Skip to main content
NCBI home page
Molecules logoLink to Molecules
. 2021 Mar 25;26(7):1855. doi: 10.3390/molecules26071855

Comparative Analysis of Chemical Constituents in Different Parts of Lotus by UPLC and QToF-MS

Haotian Pei 1, Wenyu Su 1, Meng Gui 1, Mingjie Dou 1, Yingxin Zhang 1, Cuizhu Wang 1, Dan Lu 1,*
Editor: Evagelos Gikas1
PMCID: PMC8036816  PMID: 33806084

Abstract

Six parts of lotus (seeds, leaves, plumule, stamens, receptacles and rhizome nodes) are herbal medicines that are listed in the Chinese Pharmacopoeia. Their indications and functions have been confirmed by a long history of clinical practice. To fully understand the material basis of clinical applications, UPLC-QToF-MS combined with the UNIFI platform and multivariate statistical analysis was used in this study. As a result, a total of 171 compounds were detected and characterized from the six parts, and 23 robust biomarkers were discovered. The method can be used as a standard protocol for the direct identification and prediction of the six parts of lotus. Meanwhile, these discoveries are valuable for improving the quality control method of herbal medicines. Most importantly, this was the first time that alkaloids were detected in the stamen, and terpenoids were detected in the cored seed. The stamen is a noteworthy part because it contains the greatest diversity of flavonoids and terpenoids, but research on the stamen is rather limited.

Keywords: lotus, UPLC-QToF-MS, phytochemicals, analysis, different parts, metabolomics

1. Introduction

Nelumbo nucifera Gaerth., an aquatic plant in the Nymphaeaceae family, is distributed in wetlands throughout Asia. It is also known as sacred lotus; it is not only used as a food and herb, but also deeply related to Buddhism in Asia, and its flower is used as the pedestal for divine figures. The medicinal functions of lotus were recognized earlier than its edible value and were recorded for the first time in the book “Er ya” (400 B.C.) [1]. Almost all parts of this plant have been used as food as well as herbal medicine for over 2000 years, and especially the lotus seed and rhizome are more widely used due to their delicious taste and great nutritive value [2]. The seed (Nelumbinis Semen), leaf (Nelumbinis Folium), plumule (Nelumbinis Plumula), stamen (Nelumbinis Stamen), receptacle (Nelumbinis Receptaculum) and rhizome node (Nelumbinis Rhizomatis Nodus) are listed in the official Pharmacopoeia of China (CP). Their indications and functions, which were confirmed by clinical practice for thousands of years, were recorded in the Chinese Pharmacopoeia. According to the records, lotus leaves, receptacles and rhizome nodes have a hemostatic effect; stamens can prohibit pathological spermatorrhoea and frequent urination; and the seeds and plumule can mind-tranquilize and improve sleep [3] (Figure 1).

Figure 1.

Figure 1

Open in a new tab

Schematic diagram of the whole lotus plant. The part listed in CP has a blue highlight in the background.

In recent decades, increasing research has focused on this special herb, especially the plumule and leaf, and many of its constituents have been found to possess extensive features of health benefits. The ingredients and activities of the different lotus parts, together with their applications in the food and healthcare area, have similarities and differences [2]. Without a doubt, the pharmacological effects of any plant or effective part are based on their phytochemicals. Flavonoids that have been found in the six parts of lotus are associated with a variety effects, including antioxidant [4,5,6,7], anti-inflammatory [8], antiviral [9,10], anti-obesity [11,12], and antimicrobial effects [13]. Alkaloids are effective ingredients used for treating cardiovascular diseases [14,15,16], regulating blood lipids [17], tranquilizing the mind [18] and treating cancer [19]. However, the chemical bases of their activities are far from clear compared with the medical history of thousands of years. For example, stamens and receptacles are reported to have anti-ischemic effects [20]; meanwhile, neferine and liensinine were confirmed to be anti-ischemic agents [21], but there is no proof that stamens contain alkaloids. Thus, just what do the stamens and receptacles contain that contributes to the anti-ischemic effect? Furthermore, the material basis for traditional uses as hemostatic agents is a “blank” area. Moreover, there are no index components for the quality control of seeds, stamens, receptacles and rhizome nodes in CP. Hence, this study focuses on the chemical constituents of the six parts of lotus.

UPLC is the best chromatographic method in terms of resolution, sensitivity, and speed. QToF-MS is the most sensitive quantitative and most comprehensive qualitative detector to identify and quantify the broadest range of compounds in the most complex and challenging samples. With its combined ability of high resolution and sensitivity, UPLC-QToF-MS has been successfully used for the analyses of complex samples [22]. It can be used for the rapid differentiation of different parts of a plant [23], identification of the habitats of herbs [24], and evaluation of the quality of TCMs and processed products [25]. The UNIFI information system has the ability to incorporate scientific library into with UPLC and QToF-MS data, which streamlines the process of identifying chemical structures in complex natural products. To obtain the chemical constituent profile of the cored seeds (Sem), leaves(Fol), plumule (Plu), stamens (Sta), receptacles (Rec) and rhizome nodes (RN) of lotus, we proposed a multiple ingredients identification strategy based on UPLC-QToF-MS coupled with the UNIFI informatics platform. This method can quickly identify multiple components. In this study, the constituents of the six parts were efficiently separated by ultra-performance liquid chromatography (UPLC) and detected by quadrupole time-of-flight tandem mass spectrometry (QToF-MS). Then, the data obtained by UPLC and QToF-MS were processed by the integrated information platform UNIFI.

As a result, a total of 171 components were identified from the six parts. Moreover, the differentiating components were screened by principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). These methods provide a holistic and intuitionistic description of the chemical constituents in the six parts. Twenty-three robust biomarkers were found to distinguish the six parts. The established method can be used as a standard protocol for directly discriminating between and predicting the six parts of lotus. Most importantly, data analysis provided useful information for further study and usage of the plant.

2. Results

2.1. Identification of Components

A total of 171 compounds were identified or tentatively characterized from the six parts of lotus, including alkaloids, flavonoids, terpenoids, steroids, organic acids, etc. Among them, 84 compounds were identified in positive mode, and 127 compounds were identified in negative mode. The base peak intensity (BPI) chromatograms marked with the number of compounds are shown in Figure 2. The compound identification data were listed in Table 1. The chemical structures of the compounds are shown in Figure 3. More specifically, 86, 56, 89, 87, 60 and 19 compounds were identified from Sem, Fol, Plu, Sta, Rec and RN, respectively. By comparing the numbers of the detected compounds and signal strength of the chromatographic peak observed in the UPLC-QToF-MS assay, it seems that ESI− mode is better than ESI+ for this test. However, running the ESI+ mode is still necessary because some compounds display better responses in ESI+ mode than in ESI−.

Figure 2.

Figure 2

Open in a new tab

The representative BPI chromatograms of Sem, Fol, Plu, Sta, Rec, and RN in positive and negative modes.

Table 1.

Compounds identified from different parts of lotus by UPLC-QToF-MSE.

No. tR (min) Formula Theoretical (Da) Calculated Mass (Da) Mass Error (ppm) MSE Fragmentation Identification Sources Ref.
1 0.53 C24H42O21 666.2219 666.2226 1.12 711.1808[M + HCOO]−, 665.1649[M − H]−, 485.1080[M-H-Glc]−, 179.0417[Glc-H]−,
161.0367[Glc-H-H2O]−, 689.2103[M + Na]+ 		Nystose Sem,Plu,Sta b
2 0.55 C18H32O16 504.1690 504.1678 −2.40 527.1895[M + Na]+,
505.1786[M + H]+,
503.1612[M − H]−, 485.1170[M-H-H2O]−, 179.0417[Glc-H]− 		Gentiotriose Sem,Plu,Sta b
3 0.57 C5H10O5 150.0528 150.05 0.24 195.0352[M + HCOO]−, 149.0345[M − H]− Arabinose Plu,Sta b
4 0.57 C12H22O11 342.1162 342.1166 1.09 341.0829[M − H]−, 163.0594[M-H-Glc-H2O]− Isomaltose Sem,Plu,Sta b
5 0.62 C12H22O11 342.1162 342.1152 −2.89 365.1058[M + Na]+, 163.0750[M+H-H2O]+ Sucrose Sem,Plu,Sta b
6 0.75 C6H8O7 192.0270 192.0266 −2.29 191.0192[M − H]−, 147.0065[M-COOH]−, 130.9980[M-H-COOH-CH3]− Citric acid Sem,Plu,Sta,Rec,RN b
7 0.78 C7H4O6 184.0008 184.0004 −2.00 182.9704[M − H]−, 138.9851[M-COOH]− Chelidonic acid Plu,Rec
8 1.09 C9H11NO2 165.0790 165.0785 −2.69 210.0535[M + HCOO]−, 164.0521[M − H]− Gentiatibetine Sem,Sta b
9 1.24 C9H8O3 164.0473 164.0478 2.87 209.0189[M + HCOO]−, 163.0256[M − H]−, 119.0387[M-OC2H5]− p-Coumaric acid Plu,Rec b
10 1.24 C15H18O8 326.1002 326.1008 1.80 325.0659[M − H]−, 163.0256(C9H7O3) Melilotoside Plu b
11 1.24 C16H20O10 372.1056 372.1061 1.09 371.0664[M − H]−, 165.0267[M-H-Aglc-CO2]− Deacetyl asperuloside Plu
12 1.24 C21H20O12 464.0955 464.0956 0.22 509.0485[M + HCOO]−, 463.0916[M − H]− 6-Hydroxyluteolin-7-β-d-glucopyranoside Plu [26]
13 1.45 C16H17NO3 271.1208 271.1205 −1.20 272.1302[M + H]+, 255.1011(C16H15O3), 237.0922(C16H13O2), 209.0978(C15H13O), 161.0595(C10H9O2), 143.0503(C10H7O), 107.0515(C7H7O) Higenamine Fol,Plu b
14 1.52 C17H19NO3 285.1365 285.1370 1.66 286.1447[M + H]+, 284.1104[M − H]−, 237.0922(C16H13O2), 209.0949(C15H13O), 115.0560(C9H7), 107.0515(C7H7O) Coclaurine Sem,Plu [26]
15 1.78 C11H12O5 224.0685 224.0694 4.04 223.0492[M − H]−, 205.0236[M-H-H2O]−, 179.0253[M-COOH]− Sinapic acid Sem,Fol,Plu b
16 1.95 C17H22O10 386.1213 386.1208 −1.36 431.1234[M + HCOO]−, 385.0857[M − H]− Hedyotoside Plu
17 2.10 C19H23NO3 313.1678 313.1676 −0.58 314.1734[M + H]+, 283.0286[M-CH3NH2]+, 206.1181(C12H17NO2), 107.0875(C7H7O) Armepavine Sem,Fol,Plu,Sta,Rec,RN [26]
18 3.02 C19H28O12 448.1581 448.1571 −2.27 493.0695[M + HCOO]−, 447.1161[M − H]− 6-O-Acetylshanzhiside methyl ester Plu
19 3.08 C18H26O10 402.1526 402.1522 −1.00 447.0945[M + HCOO]−, 401.1143[M − H]− Benzyl alcohol xylopyranose (1→6) glucopyranoside Sem,Plu,Sta b
20 3.31 C18H21NO3 299.1521 299.1517 −1.59 300.1602[M + H]+, 269.1184(C17H17O3), 237.1457(C16H13O2), 209.0475(C15H13O) N-Methylisococlaurine Sem,Plu,Sta b
21 3.38 C9H14O7 234.0740 234.0728 −4.83 257.0685[M + Na]+,
235.0869[M + H]+ 		Trimethyl citrate Fol
22 3.40 C9H6O4 178.0266 178.0266 0.10 177.0034[M − H]−, 133.0152 Esculetin Sem
23 3.44 C15H16O6 292.0947 292.0946 −0.31 337.0595[M + HCOO]−, 291.0453[M − H]− Cnidimol D Sta
24 3.47 C17H19NO3 285.1365 285.1352 −4.40 286.1412[M + H]+, 209.1126 Aposcopolamine Fol,Sta [26]
25 3.54 C20H30O11 446.1788 446.1790 0.52 491.1207[M + HCOO]−, 445.1222[M − H]− Hedyoside Sta
26 3.69 C21H22O11 450.1162 450.1161 −0.24 449.0625[M − H]−, 431.1064[M-H-H2O]− Miscanthoside Sta,Rec b
27 3.78 C27H30O15 594.1585 594.1580 −0.86 639.0868[M + HCOO]−, 593.1044[M − H]− Nicotiflorin Sem,Plu,Sta,Rec
28 4.00 C20H25NO3 327.1834 327.1830 −1.36 328.1913[M + H]+, 300.1531,283.6312 Leonticine Plu [26]
29 4.03 C26H28O15 580.1428 580.1422 −1.14 581.1528[M + H]+, 547.1465 Kaempferol-3-O-β-d-glucopyranoside-7-O-α-L-arabinofuranoside Plu [26]
30 4.04 C10H8OS2 208.0017 208.0009 −3.66 252.9815[M + HCOO]−, 206.9634[M − H]− 1-(5-Thiophen-2-ylthiophen-2-yl)ethanone Fol
31 4.04 C15H10O8 318.0376 318.0385 2.96 316.9868[M − H]−, 298.9766[M-H-H2O]− Myricetin Fol b
32 4.34 C32H38O19 726.2007 726.1977 −4.15 771.1393[M + HCOO]−, 725.1418[M − H]− Vaccarin Plu
33 4.47 C26H28O14 564.1479 564.1487 1.33 563.1066[M − H]−, 493.1105 Patuletin-7-O-[6′′-(2-Methylbutyryl)]-glucopyranoside Sem,Plu,Sta,Rec [26]
34 4.54 C8H8O4 168.0423 168.0425 1.64 167.0135[M − H]−, 106.9976[M-H-CH3-COOH]− Isovanillic acid Fol,Rec b
35 4.58 C26H28O14 564.1479 564.1477 −0.28 565.1638[M + H]+, 445.1074 Apiin Sem,Plu,Rec
36 4.61 C16H10O5 282.0528 282.0530 0.46 327.0220[M + HCOO]−, 281.0124[M − H]−, 237.0462[M-COOH]− Pseudobaptigenin Plu b
37 4.65 C19H21NO4 327.1471 327.1466 −1.54 328.1542[M + H]+, 297.6281[M + H-OCH3]+, 296.1195[M + H-CH3OH]+ Norisocorydin Fol,Plu b
38 4.65 C36H40N2O6 596.2886 596.2883 −0.50 641.2319[M + HCOO]−, 597.2950[M + H]+ Dauriciline Plu [26]
39 4.75 C26H28O16 596.1377 596.1378 0.16 641.0141[M + HCOO]−, 595.0060[M − H]− Isoetin-7-O-β-d-glucopyranosyl-2′-O-β-D-xyloypyranoside Sta,Rec
40 4.79 C21H22O11 450.1162 450.1154 −1.82 495.0541[M + HCOO]−, 449.0191[M-H]−, 431.1490[M-H-H2O]− Astilbin Sta b
41 4.93 C37H42N2O6 610.3043 610.3034 −1.40 609.2213[M − H]−, 503.1425(C30H35N2O5), 489.0128(C29H33N2O5) Liensinine Sem,Fol,Plu,Rec b
42 4.96 C15H26O 222.1984 222.1973 −4.95 267.1642[M + HCOO]−, 221.0231[M − H]− Pogostol Fol,Plu b
43 4.96 C18H21NO3 299.1521 299.1517 −1.48 300.1708[M + H]+, 269.1218(C17H17O3), 237.0769(C16H13O2), 209.1600(C15H13O), 107.0537(C7H7O) N-Methylcoclaurine Fol,Rec [26]
44 5.17 C37H42N2O6 610.3043 610.3047 0.61 611.3093[M + H]+, 568.2705 Dauricinoline Plu [26]
45 5.24 C15H12O7 304.0583 304.0588 1.59 349.0178[M + HCOO]−, 303.0161[M − H]− Taxifolin Fol,Rec
46 5.28 C16H28O7 332.1835 332.1835 −0.13 377.1581[M + HCOO]−, 331.1492[M − H]− Betulalbuside A Sem b
47 5.32 C26H28O14 564.1479 564.1498 3.37 609.1000[M + HCOO]−, 300.0008[A-H]− Rutin Plu [26]
48 5.35 C18H10O8 354.0376 354.0382 1.73 355.0372[M + H]+,
353.0079[M − H]− 		Mongolicumin A Sem
49 5.39 C27H30O16 610.1534 610.1525 −1.52 611.1575[M + H]+,
609.0798[M − H]− 		Nelumboroside A Sem,Plu,Sta [26]
50 5.43 C16H20O8 340.1158 340.1156 −0.56 385.0656[M + HCOO]−, 177.0334(C10H9O3) Linocinnamarin Sem,Fol b
51 5.57 C21H18O13 478.0747 478.0758 2.14 477.0278[M − H]−, 301.0090[A]−, 300.0008[A-H]− Quercetin 3-O-glucuronide Fol,Plu,Sta,Rec b
52 5.64 C15H10O7 302.0427 302.0418 −2.73 303.0519[M + H]+, 237.0421 Quercetin Fol,Plu,Sta,Rec b
53 5.67 C21H20O11 448.1006 448.1004 −0.30 447.0599[M − H]−, 429.1443(C21H17O10), 357.0284[M-H-90]−, 327.0294[M-H-120]−, 297.0076[M-H-150]− Orientin Sem,Plu,Fol b
54 5.67 C26H28O15 580.1428 580.1439 1.89 625.1139[M + HCOO]−, 579.0697[M − H]− Lutelin-7-O-[β-d-apiofuranosyl(1→6)]β-d-glucopyranoside Sem,Plu,Sta [26]
55 5.67 C27H30O14 578.1636 578.1644 1.49 577.1150[M − H]−, 431.0639, 413.0504 Kaempferitrin Plu b
56 5.70 C21H20O12 464.0955 464.0952 −0.54 463.0343[M − H]−, 300.9948[A-H]−, 299.9901[A-2H]− Isoquercetin Sem,Plu,Sta,Rec a
57 5.71 C21H20O11 448.1006 448.1008 0.50 493.0378[M + HCOO]−, 447.0382[M − H]−,
429.1273[M-H-H2O]−, 300.9948[M-H-Glc]− 		Quercitrin Fol,Sta b
58 5.74 C21H20O11 448.1006 448.0992 −3.14 449.1170[M + H]+, 287.0624(C15H11O6) Luteolin-7-O-glucoside Fol,Plu,Sta b
59 5.86 C38H44N2O6 624.3199 624.3201 0.30 625.3275[M + H]+, 594.2842[M+H-NH2CH3]+, 582.2826[M+H-CH2=N-CH3]+, 489.2331(C29H33N2O5), 206.1181(C12H16NO2), 121.0652(C8H9O) Neferine Sem,Fol,Plu b
60 5.89 C19H23NO4 329.1627 329.1621 −1.90 330.1620[M + H]+,
328.1265[M − H]− 		Sinomenine Sem,Plu
61 5.92 C22H22O10 446.1213 446.1201 −2.57 491.0754[M + HCOO]−, 427.1105[M-H-H2O]− Sissotrin Sem,Plu b
62 5.98 C18H19NO2 281.1416 281.1418 0.83 282.1552[M + H]+, 253.1186(C17H17O2), 251.1113(C17H15O2) Floribundine Fol,RN b
63 5.99 C12H15NO4 237.1001 237.0994 −3.16 282.0810[M + HCOO]−, 236.0730[M − H]− Desmodimine Plu,Sta
64 6.03 C21H20O11 448.1006 448.1013 1.59 447.0425[M − H]−, 285.0017[M-H-Glc]− Trifolin Sta b
65 6.17 C22H20O10 444.1056 444.1068 2.66 489.0943[M + HCOO]−, 443.0680[M − H]− Apigenin-7-O-glucuronide Sem
66 6.24 C20H22O6 358.1416 358.1428 3.30 357.1096[M − H]−, 339.0475[M-H-H2O]− Glicophenone Sem b
67 6.24 C28H32O16 624.1690 624.1705 2.29 669.0948[M + HCOO]−, 623.1085[M − H]−, 315.0210[A]− Isorhamnetin 3-O-rutinoside Plu,Sta,Rec b
68 6.28 C21H18O12 462.0798 462.0814 3.40 507.0176[M + HCOO]−, 461.0183[M − H]−, 285.0052(C15H9O6) kaempferol-3-O-glucuronide Fol,Sta,Rec b
69 6.29 C28H32O16 624.1690 624.1673 −2.81 625.1740[M + H]+, 607.2749[M+H-H2O]+, 317.0661(C16H13O7) Isorhamnetin 3-O-robinobioside Plu,Sta b
70 6.34 C21H20O10 432.1056 432.1066 2.27 477.0591[M + HCOO]−, 431.0469[M − H]− Cosmosiin Sem,Plu,Sta,Rec b
71 6.36 C25H33N5O7 515.2380 515.2365 −2.91 538.2259[M + Na]+,
516.1849[M + H]+ 		Asterinin D Sem
72 6.45 C27H30O14 578.1636 578.1632 −0.62 623.2516[M + HCOO]−, 577.1199[M − H]− Sophorabioside Sem,Sta b
73 6.48 C15H10O5 270.0528 270.0522 −2.44 269.0117[M − H]−,
271.0582[M + H]+ 		Apigenin Sem,Plu,Sta,Rec b
74 6.52 C43H42O22 910.2168 910.2125 −4.72 955.0959[M + HCOO]−, 909.0781[M − H]− Carthamin Sta b
75 6.57 C16H12O7 316.0583 316.0574 −2.88 361.0094[M + HCOO]−, 317.0589[M + H]+,
315.0173[M − H]−, 151.0941(C7H3O4)		 Isorhamnetin Fol,Sta,Rec b
76 6.57 C17H14O8 346.0689 346.0677 −3.49 347.0693[M + H]+, 332.0417[M+H-CH3]+ Limocitrin Sta b
77 6.57 C22H22O12 478.1111 478.1098 −2.81 479.1059[M + H]+,
477.0547[M − H]−,
459.0726[M-H-H2O]−, 315.0173[M-Glc]− 		Nepitrin Sta,Rec b
78 6.66 C19H30O7 370.1992 370.1996 1.14 415.1674[M + HCOO]−, 371.2011[M + H]+ (6R,9R)-3-Oxo-α-ionol β-d-glucoside Sem
79 6.66 C20H18O10 418.0900 418.0901 0.33 417.0346[M − H]−, 285.0052 Juglalin Sta b
80 6.66 C22H20O13 492.0904 492.0917 2.68 493.0865[M + H]+,
491.0209[M − H]− 		Quercetin-3-O-β-d-glucuronide-6″-methyl ester Fol,Sta,Rec
81 6.70 C9H16O4 188.1049 188.1045 −1.70 187.0818[M − H]−, 169.0681[M-H-H2O]−, 143.0950[M-COOH]− Azelaic acid Sem,Plu,Sta b
82 6.74 C15H12O6 288.0634 288.0641 2.31 287.0242[M − H]−, 125.0811(C6H5O3) 2-Hydroxynaringenin Sta b
83 6.74 C25H28O5 408.1937 408.1921 -3.85 453.1553[M + HCOO]−, 407.1535[M − H]− 2′,4′,7-Trihydroxy-6,8-bis(3-methyl-2-butenyl)flavanone Sem
84 6.74 C19H32O7 372.2148 372.2158 2.56 417.1852[M + HCOO]−, 371.1847[M − H]−, 209.0633[M-Glc]−,
373.2178[M + H]+ 		Blumenol C glucoside Sem,Fol,Plu b
85 6.77 C28H31ClO10 562.1606 562.1630 4.33 607.1154[M + HCOO]−, 561.1597[M − H]− Physalin H Plu b
86 6.79 C28H32O15 608.1741 608.1741 −0.02 609.1818[M + H]+, 315.1604 Kakkalide Plu
87 6.98 C22H22O10 446.1213 446.1199 −3.16 469.1024[M + Na]+, 267.0620[M+H-Glc]+ Glucoobtusifolin Sem b
88 7.02 C15H8O7 300.0270 300.0272 0.59 298.9802[M − H]−, 254.9951[M-H-CO2]− Pseudopurpurin Fol,Sta,Rec
89 7.02 C16H12O8 332.0532 332.0538 1.89 331.0454[M − H]−, 312.9961[M-H-H2O]− Patuletin Fol,Sta,Rec b
90 7.41 C28H34O9 514.2203 514.2185 −3.44 513.1503[M − H]−, 471.0800[M-COCH3]− Nomilin Sta b
91 7.53 C15H10O6 286.0477 286.0475 −0.99 287.0624[M + H]+,
285.0155[M − H]− 		Luteolin Sem,Fol,Plu,Sta,Rec,RN b
92 7.84 C19H12O7 352.0583 352.0579 −1.19 397.0275[M + HCOO]−, 351.0215[M − H]− Phellibaumin A Sem,Rec
93 7.96 C17H14O7 330.0740 330.0754 4.36 353.0627[M + Na]+,
331.2370[M + H]+ 		Jaceosidin Sem
94 8.08 C25H24O12 516.1268 516.1254 −2.65 515.0680[M − H]−, 352.9925(C16H17O9) 1,3-Dicaffeoylquinic acid Sem b
95 8.12 C19H21NO2 295.1572 295.1568 −1.35 296.1688[M + H]+, 265.1266(C18H17O2), 250.0978(C17H14O2), 235.0775(C16H11O2), 219.0819(C16H11O), 191.0874(C15H11), 179.0890(C14H11) Nuciferine Sem,Fol,Plu,Sta,Rec,RN [26]
96 8.36 C11H16O3 196.1099 196.1107 3.80 241.0896[M + HCOO]−, 197.1146[M + H]+ Loliolide Sem,Fol,Plu,Sta
97 8.43 C24H29NO6 427.1995 427.1996 0.25 450.1884[M + Na]+,
426.1630[M − H]− 		Pulchellamine D Sem,Plu
98 8.50 C15H10O7 302.0427 302.0433 2.17 301.0054[M − H]−, 245.1061(C13H9O5) Morin Sem,Fol,Sta,Rec b
99 8.53 C15H10O6 286.0477 286.0469 −3.10 287.0555[M + H]+, 269.0413[M+H-H2O]+ Citreorosein Sem b
100 8.65 C16H12O6 300.0634 300.0622 −3.94 345.0161[M + HCOO]−,
177.0334[M-C6H4OCH3]− 		Pratensein Fol b
101 8.75 C16H12O5 284.0685 284.0682 −0.95 329.0322[M + HCOO]−, 282.9966[M − H]− Biochanin A Sem b
102 8.84 C19H21NO3 311.1521 311.1527 1.93 312.1680[M + H]+, 254.1317 Thebaine Fol [26]
103 8.89 C38H42N2O6 622.3043 622.3031 −1.87 623.3239[M + H]+, 580.9211 Tetrandrine Plu,Rec [26]
104 9.32 C22H24O11 464.1319 464.1319 0.08 463.0740[M − H]−, 301.0338[M-Glc]− Hesperetin-7-glucoside Fol b
105 9.40 C21H38O9 434.2516 434.2533 3.98 479.1637[M + HCOO]−, 433.1967[M − H]− Amarantholidol A glycoside Fol
106 9.51 C18H13NO3 291.0895 291.0885 −3.47 314.0791[M + Na]+,
292.0993[M + H]+ 		Lysicamine Rec
107 9.64 C27H30O13 562.1686 562.1677 −1.69 561.1063[M − H]−, 115.9048 Kushenol O Sem
108 9.67 C20H27NO4 345.1940 345.1946 1.78 368.1962[M + Na]+,
346.2238[M + H]+ 		Saussureamine B Fol
109 9.68 C22H20O11 460.1006 460.1007 0.32 505.0414[M + HCOO]−, 459.0331[M − H]− Wogonoside Fol
110 9.74 C15H10O5 270.0528 270.0518 −3.95 271.0582[M + H]+, 253.0470[M+H-H2O]+ Genistein Sem b
111 9.82 C17H14O7 330.0740 330.0724 -4.73 375.1564[M + HCOO]−, 329.0248[M − H]− Tricin Sta,Rec
112 9.86 C11H14O5 226.0841 226.0852 4.64 249.0733[M + Na]+,
227.1603[M + H]+ 		Genipin Sem b
113 9.89 C15H10O6 286.0477 286.0481 1.21 285.0052[M − H]−, 286.0086, 243.0011, 174.9307, 106.9976 Kaempferol Fol,Sta b
114 9.89 C37H40N2O6 608.28863 608.2901 2.33 653.2299[M + HCOO]−, 607.1910[M − H]− Berbamine Plu
115 9.96 C23H18O8 422.1002 422.0996 −1.37 467.0750[M + HCOO]−, 421.0505[M − H]− Interfungin B Sem
116 10.08 C16H12O6 300.0634 300.0623 −3.59 301.0681[M + H]+, 283.1732[M+H-H2O]+, 271.0582(C15H11O5) Chrysoeriol Sem,Fol b
117 10.88 C18H34O5 330.2406 330.2403 −0.85 329.1920[M − H]−, 313.1157[M-OH]− Sanleng acid Sem,Plu,Sta,Rec,RN [26]
118 11.06 C15H28O3 256.2038 256.2038 −0.25 301.1689[M + HCOO]−, 255.7936[M − H]− Bullatantriol Sta
119 11.13 C17H14O5 298.0841 298.0833 −2.64 343.0448[M + HCOO]−, 271.1218(C15H11O5),
267.0337[M-OCH3]− 		5-Hydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one Sem b
120 11.27 C16H14O6 302.0790 302.0799 2.89 347.0313[M + HCOO]−, 301.0374[M − H]−, 283.9966[M-H-H2O]−, 164.9977(C8H5O4) Blumeatin Fol b
121 11.52 C16H12O6 300.0634 300.0636 0.86 345.0199[M + HCOO]−, 299.0297[M − H]−, 285.0086[M-CH3]−, 284.0034[M-H-CH3]− Diosmetin Sem,Fol,Plu,Sta b
122 11.70 C15H8O5 268.0372 268.0385 4.84 313.0034[M + HCOO]−, 267.0739[M − H]− Coumesterol Sem b
123 11.73 C15H28O2 240.2089 240.2096 2.74 285.1746[M + HCOO]−, 239.1349[M − H]− Isodonsesquitin A Sta
124 12.33 C17H26O2 262.1933 262.1939 2.44 307.1509[M + HCOO]−, 261.1005[M − H]− (Z)-7-Acetoxy-methyl-11-methyl-3-methylenedodeca-1,6,10-triene Sem,Plu,Sta
125 13.08 C16H12O5 284.0685 284.0673 −4.07 285.0765[M + H]+, 253.1413[M-OCH3]+ Prunetin Sem b
126 13.24 C18H23NO3 301.1678 301.1693 4.98 324.1644[M + Na]+,
302.1018[M + H]+ 		Futoamide Rec b
127 13.58 C17H19NO3 285.1365 285.1370 1.91 308.1296[M + Na]+, 288.2559 Morphine Fol,Rec,RN b
128 13.75 C17H28O2 264.2089 264.2092 0.85 309.1676[M + HCOO]−, 263.1494[M − H]−,
221.1235[M-COCH3]− 		Cedryl acetate Plu,Sta b
129 13.89 C17H30O2 266.2246 266.2248 0.85 311.1837[M + HCOO]−, 265.1130[M − H]− Cireneol G Sem,Plu,Sta,Rec
130 14.57 C17H26O4 294.1831 294.1832 0.40 339.1569[M + HCOO]−, 293.1417[M − H]− Gmelinin B RN
131 14.92 C36H58O9 634.4081 634.4058 −3.66 679.3038[M + HCOO]−, 633.3121[M − H]− Ecliptasaponin D Sta
132 14.96 C18H34O4 314.2457 314.2463 1.89 313.2063[M − H]−, 239.1380[M-H-OC4H9]− Dibutyl decanedioate Sem,Plu,Sta b
133 14.98 C34H52O9 604.3611 604.3606 −0.87 605.3710[M + H]+,
603.2665[M − H]− 		Periplocoside M Sta
134 15.04 C30H46O3 454.3447 454.3440 −1.53 455.3550[M + H]+,
437.3432[M+H-H2O]+, 409.3499[M-COOH]+ 		Ursonic acid Fol,Sta,Rec b
135 15.77 C29H46O4 458.3396 458.3388 −1.75 503.2620[M + HCOO]−, 457.2710[M − H]− Neotigogenin acetate Rec
136 15.81 C18H32O2 280.2402 280.2400 −0.93 325.2062[M + HCOO]−, 279.1994[M − H]−,
261.1038[M-H-H2O]− 		Linoleic acid Sem,Plu,Sta b
137 16.37 C15H22O4 266.1518 266.1517 −0.27 265.1230[M − H]−, 221.8184[M-H-COO]− Artemin Sem,Plu,Sta b
138 17.54 C30H46O4 470.3396 470.3375 −4.47 469.2683[M − H]−, 451.1740[M-H-H2O]−,
425.2926[M-COOH]− 		Glycyrrhetinic acid Rec,RN b
139 17.58 C32H44O9 572.2985 572.2993 1.41 573.3043[M + H]+, 555.2916[M+H-H2O]+ Ganoderic acid H Sem,Plu,Sta,Rec b
140 17.77 C30H44O8 532.3036 532.3027 −1.75 555.2916[M + Na]+, 515.3719[M+H-H2O]+,
497.3564(C30H41O6)		 Ganoderic acid G Sem,Plu,Sta,Rec b
141 18.25 C27H47N3O8 541.3363 541.3380 3.13 586.2817[M + HCOO]−, 540.2609[M − H]− 2(1H)-Isoquinolinecarboximidamide,3,4-dihydro-N-3,6,9,12,15,18,21,24-octaoxapentacos-1-yl- Sem,Plu,Sta,Rec
142 18.36 C34H46O9 598.3142 598.3119 −3.85 643.3286[M + HCOO]−, 597.2510[M − H]− Daturametelin H Sem,Sta
143 18.45 C18H28O2 276.2089 276.2076 −4.83 321.1788[M + HCOO]−, 277.2154[M + H]+,
275.1695[M − H]−, 257.1711[M-H-H2O]− 		Stearidonic acid Sem,Plu,Sta b
144 18.85 C18H34O3 298.2508 298.2512 1.26 343.2193[M + HCOO]−, 297.2159[M − H]− Ricinoleic acid Sem,Plu,Sta,Rec b
145 18.93 C39H60O15 768.3932 768.3970 4.92 813.3219[M + HCOO]−, 767.2943[M − H]− Hypoglaucin H Sem
146 19.56 C20H28O3 316.2038 316.2032 −2.08 361.1339[M + HCOO]−, 315.1700[M − H]− Saurufuran B Fol
147 19.81 C35H60O6 576.4390 576.4394 0.68 621.3870[M + HCOO]−, 464.1719[M-C8H16]− Daucosterol Fol,Plu,Sta,Rec,RN [26]
148 20.56 C30H50O3 458.3760 458.3767 1.46 459.3841[M + H]+, 441.3736[M+H-H2O]+,
423.3613(C30H47O)		 Soyasapogenol B Sem,Rec b
149 21.23 C28H46O 398.3549 398.3550 0.22 421.3392[M + Na]+,
399.3546[M + H]+ 		24-Methylenecholesterol Sem,Fol,Plu,Sta,Rec,RN b
150 21.76 C18H30O2 278.2246 278.2252 2.27 323.1931[M + HCOO]−, 277.1808[M − H]−,
233.2307[M-COOH]− 		Linolenic acid Sem,Fol,Plu,Sta,Rec b
151 23.35 C19H38O2 298.2872 298.2876 1.52 343.2383[M + HCOO]−, 279.1925[M-H-H2O]−,
253.1802[M-COOH]− 		Nonadecanoic Acid Plu,Rec b
152 23.46 C30H46O3 454.3447 454.3437 −2.29 453.2730[M − H]−, 423.2258(C29H43O2) Ganoderiol F Sta,Rec,RN b
153 23.47 C29H48O2 428.3654 428.3638 −3.76 429.3717[M + H]+, 411.3596[M+H-H2O]+ 3β-Hydroxystigmast-5-en-7-one Sta b
154 23.97 C30H46O5 486.3345 486.3337 −1.78 509.3112[M + Na]+, 469.3638[M+H-H2O]+,
423.3571[M-COOH-H2O]+ 		Ceanothic acid Sem,Fol,Plu,Sta,Rec,RN b
155 24.35 C21H30O3 330.2195 330.2195 0.12 353.2165[M + Na]+,
331.3078[M + H]+ 		Tussilagonone Fol,RN b
156 24.66 C22H42O2 338.3185 338.3173 −3.38 383.2634[M + HCOO]−, 339.3274[M + H]+,
321.3164[M+H-H2O]+, 303.3048(C22H39)		 Erucic acid Sem,Fol,Plu,Sta,Rec b
157 24.99 C16H32O2 256.2402 256.2410 3.06 255.2143[M − H]−, 237.1881[M-H-H2O]−,
211.1025[M-COOH]− 		Palmitic acid Sem,Plu,Sta b
158 24.99 C20H40O2 312.3028 312.3028 −0.15 311.2523[M − H]−, 293.1382[M-H-H2O]− Arachidic acid Sta b
159 24.99 C36H62O10 654.4343 654.4335 −1.15 699.3741[M + HCOO]−, 653.3450[M − H]− Pseudo-ginsenoside RT4 Sem,Plu,Rec
160 25.55 C30H52O4 476.3866 476.3865 −0.06 499.3885[M + Na]+,
477.3926[M + H]+ 		20(S)-Protopanaxatriol Sem,Fol,Plu,Sta
161 25.68 C22H41NO 335.3188 335.3178 −3.05 358.3636[M + Na]+,
336.3233[M + H]+ 		N-Isobutyl-2E,4E-octadecadienamide Sem,Sta,Rec
162 26.57 C29H46O 410.3549 410.3538 −2.58 411.3720[M + H]+, 393.3600[M+H-H2O]+ Corbisterol Sem,Fol,Plu,Sta,Rec,RN b
163 27.18 C57H98O6 878.7363 878.7374 1.18 923.6272[M + HCOO]−, 877.4003[M − H]− Linolein Sem,Plu
164 27.19 C29H46O2 426.3498 426.3483 −3.45 427.3565[M + H]+, 409.3457[M+H-H2O]+ Stigmast-4-ene-3,6-dione Sem,Plu,Rec b
165 27.36 C18H36O2 284.2715 284.2641 1.51 283.2275[M − H]−, 255.1946[M-C2H5]−,
237.0084[M-H-C2H5OH]− 		Ethyl hexadecanoate Sem,Fol,Plu,Sta,Rec,RN b
166 27.36 C21H42O2 326.3185 326.3195 3.12 371.2715[M + HCOO]−, 325.1361[M − H]−,
307.1581[M-H-H2O]− 		Heneicosanoic acid Plu,Sta,Rec b
167 27.38 C16H22O4 278.1518 278.1512 −2.16 301.1426[M + Na]+,
279.1635[M + H]+,
205.2028[M-OC4H9]+, 57.0751(C4H9)		 Diisobutyl phthalate Sem,Fol,Plu,Sta,Rec,RN b
168 27.38 C24H38O4 390.2770 390.2764 −1.56 413.2744[M + Na]+,
391.2936[M + H]+ 		Bis (2-ethylhexyl) phthalate Sem,Fol,Plu,Sta,RN b
169 27.38 C42H74O15 818.5028 818.5049 2.61 863.3998[M + HCOO]−, 819.5060[M + H]+ Quinquenoside L9 Plu,Sta
170 27.4 C36H64O9 640.4550 640.4522 −4.50 685.3537[M + HCOO]−, 639.3768[M − H]− 3-O-β-d-Glucopyranosyl-dammar-3β,12β,20R,25-tetraol Sem,Rec [26]
171 27.57 C55H74N4O5 870.5659 870.5654 −0.63 893.7276[M + Na]+,
871.5701[M + H]+ 		Pheophytin a Sem,Fol,Plu,Sta,Rec,RN
Open in a new tab

a Identified with the standard. b In comparison to spectral data obtained from the Human Metabolome Database (Canada).

Figure 3.

Figure 3

Figure 3

Open in a new tab

Chemical structures of compounds identified in six different parts of lotus.

As listed in Table 1, the compounds were determined according to their characteristic MS fragmentation patterns, or the retention times of reference standards. Take an alkaloid (17) and a flavonoid (56) as examples to illustrate the resolution process of the compounds. Compound 17 is a benzylisoquinoline alkaloid (tR = 2.10 min, C19H23NO3) yielded [M + H]+ ion at m/z 314.1754, and produced fragment ion at m/z 283.0286 due to parent ion peak losing CH3NH2. m/z 206.1181 and m/z 107.0875 are fragment ion peaks formed by benzyl cleavage. By comparison with literature information, this component was identified as armepavine [27]. The mass spectrogram is shown in Figure 4a. Compound 56 is an oxygen glycosides flavonoid (tR = 5.70 min, C21H20O12) yielded [M − H]− ion at m/z 463.0343, and produced fragment ion at m/z 300.9948 due to parent ion peak losing glucose moiety. By comparison with literature and reference standard information, this component was identified as isoquercetin [28]. The mass spectrogram is shown in Figure 4b.

Figure 4.

Figure 4

Open in a new tab

The possible fragment pathway for peak 17 and 56.

2.2. Metabolomics Analysis of Six Different Parts of Lotus

Metabolomics analyses of six parts of lotus included PCA and OPLS-DA. First, to separate the parts and obtain the maximum variables, PCA was used to obtain the score plots (Figure 5a) and loading plots (Figure 5b). In the score plots, the green QC points are closely gathered together to form a cluster, which indicates that the system is stable. It can be seen from the figure that the samples from the Sem, Rec, Plu, Sta, Fol and RN groups could be easily divided into six clusters, and the six parts had achieved obvious separation, indicating that the six parts could be easily distinguished. In the loading plots, 23 variables that can be distinguished among the six clusters were found.

Figure 5.

Figure 5

Open in a new tab

The PCA score plot (a) and loading plot (b) of six different parts of lotus.

Second, to further evaluate the differences between the six parts, one was distinguished from the others, the maximum separation of the six parts was achieved, the potential biomarkers that may lead to the differences were found, and OPLS-DA was carried out. Then, for the visualization of the OPLS-DA and convenient interpretation of the model, S-plots were created. At the same time, to screen the different components, the variable importance of the projection (VIP) was introduced. The metabolites with VIP values above 1.0 and p-values below 0.05 were considered as potential biomarkers [29,30,31]. Based on these two important parameters and the identification of the components from six parts (Table 1), 23 reliable known biomarkers were found to distinguish the six parts and were labeled in the S-plots (Figure 6). In addition, a heatmap (Figure 7) was drawn to systematically evaluate these biomarkers and visually display the intensity of these biomarkers. For Sem, there were three potential biomarkers, including flavonoids (110, 125) and a quinone (99). For Fol, there were three potential biomarkers, including terpenoids (105, 108) and an alkaloid (102). For Plu, there were nine potential biomarkers, including flavonoids (29, 47, 55, 86), alkaloids (28, 38, 44) an organic acid (10) and an organic acid ester (85). For Sta, there were four potential biomarkers, including steroids (133, 153), a terpenoid (131) and an organic acid (158). For Rec, there were three potential biomarkers, including an alkaloid (106), a steroid (135), and an amide (126). For RN, there was only one potential biomarker–a terpenoid (130). These robust biomarkers enabling the differentiation among Sem, Fol, Plu, Sta, Rec and RN can be used for the rapid identification of six parts of lotus.

Figure 6.

Figure 6

Figure 6

Open in a new tab

The OPLS-DA/S-plots of Sem, Fol, Plu, Sta, Rec, and RN of lotus. The points on the lower left represent the compounds in this part, and the points at the higher right represent the compounds in the other five parts. The biomarkers and their compound numbers are marked in red.

Figure 7.

Figure 7

Open in a new tab

Heatmap visualizing the intensities of potential biomarkers.

3. Discussion

Herbal medicines usually play a holistic role in maintaining health through multiple targets because they contain multiple constituents. Being a traditional Chinese herb, lotus has been used to treat various diseases. In the last decades, chemoinformatics and systems pharmacology have been successfully applied in the discovery of the active component of traditional Chinese medicines and their mechanisms of action. It is well known that the process of fully understanding the ingredients of herb using traditional methods is labor intensive, difficult and time-consuming. Fortunately, the combining of UPLC-QToF-MS technology and UNIFI platform helps researchers reveal the containing compounds in herbs in an efficient way.

The present study analysed the principal components of the lotus six parts by UPLC-QToF-MS. Combined the related literatures [6,32,33] with our previous experiments, heat reflux extraction with 80% ethanol was chosen for the samples extraction. And the detecting conditions, mobile phase elution solutions of UPLC, positive and negative ion detection modes of QToF-MS, were optimized by quality control samples. MS and MS/MS data were collected simultaneously to improve the efficiency and accuracy of data collection in the MSE model. The tolerance of tR was ± 0.1 min. The isotopic pattern was included in the peak identification. The permutation testing was the scoring function for identification and statistical analysis parameter p-value need to below 0.05. As a result, 171 compounds were identified or tentatively characterized from the six parts of lotus.

It was found that flavonoids were the most common compounds: 56 of 171 were flavonoids. Sta was ranked first due to the 31 kinds of flavonoids being detected, followed by Plu (24 kinds), Sem (23 kinds), Fol (20 kinds), and Rec (20 kinds). By comparing the species of flavonoids distributed in the six parts, it was found that 18 of the 20 in Rec are consistent with those in Sta, and half of the species in Sem are the same as those in Plu. Luteolin is the only flavonoid detected from RN, and it is also available in the other five parts.

Alkaloids are also important active components in lotus. In this experiment, 22 kinds of alkaloid compounds were detected, including isoquinoline alkaloids, aporphine alkaloids and so on. Plu contained 15 kinds, followed by Fol with 11 kinds. Armepavine and nuciferine are available in all six parts. Terpenoids were the most abundant in lotus Sta, containing 13 species. Statistical analysis was conducted on the compounds detected from the six parts, and the structure types and the numbers of compounds in the six parts are shown in Figure 8.

Figure 8.

Figure 8

Open in a new tab

Type and number of compounds in the six parts of lotus.

The established method can be used as a standard protocol for directly discriminating between and predicting the six parts of lotus. Traditional Chinese herbal therapy can be characterized by the use of a large number of multi-herb formulae. Chinese patent medicines (CPM), which come from traditional Chinese classical prescriptions, are usually prepared by modern pharmaceutical techniques with various herbs as raw material. After processing, the unique morphological characteristics of the original herbs disappeared, and the active chemical constituents were successfully preserved. So the chemical compositions and characters of CPM have been considered a reliable index of quality control. Being used as both delicious food and empirical medicine, the formulation and preparation conditions must be improved to achieve better delivery of nutritional ingredients and increased bioactivities of the food and medicinal products. Lotus is one of the commonly-used herbal drugs. Its two parts or more appeared in one prescription for some disease treatment. For example, Bai Dai Wan from Hui Zhi Tang Jing Yan Fang (Experiential Prescriptions from Hui Zhi Clinic) is comprised of eight herbs including lotus seed, stamens and rhizome nodes. The biomarkers found in this paper can provide bases for examining the raw herbs types in the finished preparations and improving the quality of products.

Most importantly, data analyses provided useful information for the further study and usage of the plant. The results of this phytochemical profile study are far more comparable because the same procedure was used during sample analysis, including during sample treatment, the detection procedure, data processing and the interpretation of testing data. Compared with the previous studies [2,33,34,35], this was the first time that alkaloids (8, 17, 20, 24, 63, 95) were detected in Sta, and terpenoids (97, 124, 139, 140, 148, 154, 159, 160, 170) were detected in Sem. Sta is a more noteworthy part than the others, for it has the greatest diversity of flavonoids and terpenoids, but relatively few studies on Sta can be found compared to the other parts.

Being a traditional Chinese herb, lotus has been used for more than 2,000 years. However, the material basis of a number of folk applications has not been illustrated clearly. For instance, being the hemostatic agent in traditional Chinese medicine, Rec and RN have the same indications. After comparison, we found that 15 of the 19 compounds identified in RN were also presented in Rec. They were three alkaloids (17, 95, 127), three terpenoids (138, 152, 154), one flavonoid (91), three steroids (147, 149,1 62), and five organic acids and esters (6, 117, 165, 167, 171). They may be the material basis of hemostatic activity of RN and Rec.

Compound 41 (liensinine) and 59 (neferine) play a major role in anti-Alzheimer disease agents [36]. However, none of them was detected in the receptacle, although Rec was reported to have an anti-Alzheimer effect [37]. In this paper, 41 and 59 were identified in Sem, Fol and Plu, as well as 41 were identified in Rec. The result provided useful information for the research and utilization of lotus seed, leaf and receptacle. Meanwhile, the discoveries of alkaloids in Sta (8, 17, 20, 24, 63, 95) and Rec (17, 41, 43, 95, 103, 106, 127) may be useful for explaining their anti-ischemic effect.

In summary, the holistic and intuitionistic description of the chemical constituents in lotus six parts in this paper contribute new information to the phytochemical research of lotus. The results will be helpful in illustrating the chemical basis of herbs activities. The established method and identified biomarkers provide valuable data and references for quality control of the CPMs who contain lotus different parts in the prescriptions.

The results of this research may be limited by the capacity of the identification database. Not compared with an analytical standard, identifications of compounds are presumptive. Therefore, the identification relies more on standard secondary spectra database. Only a fraction of compounds were included in the HMBD database, so relatively few are detected although compounds are varied and abundant in the six parts of lotus. In addition to the difference ingredients, differences in the content of common ingredients of herbs also affect their pharmacological activity. In the future, more efforts should be devoted to research on the concentrations of biological compounds and biomarkers of the herbs.

4. Materials and Methods

4.1. Materials and Reagents

The products of Sem, Fol, Plu, Sta, Rec and RN were collected from their respective cultivation areas or purchased from herbal markets in China. A total of 48 batches (each part 8 batches) were gathered and identified by Professor Ping-Ya Li (School of Pharmaceutical Sciences, Jilin University, Changchun, China). The voucher specimens (No. 2019224-2019272) had been deposited at the Research Center of Natural Drug, School of Pharmaceutical Sciences, Jilin University, Changchun, China. A site list of the samples collected is given in Table 2.

Table 2.

The collection sites of the tested samples.

Species Hunan Shandong Hubei Hebei Anhui Fujian Jiangxi Guangdong
Sem Sem1 Sem2 Sem3 Sem4 Sem5 Sem6 Sem7 Sem8
Fol Fol1 Fol2 Fol3 Fol4 Fol5 Fol6 Fol7 Fol8
Plu Plu1 Plu2 Plu3 Plu4 Plu5 Plu6 Plu7 Plu8
Sta Sta1 Sta2 Sta3 Sta4 Sta5 Sta6 Sta7 Sta8
Rec Rec1 Rec2 Rec3 Rec4 Rec5 Rec6 Rec7 Rec8
RN RN1 RN2 RN3 RN4 RN5 RN6 RN7 RN8
Open in a new tab

LC-MS grade methanol and acetonitrile were purchased from Fisher Chemical Company (Geel, Belgium). Formic acid was purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Leucine enkephalin was provided by Waters Technologies Corporation (Milford, MA, USA). All other chemicals were of analytical grade. Water was purified and deionized using a Millipore water purification system. LC-MS grade six reference standards, including hyperoside, liensinine, isoliensinine, neferine, kaempferol-3-O-rutinoside, and N-methylcoclaurine, were isolated from our own laboratory and were previously identified and confirmed by 1H-NMR and 13C-NMR. Five standard compounds, including rutin, quercetin, kaempferol, caffeic acid, and luteolin 7-glucoside, were purchased from the China National Institutes for Food and Drug Control. Six standard compounds, including isoquercitrin, isorhamnetin, catechin, epicatechin, nuciferine, and chlorogenic acid, were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan, China). The purity of all the regents was HPLC ≥ 98%.

4.2. Sample Preparation and Extraction

A mixer was used to grind each sample to generate homogeneous powders. Sample powders (0.20 g) were refluxed with 6 mL 80% (v/v) aqueous ethanol and extracted twice for 30 min each. By filtrating, the filtrate evaporated to dryness by a filtrate recovery system. The residue was dissolved with methanol; then, the mixture was filtered by a 0.22 μm syringe filter and tested by a UPLC system. A quality control (QC) solution was prepared for each part by taking 10 μL from every sample solution and then mixing. Eight QC injections were performed randomly in the testing process to ensure the stability and suitability consistency of the MS analysis. The volume injected for the samples and QC solution was 5 μL for each run.

4.3. UPLC-QToF-MS

To enable high sensitivity, selectivity, speed and precision, QToF technology and UPLC/MSE were used in this experiment. A Xevo G2-XS QToF mass spectrometer (Waters, Milford, MA, USA) connected to an UPLC system by an electrospray ionization (ESI) interface, was used for UPLC-QToF-MSE. An ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 μm) column was used for sample separation. 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). A gradient elution method was used. The elution conditions were as follows: 0-2 min, 10% B; 2–26 min, 10–100% B; 26–29 min, 100% B; 29–29.1 min, 100–10% B; 29.1–30 min, 10% B. The flow rate was 0.4 mL/min. The column temperature was 30 °C. The data were collected by mass spectrometer, the MSE continuum model was used for screening analysis, and the MSE centroid model was used for metabonomics analysis in MarkerLynx software. When running a single LC system, a low collision energy (CE) scan was quickly swithed to a high CE scan, from 6 V to 20–40 V. The capillary voltages were 2.6 kV (ESI+) and 2.2 kV (ESI−) and the cone voltage was 40 V. The source and desolvation temperatures were 150 °C and 400 °C, respectively. The flow rates of the cone gas and desolvent gas were 50 L/h and 800 L/h respectively. Leucine enkephalin (LE, m/z 556.2771 (ESI+), 554.2615 (ESI−)) was injected at a rate of 10 μL/min. The data were recorded with a MassLynx V4.1 workstation.

4.4. Data Analysis

For the screening analysis, the MS raw data were imported into the Waters’ UNIFI 1.7.0 platform to quickly identify the chemical components. Two hundred was set as the minimum peak area for two-dimensional peak detection. For three-dimensional peak detection, the low-energy peak intensity was over 1000 counts, and the high-energy peak intensity was over 200 counts. The mass error of the compound was within ± 5 ppm, and the retention time (tR) was within ± 0.1 min. The negative adducts +COOH and -H and positive adducts +H and +Na were selected.

For metabonomics analysis, the original MSE data were processed by using Waters MarkerLynx XS V4.1 software, and a table of the m/z-tR pairs with the corresponding intensities of all the peaks was obtained. The same tR and m/z values in different batches of samples were regarded as the same component. The main parameters included the following: tR range, 0–30 min; minimum intensity, 5%; mass range, 100–1500 Da; mass tolerance, 0.10. Multivariate statistical analysis was performed, including PCA and OPLS-DA.

Author Contributions

D.L. conceived and designed the experiments; H.P., M.G., M.D., Y.Z. and C.W. performed the experiments; H.P. and W.S. were responsible for data analysis. H.P. wrote the paper. D.L. assisted paper revision. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Health Commission of Jilin Province (China).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds mentioned in the materials section are available from the authors.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Guo H.B. Cultivation of lotus (Nelumbo nucifera Gaertn. ssp. nucifera) and its utilization in China. Genet. Resour. Crop Evol. 2009;56:323–330. doi: 10.1007/s10722-008-9366-2. [DOI] [Google Scholar]
  • 2.Chen G., Zhu M., Guo M. Research advances in traditional and modern use of Nelumbo nucifera: Phytochemicals, health promoting activities and beyond. Crit. Rev. Food Sci. Nutr. 2019;59:S189–S209. doi: 10.1080/10408398.2018.1553846. [DOI] [PubMed] [Google Scholar]
  • 3.The Pharmacopoeia Commission of People’s Republic of China . Pharmacopoeia of the People’s Republic of China. Volume 1. Chemical Industry Publishing House; Beijing, China: 2020. pp. 285–287. [Google Scholar]
  • 4.Liu Y., Ma S.S., Ibrahim S.A., Li E.H., Yang H., Huang W. Identification and antioxidant properties of polyphenols in lotus seed epicarp at different ripening stages. Food Chem. 2015;185:159–164. doi: 10.1016/j.foodchem.2015.03.117. [DOI] [PubMed] [Google Scholar]
  • 5.Lim S.S., Jung Y.J., Hyun S.K., Lee Y.S., Choi J.S. Rat lens aldose reductase inhibitory constituents of Nelumbo nucifera stamens. Phytother. Res. PTR. 2006;20:825–830. doi: 10.1002/ptr.1847. [DOI] [PubMed] [Google Scholar]
  • 6.Zhu M.-Z., Wu W., Jiao L.-L., Yang P.-F., Guo M.-Q. Analysis of Flavonoids in Lotus (Nelumbo nucifera) Leaves and Their Antioxidant Activity Using Macroporous Resin Chromatography Coupled with LC-MS/MS and Antioxidant Biochemical Assays. Molecules. 2015;20:10553–10565. doi: 10.3390/molecules200610553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Yi Y., Sun J., Xie J., Min T., Wang L.M., Wang H.X. Phenolic Profiles and Antioxidant Activity of Lotus Root Varieties. Molecules. 2016;21:863. doi: 10.3390/molecules21070863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Liu S.H., Lu T.H., Su C.C., Lay I.S., Lin H.Y., Fang K.M., Ho T.J., Chen K.L., Su Y.C., Chiang W.C., et al. Lotus leaf (Nelumbo nucifera) and its active constituents prevent inflammatory responses in macrophages via JNK/NF-kappaB signaling pathway. Am. J. Chin. Med. 2014;42:869–889. doi: 10.1142/S0192415X14500554. [DOI] [PubMed] [Google Scholar]
  • 9.Kuo Y.C., Lin Y.L., Liu C.P., Tsai W.J. Herpes simplex virus type 1 propagation in HeLa cells interrupted by Nelumbo nucifera. J. Biomed. Sci. 2005;12:1021–1034. doi: 10.1007/s11373-005-9001-6. [DOI] [PubMed] [Google Scholar]
  • 10.Jiang Y., Ng T.B., Liu Z., Wang C., Li N., Qiao W., Liua F. Immunoregulatory and anti-HIV-1 enzyme activities of antioxidant components from lotus (Nelumbo nucifera Gaertn.) rhizome. Biosci. Rep. 2011;31:381–390. doi: 10.1042/BSR20100062. [DOI] [PubMed] [Google Scholar]
  • 11.Wang Z., Hu J., Hamzah S.S., Ge S., Lin Y., Zheng B., Zeng S., Lin S. n-Butanol Extract of Lotus Seeds Exerts Antiobesity Effects in 3T3-L1 Preadipocytes and High-Fat Diet-Fed Mice via Activating Adenosine Monophosphate-Activated Protein Kinase. J. Agric. Food Chem. 2019;67:1092–1103. doi: 10.1021/acs.jafc.8b05281. [DOI] [PubMed] [Google Scholar]
  • 12.Ahn J.H., Kim E.S., Lee C., Kim S., Cho S.H., Hwang B.Y., Lee M.K. Chemical constituents from Nelumbo nucifera leaves and their anti-obesity effects. Bioorg. Med. Chem. Lett. 2013;23:3604–3608. doi: 10.1016/j.bmcl.2013.04.013. [DOI] [PubMed] [Google Scholar]
  • 13.Tang C., Xie B., Sun Z. Antibacterial activity and mechanism of B-type oligomeric procyanidins from lotus seedpod on enterotoxigenic Escherichia coli. J. Funct. Foods. 2017;38:454–463. doi: 10.1016/j.jff.2017.09.046. [DOI] [Google Scholar]
  • 14.Zhang J.M., Li P.Y., Wang L., Bian B.L. Study on extraction, separation and pharmacodynamics of total alkaloids from Plumula Nelumbinis. Chin. J. Exp. Tradit. Med. Formulae. 2009;15:26–28. [Google Scholar]
  • 15.Wang X.F., Lai B.C., Huang Y., Cheang W.S., Zhang M.Q., Wang N.Q. Nuciferine relaxes rat mesenteric arteries through endothelium-dependent and -independent mechanisms. Br. J. Pharmacol. 2015;172:5609–5618. doi: 10.1111/bph.13021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Zhou Y.J., Xiang J.Z., Yuan H., Liu H., Tang Q., Hao H.Z., Yin Z., Wang J., Ming Z.Y. Neferine exerts its antithrombotic effect by inhibiting platelet aggregation and promoting dissociation of platelet aggregates. Thromb. Res. 2013;132:202–210. doi: 10.1016/j.thromres.2013.05.018. [DOI] [PubMed] [Google Scholar]
  • 17.Ralf S., Stefan H., Ursula H., Söhle J., Andreas S., Raschke T., Stäb F., Wenck H., Marc W. Lotus leaf extract and L-carnitine influence different processes during the adipocyte life cycle. Nutr. Metab. 2010;7:66. doi: 10.1186/1743-7075-7-66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Yan M.Z., Chang Q., Zhong Y., Xiao B.X., Feng L., Cao F.R., Pan R.L., Zhang Z.S., Liao Y.H., Liu X.M. Lotus Leaf Alkaloid Extract Displays Sedative-Hypnotic and Anxiolytic Effects through GABAA Receptor. J. Agric. Food Chem. 2015;63:9277–9285. doi: 10.1021/acs.jafc.5b04141. [DOI] [PubMed] [Google Scholar]
  • 19.Wang W., Liu C.P., Kuo Y.C., Lin Y. Anti-lymphoproliferative Alka loids from Plumula Nelumbinis. Chin. Pharm. J. 2004;56:25–30. [Google Scholar]
  • 20.Huang G., Lin F., Gao Y. Effect of propylthiouracil on activity of thyroid NADH-cytochrome b5 reductase. Chin. Pharmacol. Bull. 2001;17:687–690. [Google Scholar]
  • 21.Kim J.-H., Kang M., Cho C., Chung H.-S., Kang C.-W., Parvezz S., Bae H. Effects of Nelumbinis Semen on contractile dysfunction in ischemic and reperfused rat heart. Arch. Pharm. Res. 2006;29:777–785. doi: 10.1007/BF02974079. [DOI] [PubMed] [Google Scholar]
  • 22.Zhang F.X., Li M., Qiao L.R., Yao Z.H., Li C., Shen X.Y., Wang Y., Yu K., Yao X.S., Dai Y. Rapid characterization of Ziziphi Spinosae Semen by UPLC/Qtof MS with novel informatics platform and its application in evaluation of two seeds from Ziziphus species. J. Pharm. Biomed. Anal. 2016;122:59–80. doi: 10.1016/j.jpba.2016.01.047. [DOI] [PubMed] [Google Scholar]
  • 23.Wang C., Zhang N., Wang Z., Qi Z., Zhu H., Zheng B., Li P., Liu J. Nontargeted Metabolomic Analysis of Four Different Parts of Platycodon grandiflorum Grown in Northeast China. Molecules. 2017;22:1280. doi: 10.3390/molecules22081280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Lin H., Zhu H., Tan J., Wang H., Wang Z., Li P., Zhao C., Liu J. Comparative Analysis of Chemical Constituents of Moringa oleifera Leaves from China and India by Ultra-Performance Liquid Chromatography Coupled with Quadrupole-Time-Of-Flight Mass Spectrometry. Molecules. 2019;24:942. doi: 10.3390/molecules24050942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lee J.W., Ji S.H., Choi B.R., Choi D.J., Lee Y.G., Kim H.G., Kim G.S., Kim K., Lee Y.H., Baek N.I., et al. UPLC-QTOF/MS-Based Metabolomics Applied for the Quality Evaluation of Four Processed Panax ginseng Products. Molecules. 2018;23:2062. doi: 10.3390/molecules23082062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Zhang Q. Studies on the Chemical Constituents and Biological Activities of Fructus Sophorae and Nelumbo nucifera. Jilin University; Changchun, China: 2018. [Google Scholar]
  • 27.Qing Z., Cheng P., Zeng J. Research progress on mass spectral Fragm. Behaviour of alkaloids in Macleaya cordata. Chin. Tradit. Herb. Drugs. 2013;44:2929–2939. doi: 10.7501/j.issn.0253-2670.2013.20.025. [DOI] [Google Scholar]
  • 28.Tian W. Study on Constituents and Activities In Vitro and Vivo of Total Flavonoids and Alkaloids from Plumula Nelumbinis. Guangdong University of Technology; Guangzhou, China: 2019. [Google Scholar]
  • 29.Wang C., Zhang N., Wang Z., Qi Z., Zheng B., Li P., Liu J. Rapid characterization of chemical constituents of Platycodon grandiflorum and its adulterant Adenophora stricta by UPLC-QTOF-MS/MS. J. Mass Spectrom. 2017;52:643–656. doi: 10.1002/jms.3967. [DOI] [PubMed] [Google Scholar]
  • 30.Wang Y., Wang C., Lin H., Liu Y., Li Y., Zhao Y., Li P., Liu J. Discovery of the Potential Biomarkers for Discrimination between Hedyotis diffusa and Hedyotis corymbosa by UPLC-QTOF/MS Metabolome Analysis. Molecules. 2018;23:1525. doi: 10.3390/molecules23071525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Zhu H., Lin H., Tan J., Wang C., Wang H., Wu F., Dong Q., Liu Y., Li P., Liu J. UPLC-QTOF/MS-Based Nontargeted Metabolomic Analysis of Mountain- and Garden-Cultivated Ginseng of Different Ages in Northeast China. Molecules. 2018;24:33. doi: 10.3390/molecules24010033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Zou S., Ge Y., Chen X., Li J., Yang X., Wang H., Gao X., Chang Y.X. Simultaneous Determination of Five Alkaloids by HPLC-MS/MS Combined With Micro-SPE in Rat Plasma and Its Application to Pharmacokinetics After Oral Administration of Lotus Leaf Extract. Front. Pharmacol. 2019;10:1252. doi: 10.3389/fphar.2019.01252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Limwachiranon J., Huang H., Shi Z., Li L., Luo Z. Lotus Flavonoids and Phenolic Acids: Health Promotion and Safe Consumption Dosages. Compr. Rev. Food Sci. Food Saf. 2018;17:458–471. doi: 10.1111/1541-4337.12333. [DOI] [PubMed] [Google Scholar]
  • 34.Sharma B., Gautam L., Adhikari D., Karki R. A Comprehensive Review on Chemical Profiling of Nelumbo nucifera: Potential for Drug Development. Phytother. Res. PTR. 2017;31:3–26. doi: 10.1002/ptr.5732. [DOI] [PubMed] [Google Scholar]
  • 35.Bhardwaj A., Modi K. A Review on Therapeutic Potential of Nelumbo Nucifera (Gaertn): The Sacred Lotus. Int, J. Pharm. Sci. Res. 2016;7:42–54. doi: 10.13040/IJPSR.0975-8232. [DOI] [Google Scholar]
  • 36.Jung H.A., Karki S., Kim J.H., Choi J.S. BACE1 and cholinesterase inhibitory activities of Nelumbo nucifera embryos. Arch. Pharm. Res. 2015;38:1178–1187. doi: 10.1007/s12272-014-0492-4. [DOI] [PubMed] [Google Scholar]
  • 37.Gong Y.S., Guo J., Hu K., Gao Y.Q., Xie B.J., Sun Z.D., Yang E.N., Hou F.L. Ameliorative effect of lotus seedpod proanthocyanidins on cognitive impairment and brain aging induced by D-galactose. Exp. Gerontol. 2016;74:21–28. doi: 10.1016/j.exger.2015.11.020. [DOI] [PubMed] [Google Scholar]

Articles from Molecules are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

RESOURCES