +HCOO 358.1066, 243.0306, 231.0308, 178.9997, 151.0047, 121.0304 Cimicifugic acid D a D
73 6.70 C21H24O10 436.1373 436.1369 0.76 −H 273.0781, 255.0666, 179.0358, 149.0248, 123.0457 Epiafzelechin-3-O-β-d-allopyranoside a D
74 6.75 C42H68O16 828.4491 828.4507 −1.99 +H 667.4052, 651.4104, 505.3529, 487.3428, 469.3321, 421.3113 Platycosaponin A R
75 6.79 C22H22O10 446.1231 446.1213 3.66 +HCOO 285.0424, 187.0053, 163.0414, 124.0179 Rhamnocitrin-3-O-rhamnoside a S
76 6.81 C20H28O8 396.1793 396.1784 2.03 +HCOO 215.1094, 185.0984, 159.0826, 143.0724, 125.0616 Lobetyolin R
77 * 6.85 C64H104O34 1416.6388 1416.6409 −1.49 +H 811.4487, 763.42581, 647.37911, 485.3261 Deapio platycoside E R
78 6.93 C35H58O6 574.4227 574.4233 −1.03 +H 472.3166, 463.3096, 378.2044, 302.1716 α-Spinasterol glucoside R
79 * 6.98 C69H112O38 1548.6799 1548.6832 −2.13 +H 1007.5104, 845.4571, 683.4034, 521.3493, 485.3282 Platycoside E R
80 6.99 C23H24O11 476.1314 476.1319 −0.84 +HCOO 433.1097, 345.0819, 313.0554, 183.0309, 151.0041 5-Hydroxy-6,4′-dimethoxy-flavone-7-O-β-d-gluco-pyranoside S
81 7.35 C29H46O4 458.3396 458.3396 −0.05 +H 341.2455, 217.1953, 149.1333, 121.1027 Neotigogenin acetate a R
82 7.57 C58H94O29 1254.5905 1254.5881 1.95 +H 931.4894, 845.4518, 799.4485, 295.1007 Deapioplatycodin D3 R
83 * 7.68 C63H102O33 1386.6326 1386.6303 1.65 +H 1255.5937, 931.4894, 845.4518, 799.4484, 665.3879, 441.1585 Platycodin D3 R
84 7.69 C15H12O6 288.0629 288.0634 −1.59 +H 255.0652, 179.0353, 163.0400, 153.0196, 145.0295 3-Hydroxynaringenin a D
85 * 7.77 C15H10O7 302.0422 302.0427 −1.40 +H 243.0319, 151.0055, 125.0260, 107.0157 Quercetin S, L
86 7.86 C15H10O6 286.0488 286.0477 3.61 +H 269.0460, 257.0450, 241.0490, 161.0239, 135.0453 6-Hydroxyaloeemodin a D
87 7.91 C30H26O13 594.1373 594.1373 −0.14 −H 447.0966, 429.0832, 285.0440, 145.0316, 119.0513 Buddlenoid A a S, L
88 7.92 C47H76O20 960.4934 960.4930 0.39 +HCOO 869.4537, 715.3371, 529.2698, 295.2034 Platycoside F R
89 * 7.94 C63H102O32 1370.6373 1370.6354 1.40 +H 827.4398, 783.4476, 637.3944, 459.3430, 409.3090, 325.1130 Platycoside G3 R
90 8.33 C57H90O29 1238.5577 1238.5568 0.71 +H 1107.5237, 957.4692, 895.4676, 811.4125, 697.3760, 661.3582, 485.3245, 409.3094 Platyconic acid A D
91 * 8.46 C52H84O24 1092.5397 1092.5353 4.07 −H 959.4846, 941.4753, 681.3871, 663.3768, 649.3607, 503.3364, 485.3366, 295.1038, 277.0942 Deapioplatycodin D R
92 8.48 C59H92O30 1280.5649 1280.5673 −1.90 +H 1017.4875, 999.4760, 931.4860, 829.4192, 697.3796, 679.3651, 651.3761, 519.3316, 503.3334, 487.3377 Platycodin L R
93 * 8.51 C58H94O29 1254.5847 1254.5881 −2.65 +H 931.4894, 845.4518, 799.4485, 483.3065, 457.1533, 427.1433, 325.1116, 295.1007 Deapioplatycodin D2 R
94 * 8.62 C63H102O33 1386.6300 1386.6303 −0.26 +H 977.4981, 845.4558, 829.4604, 683.4031, 667.4073, 653.3919, 521.3488, 485.3273 Platycodin D2 R
95 * 8.68 C57H92O28 1224.5778 1224.5775 0.23 +H 799.4485, 683.3961, 667.4052, 521.3444, 503.3364, 485.3257 Platycodin D R, D
96 * 8.73 C65H104O34 1428.6407 1428.6409 −0.15 +H 1297.6065, 1165.5621, 845.4520, 841.4580, 681.3837, 665.3903, 653.3884, 617.3663, 519.3298, 485.3243 2′-O-Acetylplatycodin D2 R, D
97 8.78 C59H94O29 1266.5869 1266.5881 −0.93 +H 1003.5108, 841.4569, 823.4458, 683.3979, 189.0749, 171.0641 Platycodin A R, D
98 8.80 C65H104O33 1412.6458 1412.6460 −0.16 +H 985.4990, 823.4461, 635.3794, 617.3695, 503.3369, 453.1605, 321.1182, 303.1076, 189.5707 3′′-O-Acetylpolygalacin D2 R
99 8.86 C15H10O5 270.0539 270.0528 4.15 −H 151.0043, 123.0099, 117.0359, 107.0154 Apigenol D
100 8.87 C52H82O25 1106.5163 1106.5145 1.57 +H 975.4806, 931.4908, 829.4243, 811.4113, 697.3814, 679.3695, 517.3151, 503.3373, 455.3161 Platyconic acid C R
101 8.94 C59H92O30 1280.5705 1280.5673 2.47 +H 1017.4875, 829.4192, 697.3796, 637.3939, 519.3316, 321.1178 Platycodin K R, D
102 9.04 C54H86O25 1134.5444 1134.5458 −1.23 +H 1003.5108, 841.4569, 823.4458, 683.3979, 321.1160, 189.0749 Platycoside B R
103 * 9.10 C65H104O34 1428.6370 1428.6409 −2.71 +H 1297.6065, 955.4894, 841.4580, 813.4279, 797.4332, 681.3837, 665.3903, 653.3884, 635.3780 3′-O-acetyl-platycodin D2 R
104 9.11 C15H10O6 286.0483 286.0477 1.85 +H 231.0662, 229.0504, 195.0289, 153.0187, Kaempferol L
105 9.14 C20H24O11 440.1314 440.1319 −1.01 −H 393.0860, 303.0523, 257.0104, 231.0303, 177.0204 (-)-Chebulic acid triethyl ester a S, L
106 9.18 C65H104O33 1412.6430 1412.6460 −2.14 +H 823.4461, 503.3369, 485.3255, 455.3156, 321.1182, 189.0757 2′′-O-acetylpolygalacin D2 R, D
107 9.23 C59H94O28 1250.5904 1250.5932 −2.23 −H 1208.5857, 1159.5571, 635.3812, 499.3046, 131.0337 2′-O-acetyl Polygalacin D R
108 9.32 C20H22O11 438.1170 438.1162 1.78 −H 419.0956, 235.0654, 163.0050 6′-O-Galloyl-homoarbutin a S, L
109 9.37 C54H84O26 1148.5293 1148.5251 3.63 +H 1017.4908, 999.4786, 535.3279, 631.3477, 517.3170, 499.3050, 453.3001, 321.1190, 189.0764 Platyconic acid D R
110 9.45 C35H54O11 650.3666 650.3666 0.04 +HCOO 451.2830, 441.2997, 197.1183, 149.0465, 131.0354 15α-Hydroxy-ximicifugoside H2 a R
111 9.59 C37H60O12 696.4087 696.4085 0.28 −H 487.3424, 469.3302, 425.3438 3-O-d-glucopyranosyl platycodigenin methyl ester S
112 9.80 C30H42O7 514.2938 514.2931 1.41 −H 436.2610, 319.1910, 301.1814, 265.1468 Marstenacigenin A R
113 9.91 C36H58O12 682.3893 682.3928 −4.81 +HCOO 635.3797, 449.3263, 407.2948, 179.0565 3-O-d-glucopyranosyl platycodigenin R
114 9.94 C19H16O7 356.0886 356.0896 −2.55 +HCOO 401.0868, 313.0718, 121.0297 6-Formyl-isoophiopogonanone A a R
115 10.17 C15H18O3 246.1258 246.1256 0.84 +H 229.1220, 163.0756, 149.0598, 119.0865, 105.0713 Curcolone a S, L
116 10.25 C18H34O5 330.2418 330.2406 3.57 −H 311.2224, 293.2140, 211.1348, 185.1189, 129.0928 Sanleng acid a R, S, D
117 10.91 C15H14O4 258.0901 258.0892 3.45 −H 239.0705, 163.0397, 151.0421, 133.0313, 121.0296 Benzyl-2-hydroxy-6-methoxybenzoate D
118 * 10.95 C15H20O3 248.1413 248.1412 0.27 +H 231.1379, 219.1381, 203.1425, 119.0864, 107.0867 Atractylenolide ІІІ L
119 11.13 C15H20O2 232.1464 232.1463 0.24 +H 215.1424, 187.1486, 159.1172, 135.1174, 107.0867 Atractylenolide ІІ S, L
120 12.19 C16H12O6 300.0637 300.0634 1.18 +H 285.0761, 242.0571, 167.0340, 136.0162, 108.0215 5-Methyl kaempferol S, L
121 12.26 C17H14O6 314.0794 314.0790 1.05 +H 299.0552, 275.0673, 257.0445, 161.0597, 139.0397 3′,5-Dihydroxy-7,4′-dimethoxy flavone S
122 12.94 C17H26O4 294.1833 294.1831 0.56 −H 235.1341, 141.0919, 129.0924 6-Gingerol a R
123 13.46 C36H58O12 682.3905 682.3928 −3.36 −H 635.3787, 473.3258, 443.3119, 425.3020, 179.0553 Trachelosperoside B-1 a D
124 13.68 C30H48O5 488.3514 488.3502 2.47 −H 455.3548, 439.3599, 281.2503, 293.2127, 171.1035 2α,19α-Dihydroxyursolic acid L
125 13.91 C18H16O6 328.0949 328.0947 0.72 +H 314.0777, 296.0677, 184.0737, 136.0166 4′,7-Dimethyltectorigenin a S, L
126 * 14.58 C18H34O4 314.2466 314.2457 2.86 −H 201.1140, 199.0980, 155.1082, 127.1135 Dibutyl sebacate R
127 14.85 C19H18O7 358.1051 358.1053 −0.47 +H 343.0809, 326.0778, 301.0705, 283.0599 3,4-Dihydro-6,8-dihydroxyl-3-(2′-acetyl-3′-hydroxyl-5′-methoxyphenyl)methyl-1H-[2] benzoplyran-1-one a S, L
128 14.86 C17H30O2 266.2258 266.2246 3.76 +HCOO 311.2240, 155.1083, 139.1137 Methyl 7, 10-hexadecadienoate R
129 15.36 C30H48O7 520.3385 520.3400 −2.93 −H 476.2774, 473.3256, 443.3168, 425.3093, 407.2940, 395.2941 Platycodigenin D
130 15.39 C17H14O5 298.0843 298.0841 0.51 +H 284.0679, 256.0730, 241.0495, 167.0339, 133.0648 5-Hydroxy-7, 4′-dimethoxyflavanone S, L
131 15.57 C26H40O6 448.2818 448.2825 −1.59 +H 393.2636, 350.1875, 242.1877 Tenasogenin a R
132 15.89 C14H20O 204.1513 204.1514 −0.51 +H 163.1118, 159.1169, 149.0956, 119.0863, 107.0502 2-(p-Anisyl)-5-methyl-1-hexen L
133 16.28 C18H16O6 328.0957 328.0947 2.95 +H 314.0790, 299.0550, 286.0830, 271.0604, 150.0314 5-Hydro-7, 8, 2′-trimethoxyflavanone S, L
134 16.57 C32H44O9 572.2965 572.2985 −3.51 −H 481.2572, 429.2997, 227.0350, 183.1043 Ganoderic acid H a L
135 17.23 C30H48O4 472.3550 472.3553 −0.49 −H 471.3448, 437.3061, 419.2937, 339.2705, 253.2187 2α-Hydroxybetulinic acid S, L
136 17.62 C16H30O2 254.2252 254.2246 2.21 +Na 207.1743, 165.1274, 143.1067, 125.0961 Palmitoleic acid R
137 17.78 C18H34O3 298.2505 298.2508 −1.05 −H 217.1615, 195.1391, 183.1401, 113.0984 Ricinoleic acid D
138 18.00 C18H30O3 294.2203 294.2195 2.51 +Na 277.2177, 165.1284, 151.1127, 109.1035 (E,E)-9-Oxooctadeca-10,12-dienoic acid a R
139 18.01 C18H28O2 276.2100 276.2089 3.85 +H 179.1424, 135.1180, 119.0862 Stearidonic acid R
140 18.26 C28H42N4O6 530.3100 530.3104 −0.77 −H 529.3027, 511.2928, 293.2163 Kukoamine A a R
141 19.02 C18H32O3 296.2358 296.2351 2.19 +Na 279.2312, 161.1323, 147.1165, 133.1018, 121.1023 Coronaric acid R
142 19.23 C28H40O5 456.2878 456.2876 0.46 −H 409.2359, 343.1925, 339.2004, 275.2022 Siraitic acid D a R
143 20.35 C32H50O5 514.3662 514.3658 0.81 −H 495.3495, 469.3702, 451.3596, 449.3449 19α-Hydroxy-3-acetyl-ursolic acid S
144 20.39 C30H46O3 454.3452 454.3447 1.03 +H 437.3422, 409.3470, 247.1695, 203.1796, 189.1642 Oleanonic acid S
145 20.77 C30H48O3 456.3604 456.3603 0.13 −H 455.3531, 443.3528, 233.1561 3-Epioleanolic acid S
146 20.78 C33H36N4O6 584.2660 584.2635 4.08 +Na 567.2589, 535.2340, 501.2257, 467.20432, 417.1830 Bilirubin a L
147 21.49 C15H30O 226.2309 226.2297 4.48 +HCOO 271.2302, 197.1911, 195.1754 n-Pentadecanal S
148 22.20 C30H50O2 442.3803 442.3811 −1.76 +H 425.3776, 407.3666, 217.1950, 203.1791, 189.1641 Betulin R
149 * 22.93 C18H30O2 280.2402 280.2400 −0.25 −H 149.0972 Linolenic acid R
150 * 22.95 C19H38O4 330.2774 330.2770 1.00 +Na 313.2738, 239.2368 1-Monopalmitin S
151 22.98 C16H32O 240.2452 240.2453 −0.47 +Na 263.2344, 125.1317, 111.1175 n-Hexadecanal D
152 24.06 C21H42O 310.3240 310.3236 1.28 +HCOO 355.3214, 125.0972 n-Henicosanal S
153 24.40 C16H32O2 256.2401 256.2402 −0.49 −H 241.2176, 237.226, 227.2019, 125.0976 Palmitic acid S
154 24.74 C18H34O2 282.2569 282.2559 3.70 −H 253.2185, 163.1132, 125.0982, 111.0825 Ethyl palmitate R
155 25.73 C29H46O 410.3565 410.3549 4.03 +H 395.3680, 203.1799, 145.1021, 133.1019 Δ7-stigmasterol R
156 26.87 C24H38O4 390.2771 390.2770 0.21 +H 301.1413, 189.0156, 165.0905, 149.0235 Bis(2-ethylhexyl)phthalate R
157 27.09 C22H43NO 337.3356 337.3345 3.47 +H 321.3149, 212.2014, 198.1857, 153.1275 Erucic amide a R
158 27.63 C20H40O 296.3093 296.3079 4.10 +HCOO 251.2393, 179.1459, 113.0987 Phytol S
159 * 28.49 C29H48O 412.3695 412.3705 −2.48 +H 135.1178, 109.1025 Stigmasterol R
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* Identified with a reference standard. a Tentatively new identifications in Campanulaceae. The fragment ion mass highlighted as bold font is the characteristic MS fragmentation for each compound.

For the compounds which have isomers, they may be distinguished by their characteristic MS fragmentation patterns reported in literature, or may be compared with the retention times of reference standards. Taking compounds 98 and 106 as example, both have the same protonated ion [M + H]+ at m/z 1413.6530 and 1413.6530. In the results, they matched 3″-O-acetylpolygalacin D2 and 2″-O-acetylpolygalacin D2, respectively.

Their identical MS fragment pattern were similar. But according to the literature, the C3-glucoside was eluted earlier than the C2-glucoside [26,27,28] in the ESI-BPI chromatogram, so the compound with the earlier RT was identified as the C3-glucoside, 3″-O-acetylpolygalacin D2, and the other one with the later RT was identified as the C2-glucoside, 2″-O-acetylpolygalacin D2.

2.2. Biomarker Discovery for Differentiating Four Parts of PG

The PCA 2D plots of the samples from the root, stem, leaf and seed groups were classified in four clusters according to their common spectral characteristics (Figure 3). That means the four parts of PG could be easily differentiated.

Figure 3.

Figure 3

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PCA of root (R), stem (S), leaf (L) and seed (D) of PG in positive mode and negative mode.

In order to differentiate one part from other three parts, the OPLS-DA models were built in both positive and negative modes. Then, OPLS-DA score plot, S-plot, variable trend and VIP (variable importance in the projection) values were obtained to understand which variables are the responsible for this sample separation [29]. Based on VIP values (VIP > 4) (Figure 4) and p values (p < 0.05) [30] from univariate statistical analysis, 38 robust known biomarkers enabling the differentiation among root, stem, leaf and seed, were discovered and marked in S-plots (Figure 5). In order to systematically evaluate the biomarkers, a heatmap was generated from these biomarkers (shown in Figure 6), which shows distinct segregation among the four parts.

Figure 4.

Figure 4

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VIP value obtained from OPLS-DA model of the potential markers in root (R), stem (S), leaf (L) and seed (D) of PG.

Figure 5.

Figure 5

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The OPLS-DA/S-plots of root (I), stem (II), leaf (III) and seed (IV) of PG in positive mode and negative mode.

Figure 6.

Figure 6

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Heatmap visualizing the intensities of potential biomarkers.

3. Discussion

There are 73, 42, 35, 44 compounds that were characterized from the root, stem, leaf and seed, respectively. As the results show, 95 compounds were identified in ESI(−) mode and 64 compounds were identified in ESI(+) mode. According to the BPI chromatograms of the four parts of PG, it seems that ESI(−) ionization mode is better than ESI(+) based on the quantity and the responses of the identified compounds, but it is still necessary to run the ESI(+) mode because some compounds showed better respond than in ESI(−) mode.

Compared with the results from previous studies [2,8,16,31,32], 56 chemical components were identified for the first time in Campanulaceae. The stem, leaf and seed contain more flavonoids but few saponins that can be easily discriminated from the root. In previous study, various metabolites in Korean Platycodon grandiflorum were profiled by UPLC-QTOF/MS [16]. Compared with the root of PG in Korea, there were only nine constituents (compounds 5, 31, 76, 79, 83, 91, 94, 95, 97) in common. Meanwhile, the stems and leaves of PG in Korea and in China are both rich in natural components with various structural patterns, including triterpenoid saponins, flavonoids, organic acids, phenols, alcohols, amino acids, coumarins and amino acids, etc., but there are only two similar chemical components (compounds 99, 104). It is also interesting that there are eleven components (compounds 5, 14, 17, 21, 23, 31, 52, 83, 94, 95, 97) reported in stems and leaves of PG in Korea that were found in the root of PG in China. The reason for this phenomenon may be the different analytical methods and the different growing locations.

In this paper, 38 robust known biomarkers enabling the differentiation among root, stem, leaf and seed, were discovered. For the root part, there are 15 potential biomarkers including triterpenoid saponins (77, 79, 82, 83, 89, 91, 94, 95, 96, 97, 101, 102, 106), an organic acid (116) and a phenyl-propanoid (42). For stem part, there are five potential biomarkers including flavonoids (53, 61, 87), a tannin (7) and a triterpenoid saponin (144). For leaf part, there are seven potential biomarkers including flavonoids (47, 59, 125), sesquiterpenoids (115, 119) and tannins (26, 60). For seed part, there are 11 potential biomarkers including flavonoids (8, 18, 37, 57, 69, 73, 84, 99), quinones (55, 86) and an organic acid (117). These robust biomarkers enabling the differentiation among root, stem, leaf and seed can be used for rapid identification of four different parts of PG grown in northeast China.

Even so, there are still some unresolved issues. Firstly, pharmaceutical effects associated with these robust biomarkers or these identified compounds should be screened in the future. Additionally, as shown in BPI chromatograms, though 159 compounds were identified there are still many unidentified components. Further research should be carried on based on the formula of these unknown compounds [13]. Most importantly, the stems and leaves of PG should be developed and utilized due to the presence of so many different components from the root. This comprehensive and unique phytochemical profile study revealed the structural diversity of secondary metabolites and the different patterns in various parts of PG. The method developed in this study can be used as a standard protocol for discriminating and predicting parts of PG directly.

4. Experimental Section

4.1. Materials and Reagents

All samples were harvested from Jilin Province, China, as listed in Table 2, and identified by Professor Ping-Ya Li (School of Pharmaceutical Sciences, Jilin University, Changchun, China). The voucher specimens (No. 2016121-2016144) had been deposited at the Research Center of Natural Drug, School of Pharmaceutical Sciences, Jilin University, Changchun, China. The cultivation ages of the roots are all 2 years, while the others are all 1 year old.

Table 2.

Information of samples from Jilin Province, China.

Collection Region Mark of Samples Collection Date Collection Region Mark of Samples Collection Date
Antu County S1 2 October 2016 Fusong County S4 4 October 2016
L1 2 October 2016 L4 4 October 2016
R1 26 October 2016 R4 30 October 2016
D1 2 October 2016 D4 4 October 2016
Hunchun City S2 1 October 2016 Tonghua City S5 5 October 2016
L2 1 October 2016 L5 5 October 2016
R2 27 October 2016 R5 28 October 2016
D2 1 October 2016 D5 5 October 2016
Changbai County S3 30 September 2016 Jiaohe City S6 3 October 2016
L3 30 September 2016 L6 3 October 2016
R3 29 October 2016 R6 25 October 2016
D3 30 September 2016 D6 3 October 2016
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S: stem, L: leaf, R: root; D: seed.

Acetonitrile and methanol suitable for UHPLC-MS purchased from Fisher Chemical Company (Geel, Belgium). Formic acid for UPLC was purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was purified using a Millipore water purification system (Millipore, Billerica, MA, USA). All other chemicals were of analytical grade. Fourteen standard compounds including platycodin D (111851-201607), mannitol (100533-201304), citric acid (111679-201602), phenylalanine (140676-201405), tryptophan (140686-201303), chlorogenic acid (110753-201716), caffeic acid (110885-201102), dibutyl sebacate (190102-201501), linolenic acid (111631-201605), sucrose (111507-201303), adenosine (110879-201202), monopalmitin (190011-201302), rutin (100080-201610), quercetin (100081-201610), were purchased from the National Institutes for Food and Drug Control (Beijing, China). Seven standard compounds including gallocatechin (201512013), quinine acid (20150321), brusatol (20150410), stigmasterol (20150111), xanthotoxol (20109376), delphinidin (20159567), and atractylenolide ІІІ (2014712) were purchased from Beijing Putian Genesis Biotechnology Co., Ltd. (Beijing, China). Nine standard compounds including deapioplatycoside E (160712), deapioplatycodin D (160518), -D2 (160407), platycoside E (160112), platycodin D2 (160721), -D3 (160909), platycoside G3 (160921), 2′-O-acetyl-platycodin D2 (160112), 3′-O-acetylplatycodin D2 (160923) were provided by Institute of Frontier Medical Science of Jilin University (Changchun, China).

4.2. Sample Preparation and Extraction

The roots, stems, leaves and seeds of PG from the different sites were respectively air dried, ground and sieved (40 mesh) to give a homogeneous powder. Then 200 mg of the powder was respectively extracted thrice with 80% methanol at 80 °C for 3 h each time. After filtering, the extracts were combined, concentrated and evaporated to dryness. Finally, the desiccated extracts were dissolved and diluted with 80% methanol to 10.0 mL. The solution was filtered through a syringe filter (0.22 µm) and injected directly into the UPLC system. The volume injected was 2 μL for each run.

4.3. UPLC-QTOF-MSE

The UPLC analysis was performed by a Waters ACQUITY UPLC System. The column used was an ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) from Waters Corporation (Milford, MA, USA). The mobile phases consisted of eluent A (0.1% formic acid in water, v/v) and eluent B (0.1% formic acid in acetonitrile, v/v) with flow rate of 0.4 mL/min with a liner gradient program: 10% B from 0 to 2 min, 10–90% B from 2 to 26 min, 90% B from 26 to 28 min, 90–10% B from 28 to 28.1 min, 10% B from 28.1 to 30 min. The temperature of the UPLC column and autosampler were set at 30 °C and 15 °C. Mixtures of 10/90 and 90/10 water/acetonitrile were used as the strong wash and the weak wash solvent respectively.

The MS experiments were performed on a Waters Xevo G2-S QTOF mass spectrometer (Waters Co., Milford, MA, USA.) connected to the UPLC system through an electrospray ionization (ESI) interface. The optimized instrumental parameters were as follows: capillary voltage floating at 2.6 kV (ESI+) or 2.2 kV (ESI−); cone voltage at 40 V; source temperature at 120 °C, desolvation temperature at 300 °C and cone gas flow was 50 L/h, desolvation gas flow was 800 L/h. In MSE mode, collision energy of low energy function was set at 6 V, while ramp collision energy of high energy function was set at 20–40 V. To ensure mass accuracy and reproducibility, the mass spectrometer was calibrated over a range of 100–1600 Da with sodium formate. Leucine-enkephalin (m/z 556.2771 in positive ion mode; m/z 554.2615 in negtive ion mode) was used as the lockmass at a concentration of 200 ng/mL and flow rate of 20 μL/min. Data were collected in continuum mode, all the acquisition of data were controlled by the Waters MassLynx v.4.1 software ( waters, Milford, MA, USA).

4.4. Data Analysis

For the screening analysis, the raw data were processed using the streamlined workflow of UNIFI 1.7.0 software (Waters, Manchester, UK) to quickly identify the chemical components [15]. Besides the Waters Traditional Medicine Library in the UNIFI software, a self-built database was created including the information of chemical components from PG based on the literature and on-line databases such as China Full-text Journals Database (CNKI), PubMed, Medline, Web of Science and ChemSpider. Minimum peak area of 200 was set for 2D peak detection.The peak intensity of high energy over 200 counts and over 1000 counts for low energy were the selected parameters in 3D peak detection. A margin of error up to 5 ppm for identified compounds was allowed. Positive adducts containing +H, +Na, and negative adducts including +COOH and −H were selected. The verification of compounds was carried out by comparison with retention time of reference standards and characteristic MS fragmentation patterns reported in literature.

For metabonomics analysis, the raw data were processed by MarkerLynx XS V4.1 software for alignment, deconvolution, data reduction, etc. [33]. As a result, the list of mass and retention time pairs with corresponding intensities for all the detected peaks from each data file. The main parameters were as follows: retention time range 0–28 min, mass range 100–1600 Da, mass tolerance 0.10, minimum intensity 5%, marker intensity threshold 2000 counts, mass window 0.10, retention time window 0.20, and noise elimination level 6. The resulting data were analyzed by principle component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). S-plots and VIP-plots were obtained via OPLS-DA analysis to find potential biomarkers that significantly contributed to the difference among the groups.

5. Conclusions

In the present study, UPLC-QTOF-MSE coupled with UNIFI platform and precise multivariate statistical analyses was used to profile the four parts of PG. For the constituent screening under the optimized conditions, a total of 159 chemical compounds (73, 42, 35, 44 compounds characterized from root, stem, leaf and seed, respectively) were identified from PG. The results showed various structural patterns including triterpenoid saponins, organic acids, steroids, phenols, flavonoids, alcohols, amino acids, coumarins, terpenoids, alkaloids and amides. The stem, leaf and seed contain more flavonoids but few saponins that can be easily discriminated from the root.

For the metabolomic analysis, four parts of PG were successfully discriminated into four different clusters. A total of 38 robust biomarkers were discovered. That is to say, 15, 5, 7, and 11 robust biomarkers enabling the differentiation among root, stem, leaf and seed, were characterized. These biomarkers can be suitable for the simultaneous differentiation of four different parts of PG, which is reported for the first time. In a word, these results provided the reliable characterization profiles and the differentiate components among root, leaf, stem and seed of PG grown in northeast China. The method developed in this study can be used as a standard protocol for discriminating and predicting the different parts of PG directly.

Acknowledgments

This work was supported by Talents Team Major Program of Jilin Province of China (JRCBTZ. [2016] No. 3).

Author Contributions

Pingya Li and Jinping Liu conceived and designed the experiments; Cuizhu Wang, Nanqi Zhang and Zhenzhou Wang performed the experiments; Cuizhu Wang, Zeng Qi, Hailin Zhu and Bingzhen Zheng were responsible for data analysis. Cuizhu Wang wrote the paper. Jinping Liu and Pingya Li assisted paper revision.

Conflicts of Interest

The authors declare that they have no conflicts of interest concerning this article.

Footnotes

Sample Availability: Samples of the compounds are available from the authors.

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