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. 2021 Jun 29;12:685575. doi: 10.3389/fphar.2021.685575

Microscopic Mass Spectrometry Imaging Reveals the Distribution of Phytochemicals in the Dried Root of Isatis tinctoria

Li-Xing Nie 1,2, Jing Dong 3, Lie-Yan Huang 2, Xiu-Yu Qian 2, Chao-Jie Lian 2, Shuai Kang 2,4,*, Zhong Dai 2, Shuang-Cheng Ma 1,2,*
PMCID: PMC8276017  PMID: 34267659

Abstract

The dried root of Isatis tinctoria L. (Brassicaceae) is one of the most popular traditional Chinese medicines with well-recognized prevention and treatment effects against viral infections. Above 300 components have been isolated from this herb, but their spatial distribution in the root tissue remains unknown. In recent years, mass spectrometry imaging (MSI) has become a booming technology for capturing the spatial accumulation and localization of molecules in fresh plants, animal, or human tissues. However, few studies were conducted on the dried herbal materials due to the obstacles in cryosectioning. In this study, distribution of phytochemicals in the dried root of Isatis tinctoria was revealed by microscopic mass spectrometry imaging, with application of atmospheric pressure–matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap–time-of-flight mass spectrometry (IT-TOF/MS). After optimization of the slice preparation and matrix application, 118 ions were identified without extraction and isolation, and the locations of some metabolites in the dried root of Isatis tinctoria were comprehensively visualized for the first time. Combining with partial least square (PLS) regression, samples collected from four habitats were differentiated unambiguously based on their mass spectrometry imaging.

Keywords: mass spectrometry imaging, Isatis tinctoria, atmospheric pressure–matrix-assisted laser desorption/ionization, ion trap–time-of-flight mass spectrometry, traditional Chinese medicine

Introduction

Natural products have always benefited the health care of people worldwide and are used as herbal medicines commonly (He et al., 2020). Among them, traditional Chinese medicine (TCM) has made significant contributions to the treatment of human disease from ancient times to present (Wang et al., 2021). For instance, the dried root of Isatis tinctoria L. (Isatis indigotica Fortune ex Lindl.) (Isatidis Radix in Latin, Isatis root in English, and Banlangen in Chinese) has been widely used as the remedy for fever and infection in China and other countries (Zhou and Zhang, 2013). It is well recognized for prevention and treatment effects against a variety of viral infections, such as seasonal flu (Speranza et al., 2020), severe acute respiratory syndrome (Lin et al., 2005), and H1N1 flu epidemic (Li and Tao, 2013; Luo et al., 2019). As the important ingredient in the so-called Three Drugs and Three Prescriptions, the dried root of Isatis tinctoria has also been playing an active role in fighting against the novel corona virus disease 2019 (COVID-19) (Li et al., 2020b; Li and Xu, 2021). Till now, more than 300 components have been isolated from the root of Isatis indigotica, including alkaloids, sulfur-containing compounds, phenylpropanoids, amino acids, nucleosides, organic acids and esters, flavonoids, quinones, terpenes, sterols, saccharides, aromatics, peptides, alcohols, aldehydes and ketones, nitriles, and sphingolipids. Although chemical composition and pharmacological activities of phytochemicals in the dried root of Isatis tinctoria have been extensively investigated by Lin et al. (2005); Speranza et al. (2020), the analysis of their spatial distribution in tissue has not yet been done.

Unraveling the tissue-specific localization of molecules in medicinal herbs can provide straightforward clues to understand their biological functions. Technologies for this aim face challenges and are still under development. Conventional investigations have enabled the comprehensive chemical profiling of metabolites from the dried root of Isatis tinctoria, applying separation and identification techniques such as high-performance liquid chromatography (HPLC) (Zou et al., 2005), ultra-performance liquid chromatography (UPLC) (Shi et al., 2012), and ultra-performance liquid chromatography–quadrupole–time-of-flight mass spectrometry (UPLC-Q-TOF/MS) (Guo et al., 2020a). However, the microscopic localizations of components in samples are largely ignored during the homogenization and purification process.

By directly detecting the ion beams of components on a sample surface, mass spectrometry imaging (MSI) can achieve the chemical distribution information. With the aid of the optical microscope, MSI can link morphological features with chemical profiling, thus providing untargeted, label-free, and multiplexed approach for molecular imaging. In recent years, it has become a fascinating tool for capturing the spatial accumulation and localization of metabolites in plants (Shimma and Sagawa, 2019; Li et al., 2020a; Li et al., 2020c), but only a few studies had been conducted on the dried herbal materials (Ng et al., 2007; Wu et al., 2007; Yi et al., 2012; Liang et al., 2014a; Liang et al., 2014b), which are extremely difficult to sectioning.

In the present study, distribution of phytochemicals in the dried root of Isatis tinctoria was revealed by microscopic mass spectrometry imaging, using atmospheric pressure–matrix-assisted laser desorption/ionization (AP-MALDI) combined with ion trap–time-of-flight mass spectrometry (IT-TOF/MS). In particular, the slice preparation method directly associated with the quality of the mass spectrometry imaging (MSI) results was optimized for the dried woody sample. Numerous constituents, including alkaloids, sulfur-containing compounds, phenylpropanoids, amino acids, nucleosides, organic acids, flavonoids, terpenes, saccharides, aromatics, peptides, and sphingolipids, were comprehensively visualized in the dried root of Isatis tinctoria for the first time. Moreover, samples from different habitats were distinguished based on their mass spectrometry imaging and the partial least square (PLS) regression.

Material and Methods

Chemicals and Samples

2′, 5′-Dihydroxyacetophenone (2, 5-DHAP), 2, 5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), 1, 5-naphthalenediamine (1, 5-DAN), 9-aminoacridine (9-AA), 1, 8-bis(tetramethylguanidino)naphthalene (TMGN), 1, 2-bis (trimethoxysilyl)ethane (BTME), gelatin, formic acid (LC-MS grade), ethanol (HPLC grade), and methanol (LC-MS grade) were purchased from Sigma-Aldrich (St. Louis, MO, United States ). De-ionized water was purified by a Milli-Q system (Millipore, Bedford, MA, United States). Optimum cutting temperature (OCT) compound was purchased from Leica (Nussloch, Germany). The samples of the dried root of Isatis tinctoria were collected from four main habitats in China, Gansu, Heilongjiang, Xinjiang, and Neimenggu. The sample collection information could be found in Table 1, and the typical macroscopic images of the herb and its transverse section are shown in Figure 1. All samples were authenticated by Associate Professor Shuai Kang, who is in charge of the Traditional Chinese Medicine Herbarium, National Institutes for Food and Drug Control. The voucher specimens were deposited in National Institutes for Food and Drug Control (NIFDC), Beijing, China.

TABLE 1.

Sample collection information of the dried root of Isatis indigotica.

Sample no Habitat Collection time
GS1 Gansu Sep., 2019
GS2 Gansu Sep., 2019
GS3 Gansu Sep., 2019
HLJ1 Heilongjiang Oct., 2019
HLJ2 Heilongjiang Oct., 2019
HLJ3 Heilongjiang Nov., 2019
XJ1 Xinjiang Sep., 2019
XJ2 Xinjiang Oct., 2019
XJ3 Xinjiang Oct., 2019
NMG1 Neimengu Nov., 2019
NMG2 Neimengu Oct., 2019
NMG3 Neimengu Oct., 2019
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FIGURE 1.

FIGURE 1

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A photograph of the dried root of Isatis indigotica (A) and the magnified image of its transverse section (B).

Slice Preparation and Optical Imaging

A piece 1 cm in length was cut from the dried root of Isatis tinctoria using a blade, and then embedded in 0.1 g/ml gelatin solution before freezing at −20°C. The frozen sample embedded with gelatin was axially fixed on a cryomicrotome (Leica CM 1950, Nussloch, Germany) by OCT compound carefully to avoid contamination on the surface of the sample. The gelatin on the top was peeled off to expose the surface of the cross section of the root before one side of the double-sided conductive tape (3M, St. Paul, MN, United States) was adhered to the root surface. Then the tissue was sectioned into a 40-μm slice with the tape at −18°C. Finally, the slice was fixed carefully on an indium tin oxide (ITO)-coated glass slide (Matsunami Glass, Osaka, Japan) with another side of the tape. Before matrix coating, the optical image of the tissue was captured by a charge-coupled device (CCD) camera of the optical microscope embedded to the imaging mass microscope system (Shimadzu iMScope, Kyoto, Japan).

Matrix Application

After optimization, 2′, 5′-dihydroxyacetophenone (2, 5-DHAP) and 1, 5-naphthalenediamine (1, 5-DAN) were chosen as the matrix for positive and negative ion detection, respectively, and were applied in the mode of spraying. The matrix solution of 2′, 5′-dihydroxyacetophenone (2, 5-DHAP) was prepared at a concentration of 10 mg/ml in methanol and water (all containing 0.1% formic acid) at a ratio of 8:2, and 1, 5-naphthalenediamine (1, 5-DAN) was prepared as a saturated solution in ethanol–water (70:30). For spraying, 1 ml of matrix solution was added to the cavity of the handle airbrush. Then the solution was sprayed on the sample surface using the airbrush, keeping a distance at about 10 cm. For each glass, the airbrushing was repeated 10 cycles every 60 s. Finally, the sprayed glass slide was kept in the fume hood for 5 min to vaporize the solvent.

Microscopic Mass Spectrometry Imaging

Microscopic mass spectrometry imaging of the tissue was performed using the iMScope instrument (Shimadzu, Kyoto, Japan) equipped with an optical microscope, an atmospheric pressure chamber for matrix-assisted laser desorption/ionization (AP-MALDI) source, and an ion trap–time-of-flight mass spectrometer. The acquisition region was defined with the help of the optical microscope, and the tissue was irradiated with a diode-pumped 355 nm Nd:YAG laser with 5 ns pulse width. The laser diameter was 80 μm, and the data were collected at an interval of 80 μm. The tissue surface was laser-irradiated with 100 shots (1,000 Hz repetition rate) for each pixel. All the data were acquired in the positive and negative modes with sample voltage of 3.5 and 3.0 kV, respectively. The mass ranges were m/z 100–500 and m/z 500–1,000, while the detector voltage was 1.97 kV for all the samples. Three repetitions for each sample were performed, and two slices were prepared for each repetition in order to measure the positive and negative ions separately.

Data Analysis

Mass image reconstruction and data analysis were performed using IMAGEREVEAL™ MS (Shimadzu, Kyoto, Japan). All images were reconstructed by linear smoothing and displayed in absolute intensity after total ion current (TIC) normalization. Statistical analysis including principal component analysis (PCA) and partial least squares (PLS) regression was carried out by IMAGEREVEAL™ MS for differentiation of samples from different habitats.

Results

Phytochemical Profiles of the Dried Root of Isatis tinctoria

Figure 2 showed the typical overall average mass spectra of the dried root of Isatis tinctoria gained by matrix-assisted laser desorption/ionization and ion trap–time-of-flight (MALDI-IT-TOF) mass spectrometry imaging (MSI) under positive and negative/ionization modes. Positive ions were mainly detected in the mass range of m/z 100–400 and m/z 400–800, while negative ions were mainly observed in the mass range of m/z 100–200, m/z 400–500, and m/z 400–700. The putative identification of the components was based on the accurate mass-to-charge ratio with reference to the isotopic peak, the reference standards, and/or the literatures and data bases. As could be seen from Table 2, the detected phytochemicals belong to a wide range of chemical compound classes such as alkaloids, sulfur-containing compounds, phenylpropanoids, amino acids, nucleosides, organic acids, flavonoids, terpenes, saccharides, aromatics, peptides, and sphingolipids. In the positive ion mode, the detected ions were prominently in the protonated adduct form of all amino acids, most of the alkaloids, some of the phenylpropanoids and the nucleosides, majority of the aromatics, minority of the sulfur-containing compounds, several sphingolipids, as well as organic acids with basic group. Also sodium or potassium adducts of some nucleosides and a few alkaloids, peptides, and sulfur-containing compounds were found. In the negative ion mode, majority of the sulfur-containing compounds and the organic acids, some of the phenylpropanoids, minority of the aromatics, a few saccharides, flavonoids, nucleosides, as well as alkaloids with acid group were readily detected as [M−H]− form of ions.

FIGURE 2.

FIGURE 2

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Typical overall average mass spectra acquired from a cross section of the dried root of Isatis indigotica by matrix-assisted laser desorption and ion trap–time-of-flight (MALDI-IT-TOF) mass spectrometry imaging (MSI) in the spectral ranges of m/z 100–500 in a positive mode (A), m/z 500–1,000 in a positive mode (B), m/z 100–500 in a negative mode (C), and m/z 500–1,000 in a negative mode (D).

TABLE 2.

Assignment of ions observed in the matrix-assisted laser desorption and ion trap–time-of-flight (MALDI-IT-TOF) mass spectrometry imaging (MSI) of the dried root of Isatis indigotica with references regarding shown compounds.

No Compound Chemical class Ion formula Theoretical m/z Observed m/z Mass accuracy (ppm) Refs
1 r-Aminobutyric acid Amino acids C4H9NO2+H 104.0712 104.0709 2.9 Pan, (2014)
2 Choline Alkaloids C5H14NO+ 104.1075 104.1066 8.6 The Metabolomics Innovation Center, (2020)
3 Proline Amino acids C5H9NO2+H 116.0712 116.0709 2.6 Wu et al. (1997)
4 Valine Amino acids C5H11NO2+H 118.0869 118.0865 3.4 Chen et al. (2012)
5 Leucine/isoleucine Amino acids C6H13NO2+H 132.1025 132.1018 5.3 Zhao, (2015)
6 Adenine Nucleosides C5H5N5+H 136.0624 136.0613 8.1 Chen et al. (2012)
7 Aminobenzoic acid Organic acids C7H7NO2+H 138.0556 138.0545 8.0 Wang et al. (2013)
8 4-(2-Hydroxyethyl) phenol Aromatics C8H10O2+H 139.0760 139.0773 9.3 Wang et al. (2013)
9 Hexyl isothiocyanate Alkaloids C7H13NS + H 144.0848 144.0853 3.5 Condurso et al. (2006)
10 3-Formylindole Alkaloids C9H7NO + H 146.0607 146.0614 4.8 Zhao, (2015)
11 Glutamine Alkaloids C5H10N2O3+H 147.0770 147.0758 8.2 Pan, (2014)
12 Lysine Amino acids C6H14N2O2+H 147.1154 147.1132 1.4 Pan, (2014)
13 Uracil Nucleosides C4H4N2O2+K 151.1256 151.1243 8.6 Pan et al. (2013)
14 Guanine Nucleosides C5H5N5O + H 152.0573 152.0588 9.9 Pan et al. (2013)
15 Dopamine Alkaloids C8H11NO2+H 154.0791 154.0790 0.6 HighChem LLC, (2020)
16 Oxindole Alkaloids C8H7NO + Na 156.0426 156.0416 6.4 Wang et al. (2013)
17 Histidine Amino acids C6H9N3O2+H 156.0774 156.0786 7.7 Pan, (2014)
18 Hypoxanthine Nucleosides C5H4N4O + Na 159.0283 159.0275 5.0 Xiao et al. (2003)
19 3-Indoleformic acid Alkaloids C9H7NO2+H 162.0556 162.0553 1.9 Yang et al. (2014b)
20 Phenylalanine Amino acids C9H11NO2+H 166.0869 166.0853 9.6 Pan, (2014)
21 Acetovanillone Aromatics C9H10O3+H 167.0709 167.0709 0.0 Wang et al. (2013)
22 Isatin Alkaloids C8H5NO2+Na 170.0218 170.0221 1.8 Zou and Koh, (2017)
23 2,5-Dihydroxyindole Alkaloids C8H7NO2+Na 172.0375 172.0388 7.6 Li, (2010a)
24 Arginine Amino acids C6H14N4O2+H 175.1196 175.1188 4.6 Zeng et al. (2010)
25 3-Indoleacetonitrile Alkaloids C10H8N2+Na 179.0585 179.0592 3.9 Wang et al. (2013)
26 Tyrosine Amino acids C9H11NO3+H 182.0818 182.0800 9.9 Pan, (2014)
27 Dihydroconiferyl alcohol Phenylpropanoids C10H14O3+H 183.1022 183.1036 7.6 Wang et al. (2013)
28 4-Hydroxyindole-3-carboxaldehyde Alkaloids C9H7NO2+Na 184.0375 184.0368 3.8 Li et al. (2010b)
29 2,4(1H,3H)-Quinazolinedione Alkaloids C8H6N2O2+Na 185.0327 185.0326 0.5 Wang and Liu, 2008
30 Deoxyvasicinone Alkaloids C11H10N2O + H 187.0872 187.0864 4.3 Zhao, 2015
31 1-Methoxy-3-indoleformic acid Alkaloids C10H9NO3+H 192.0661 192.0648 6.8 Yang et al. (2014b)
32 (1′R,2′R,3′S,4′R)-1,2,4-triazole Nucleosides C7H11N3O4+H 202.0829 202.0841 5.9 Pan, (2014)
33 Tryptophan Amino acids C11H12N2O2+H 205.0978 205.0959 9.3 Pan, (2014)
54 L-targinine Peptides C7H16N4O2+Na 211.1165 211.1150 7.1 The Metabolomics Innovation Center, (2020)
35 (−)-(R)-2-(4-Hydroxy-2-oxoindolin-3-yl)-acetamide Alkaloids C10H10N2O3+Na 229.0589 229.0576 5.7 Chen et al. (2012)
36 2′-Deoxyuridine Nucleosides C9H12N2O5+H 229.0825 229.0842 7.4 Pan, (2014)
37 (+)-(S)-2-(3-Hydroxy-4-methoxy-2-oxoindolin-3-yl)-acetamide Alkaloids C11H12N2O4+H 237.0876 237.0899 9.7 Chen et al. (2012)
38 2′-Deoxycytidine Nucleosides C9H13N3O4+Na 250.0804 250.0824 8.0 Pan, (2014)
39 (S)-(−)-Spirobrassinin Sulfur-containing compounds C11H10N2OS2+H 251.0314 251.0335 8.4 Zhang et al. (2020b)
40 Pyrraline Amino acids C12H18N2O4+H 255.1339 255.1332 2.7 The Metabolomics Innovation Center, (2020)
41 Indiforine C Alkaloids C12H14N2O3+Na 257.0902 257.0918 6.2 Liu et al. (2018b)
42 Indirubin/indigotin Alkaloids C16H10N2O2+H 211.0813 211.0821 3.0 Chen et al. (2012)
43 Thymidine Nucleosides C10H14N2O5+Na 265.0801 265.0824 8.7 Pan, (2014)
44 Adenosine Nucleosides C10H13N5O4+H 268.1047 268.1011 6.0 Zhao, (2015)
45 Inosine Nucleosides C10H12N4O5+H 269.0887 269.0905 6.7 Pan et al. (2013)
46 Tryptanthrin Alkaloids C15H8N2O2+Na 271.0484 271.0488 1.5 Chen et al. (2012)
47 2′-O-Methyladenosine Nucleosides C11H15N5O4+H 282.1203 282.1221 6.4 Zhang et al. (2019a)
48 Indican Alkaloids C14H17NO6+H 296.0925 296.0935 3.4 Zou and Koh, (2017)
49 Guanosine Nucleosides C10H13N5O5+Na 306.0815 306.0824 2.9 Xiao et al, (2014)
50 (−)-(2′S)-Isatiscaloids E/(+)-(2′R)-isatiscaloids E Alkaloids C14H19N3O5+H 310.1404 310.1428 7.7 Wang et al. (2009)
51 Isatiscaloids A Alkaloids C15H22N2O5+H 311.1608 311.1627 6.1 Wang et al. (2009)
52 Indiforine F Alkaloids C14H18N2O5+Na 317.1114 317.1138 7.6 Liu et al. (2018b)
53 Evofolin-B Phenylpropanoids C17H18O6+H 319.1182 319.1160 6.9 Wang et al. (2013)
54 Isatiscaloids B Alkaloids C16H20N2O5+H 321.1451 321.1458 2.2 Wang et al. (2013)
55 Adenosine-3′,5′-cyclic monophosphate Nucleosides C10H12N5O6P + H 330.0604 330.0618 4.2 Pan, (2014)
56 Indole-3-acetonitrile-6-O-β-D-glucopyranoside Alkaloids C16H18N2O6+H 335.1244 335.1240 1.2 He et al. (2006a)
57 Isatisindigoticanine K Alkaloids C19H13N3O2+Na 338.0906 338.0927 6.2 Zhang et al. (2020c)
58 Coniferin Phenylpropanoids C16H22O8+H 543.1394 543.1371 6.7 Zhang et al. (2019a)
59 Isaindigodione Alkaloids C18H18N2O4+Na 549.1165 549.1133 9.2 Xiao et al. (2014)
60 Cyclo (L-Phe–L-Tyr) Peptides C18H18N2O3+Na 549.2300 549.2320 5.7 Wang et al. (2013)
61 Indole-3-acetonitrile-2-S-β-D-glucopyranoside Sulfur-containing compounds C16H18N2O5S + H 351.1015 351.1005 2.8 Yang et al. (2014b)
62 Qingdainone Alkaloids C23H13N3O2+ H 364.1087 364.1079 2.2 He et al. (2006a)
11 Isatindigotindoline C Alkaloids C23H21N3O4+H 404.1611 404.1602 2.2 Liu et al. (2018a)
64 Isatisindigoticanine A Alkaloids C22H18N2O6+H 407.1244 407.1215 7.1 Zhang et al. (2019b)
65 Isatindigobisindoloside G Sulfur-containing compounds C24H21N3O5S + H 455.1278 455.1238 8.8 Zhang et al. (2020a)
66 3-[2′-(5′-hydroxymethyl)furyl]-1(2H)-isoquinolinone-7-O-β-D-glucoside Alkaloids C20H21NO9+K 458.2199 458.2167 7.0 He et al. (2006b)
67 Isatisindigoticanine I Sulfur-containing compounds C24H21N3O5S + H 464.1281 464.1245 7.8 Zhang et al. (2020a)
68 Isatindigoside F Sulfur-containing compounds C25H23N3O5S-H 476.1279 476.1258 4.4 Zhang et al. (2020a)
69 Isatigotindolediosides B Alkaloids C20H25NO11 + Na 478.1326 478.1307 4.0 Meng et al. (2017b)
70 Isatigotindolediosides A Alkaloids C21H27NO12 + H 486.1612 486.1579 6.8 Meng et al. (2017b)
71 Isatindigoside J Alkaloids C25H27N3O8+H 498.1877 498.1892 3.0 Zhang et al. (2020c)
72 Isatithioetherin A/isatithioetherin B Sulfur-containing compounds C20H26N4O4S3+Na 505.1014 505.1058 8.7 Guo et al. (2020b)
73 Bisindigotin Alkaloids C32H18N4O2+Na 513.1328 513.1278 9.7 Wei et al. (2005)
74 Isatigotindolediosides D Alkaloids C22H28N2O13 + H 529.1670 529.1111 7.4 Meng et al. (2017b)
75 Isatithioetherin C/isatithioetherin E Sulfur-containing compounds C20H26N4O4S4+Na 537.0735 537.0784 9.1 Guo et al. (2020b)
76 (+)-(7R,7′R,8S,8′S)-Neo-olivil Phenylpropanoids C26H54O12 + H 539.2129 539.2143 2.6 Kikuchiand Kikuchi, (2005)
77 (2S,3R)-3-Hydroxymethyl-N-(2′-hydroxynonacosanoyl)-trideca-9E-sphingenine Sphingolipids C43H85NO5+H 696.6507 696.6565 8.3 Li et al. (2007)
78 1-O-β-D-Glucopyranosyl-(2S,3R)-N-(2′-hydroxyhe xacosanoyl)-octadeca-11E-sphingenine Sphingolipids C50H97NO9+H 856.7242 856.7292 5.8 Sun et al. (2009)
79 Propanedioic acid Organic acids C3H4O4-H 103.0031 103.0040 8.7 Li, (2010)
80 Pyrocatechol Aromatics C6H6O2-H 109.0289 109.0294 4.6 Wang et al. (2013)
81 Maleic acid/fumaric acid Organic acids C4H4O4-H 115.0031 115.0040 7.8 Peng et al. (2005b)
82 Nicotinic acid Organic acids C6H5NO2-H 122.0241 122.0254 5.7 Li, (2010)
83 3-Methylfuran-2-carboxylic acid Organic acids C6H6O3-H 125.0238 125.0243 4.0 Zeng et al. (2010)
84 Goitrin/epigoitrin Sulfur-containing compounds C5H7NOS-H 128.0169 128.0162 5.5 Wang et al. (2014)
85 Malic acid Organic acids C4H6O5-H 133.0136 133.0147 8.3 Liu et al. (2010)
86 Salicylic acid Organic acids C7H6O3-H 137.0238 137.0241 2.2 Zhao, (2015)
87 Vanillin Aromatics C8H8O3-H 151.0394 151.0388 4.0 Sun et al., 2007
88 Fructose/glucose Saccharides C6H12O6-H 179.0555 179.0554 0.6 Liu et al. (2010)
89 Mannitol Saccharides C6H14O6-H 181.0711 181.0697 7.7 He et al., 2006b
90 2-Amine-4-quinlinecarboxylic acid Alkaloids C10H8N2O2-H 187.0507 187.0517 5.3 Pan, (2014)
91 Citric acid Organic acids C6H8O7-H 191.0191 191.0191 0.0 Liu et al. (2010)
92 Glucuronic acid Organic acids C6H10O7-H 193.0548 193.0338 5.2 Liu et al. (2010)
93 Syringic acid Organic acids C9H10O5-H 197.0461 197.0449 6.1 Wang et al. (2009)
94 Isatindosulfonic acid E Sulfur-containing compounds C9H9NO3S-H 210.0224 210.0233 4.3 Meng et al. (2017a)
95 Isatindosulfonic acid C Sulfur-containing compounds C10H11NO4S-H 240.0330 240.0318 5.0 Meng et al. (2017a)
96 Palmitic acid Organic acids C16H32O2-H 255.2323 255.2542 7.4 Kizil et al. (2009)
97 Emodin Flavonoids C15H10O5-H 269.0449 269.0450 0.4 Li, (2010)
98 Linolenic acid Organic acids C18H30O2-H 277.2167 277.2172 1.8 Kizil et al. (2009)
99 Calycosin Flavonoids C16H12O5-H 283.0606 283.0111 8.8 Wang et al. (2013)
100 Stearic acid Organic acids C18H36O2-H 283.2116 283.2646 3.5 Kizil et al. (2009)
101 Sucrose Saccharides C12H22O11-H 541.1083 541.1089 1.8 Liu et al. (2010)
102 Sinensetin Flavonoids C20H20O7- H 371.1130 371.1103 7.3 Li, (2010)
103 Gluconapin Sulfur-containing compounds C11H19NO9S2-H 372.0422 372.0429 1.9 Mohn et al. (2007)
104 Isatindigotindoloside C/Isatindigotindoloside D Sulfur-containing compounds C17H20N2O6S-H 379.0911 379.0938 6.6 Liu et al. (2015b)
105 Progoitrin/epiprogoitrin Sulfur-containing compounds C11H19NO10S2-H 388.0371 388.0355 4.1 Mohn et al. (2007)
106 Glucotropaeolin Sulfur-containing compounds C14H19NO9S2-H 408.0422 408.0418 1.0 Mohn et al. (2007)
107 Isovitexin Flavonoids C21H20O10-H 431.0977 431.1012 8.1 Zhao, (2015)
108 Glucobrassicin Sulfur-containing compounds C16H19N2O9S2-H 447.0531 447.0537 1.3 Guo et al. (2020a)
109 Isatindigobisindoloside A/isatindigobisindoloside B Alkaloids C24H23N3O6-H 448.1508 448.1554 5.8 Liu et al. (2015a)
110 Isatindigoside F Sulfur-containing compounds C25H23N3O5S-H 476.1279 476.1258 4.4 Zhang et al. (2020a)
111 Isatigotindolediosides F Sulfur-containing compounds C21H27NO12S-H 516.1175 516.1153 4.3 Meng et al. (2017b)
112 Isatigotindolediosides E Sulfur-containing compounds C22H28N2O11S-H 527.1335 527.1284 9.7 Meng et al. (2017b)
113 Isatigotindolediosides D Sulfur-containing compounds C22H28N2O13-H 527.1512 527.1538 4.9 Meng et al. (2017b)
114 Glucoisatisin/epiglucoisatisin Sulfur-containing compounds C21H26N2O12S2-H 561.0848 561.0830 3.2 Mohn and Hamburger, (2008)
115 Isovitexin Flavonoids C21H20O10-H 431.0977 431.1012 8.1 Zhao, (2015)
116 Linarin Flavonoids C28H32O14-H 591.1713 591.1674 6.6 Peng et al. (2005a)
117 Neohesperidin Flavonoids C28H54O15-H 609.1819 609.1801 3.0 Peng et al. (2005a)
118 Clemastanin B Phenylpropanoids C32H44O16-H 683.2550 683.2489 8.9 Yang et al. (2014a)
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Visualization of the Distribution of Phytochemicals in the Dried Root of Isatis tinctoria

The optical images in Figures 3, 4 showed the main compartments of the cross section of the dried root of Isatis tinctoria: cork and cortex, phloem, cambium, and xylem as well as the distinctive spatial distribution of various kinds of characteristic constituents. The most abundant class of chemical components isolated from Isatis tinctoria is alkaloid. They were mostly detected as the positive ions and presented a variety of distributions. As could be seen from Figure 3B, sodium adduct of oxindole (m/z 156.0416), an indole alkaloid, was located exclusively in xylem. Another ion of m/z 458.2167 (Figure 3C) was found to have a different distribution in the specific region of phloem. This ion was assigned to the potassium adduct of 3-[2′-(5′-hydroxymethyl) furyl]-1(2H)-isoquinolinone-7-O-β-D-glucoside, an isoquinolinone alkaloid glycoside. Sulfur-containing compounds are characteristic secondary metabolites occurring in cruciferous plant, and they are the second abundant class of chemical components in Isatis tinctoria. Taking isatindigoside F (m/z 476.1258), a typical glucosinolate, as an example (Figure 4B), sulfur-containing compounds were mainly detected as the negative ions which accumulated mostly in phloem of the dried root of Isatis tinctoria. Dozens of phenylpropanoids are also found in Isatis indigotica, and they can be detected in positive or negative modes. Figure 3D and Figure 4C showed the spatial distribution of the ions of m/z 343.1371 and m/z 683.2479, which corresponded to the [M+H]+ and [M−H]− forms of the ions of coniferin and clemastanin B, a phenylpropanoid glycoside and a lignan diglucoside, respectively. Regardless of the form of the ions, the majority of the phenylpropanoids presented the highest abundance near the lateral area of the root, including phloem, cortex, and cork. Isatis root produces several nucleosides, which are normally observed as positive ions. MSI results suggested that the ion of m/z 152.0588 was assigned to the protonated adduct of guanine. As noticeable in the ion, it was located almost exclusively in xylem of the root. Moreover, mass spectrometry imaging (MSI) study on the constituents of the dried root of Isatis tinctoria revealed the presence of a variety of organic acids. The negative ions assigned as [M−H]− form of maleic acid (m/z 115.004), malic acid (m/z 133.0147, Figure 4E), and citric acid (m/z 191.0191, Figure 4F) were found at very high intensities in the area of phloem. Amino acids are widespread primary metabolites in plants, and they showed diversified distribution in the dried root of Isatis tinctoria in the form of the proton adducts. The protonated adduct of histidine at m/z 156.0786 (Figure 3F) was more abundant at the inner side of the cambium. On the contrary, another ion image concerned the distribution of proline at m/z 116.0709 (Figure 3G) with the highest abundance at the outer side of the cambium. Interestingly, the ion of arginine at m/z 175.1188 (Figure 3H) was located with prominent abundance in all tissues except regions of cambium, cortex, and cork. Ions based on phytochemicals belonging to minor groups in Isatis root like peptides, saccharides, flavonoids, and aromatics were also analyzed. It could be judged from Figure 3I that the highest concentration of a cyclic peptide named cyclo (L-Phe–L-Tyr) in the form of potassium adduct at m/z 349.2320 was in xylem and phloem. Sucrose is a nutritional ingredient that naturally occurs in many plants. Its negative ion at m/z 341.1089 (Figure 4G) was located mainly in the outer part of the root corresponding to the location of phloem, cortex, and cork. Similar distribution pattern was observed for the ion assigned to isovitexin (m/z 431.1012, Figure 4H), a representative flavone glycoside from Isatis root. Compounds belonging to aromatics are also isolated from Isatis root. This group includes vanillin, whose negative ion could be found at m/z 151.0388. In contrast to the above-discussed localization of two ions, this component accumulated at the area corresponding to xylem mainly (Figure 4I).

FIGURE 3.

FIGURE 3

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Optical image of the dried root of Isatis indigotica (A) and the mass spectrometry images of the positive ions of oxindole (B), 3-[2′-(5′-hydroxymethyl)furyl]-1(2H)-isoquinolinone-7-O-β-D-glucoside (C), coniferin (D), guanine (E), histidine (F), proline (G), arginine (H), and cyclo (L-Phe–L-Tyr) (I).

FIGURE 4.

FIGURE 4

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Optical image of the dried root of Isatis indigotica (A), and the mass spectrometry images of the negative ions of isatindigoside F (B), clemastanin B (C), maleic acid (D), malic acid (E), citric acid (F), sucrose (G), isovitexin (H), and vanillin (I).

Differentiation of the Habitats of the Dried Root of Isatis tinctoria

Three repetitions for twelve samples of the dried root of Isatis tinctoria from Gansu, Xinjiang, Heilongjiang, and Neimenggu were first classified according to their habitats as groups 1, 2, 3, and 4, respectively. Mass spectrometry imaging (MSI) data of the whole tissues within two spectral ranges (m/z 100–500 and m/z 500–1,000) in positive and negative modes were input to establish four partial least square (PLS) regression models separately. Partial least square (PLS) regression was performed by importing the information of all detected ions to the X-matrix, while the actual groups of habitats were imported to the Y-matrix. As shown in Figure 5, good correlation between the predicted and actual groups of habitats of the dried root of Isatis tinctoria was found with all regression coefficients (R 2) above 0.99, which indicated an excellent discrimination ability of the mass spectrometry imaging (MSI) method.

FIGURE 5.

FIGURE 5

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Results of partial least square (PLS) regression models for samples of the dried root of Isatis indigotica from four habitats based on mass spectrometry imaging (MSI) data in the spectral ranges of m/z 100–500 in a positive mode (A), m/z 500–1,000 in a positive mode (B), m/z 100–500 in a negative mode (C), and m/z 500–1,000 in a negative mode (D).

Discussion

In this study, iMScope, the optical microscope, in combination with the atmospheric pressure–matrix-assisted laser desorption/ionization (AP-MALDI) and the ion trap–time-of-flight mass spectrometry (IT-TOF/MS), was first applied to visually clarify the distribution of phytochemicals in the dried root of Isatis tinctoria. Nowadays, there have been increasing reports on the applications of mass spectrometry imaging (MSI) in the investigation of animal or human tissues (Wu et al., 2019), but with less focus on plants (Davison et al., 2019; Sun et al., 2020; Zhang et al., 2020d), not to mention traditional Chinese medicine (TCM). In addition, most of the research works were performed with the fresh herbs (Fowble et al., 2017; Lange et al., 2017; Freitas et al., 2019) due to the obstacles in the sectioning of the dried material. To reveal the spatial localization of phytochemicals in traditional Chinese medicine (TCM) in the form of actually sold in the market and used in clinic, the dried root of Isatis tinctoria was chosen as the imaging subject. As expected, the cryosectioning of the hard and fragile woody root presented great challenges.

First, the thickness of the tissue was optimized in the range from 20 to 80 μm. It was apparent that the thicker the slice, the more integrated the tissue, but thinner slice posed more sensitive detection of ions. Taking a comprehensive consideration of the integrity of the tissue and the quality of the mass data, a thickness of 40 μm was selected. Next, the temperature for cryosectioning of the frozen tissue was assessed in the range of −12–−22°C. It was found that the section would rupture when the temperature was too low, while when the temperature was too high, the section would wrinkle. Numerous trials indicated that satisfactory result could be obtained with a temperature of −18°C, which was coincident with the reported optimum cryosectioning parameter for the roots of Panax genus (Wang et al., 2016). Since the thin slice of the dried root of Isatis tinctoria was easy to fall off from the glass slide, a double-sided adhesive tape was utilized. To better avoid tissue break and movement during the sectioning, the sample surface was adhered by one side of the tape before cutting.

Subsequently, several matrices were evaluated as follows: 2′, 5′-dihydroxyacetophenone (2, 5-DHAP), 2, 5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and 1, 5-naphthalenediamine (1, 5-DAN) for the positive ion mode, and 1, 5-naphthalenediamine (1, 5-DAN), 9-aminoacridine (9-AA), 1, 8-bis(tetramethylguanidino) naphthalene (TMGN), and 1, 2-bis(trimethoxysilyl)ethane (BTME) for the negative ion mode. Briefly, comprehensive detection of molecules was achieved when using 2′, 5′-dihydroxyacetophenone (2, 5-DHAP) and 1, 5-naphthalenediamine (1, 5-DAN) as the matrixes in positive and negative ion modes, respectively. It was unexpected that DHB, the regular matric used in MALDI MSI of small molecules in plants, was not the most fitted matric for the dried root of Isatis tinctoria. In addition, two matrix-coating modes, air-assisted spraying and sublimation, were compared, and the results indicated that spraying presented stronger signal intensity and miner analyte delocalization.

As a result, 118 ions in the dried root of Isatis tinctoria were assigned as 10 classes of components including some bioactive molecules. Not surprisingly, the majority of the identified phytochemicals belonged to alkaloids and sulfur-containing compounds. The second most detected components were nucleosides, organic acids, and amino acids. A few aromatics, flavonoids, phenylpropanoids, saccharides, peptides, and sphingolipids were also found. On the contrary, esters, quinones, terpenes, sterols, alcohols, aldehydes, ketones, and nitriles from Isatis indigotica were not detected by mass spectrometry imaging (MSI) this time. Like fructose and glucose, several alkaloids, sulfur-containing compounds, amino acids, and saccharides were grouped together in Table 2 since they hold isomeric relationship and could not be differentiated by their exact mass. Hence, further work should be done for the separation of the detected isomers. The presence of most of the identified components was previously found in Isatis indigotica except choline, dopamine, L-targinine, and pyrraline, which might be caused by the loss during the extraction and separation process in routine methods. Therefore, the matrix-assisted laser desorption/ionization and ion trap–time-of-flight (MALDI-IT-TOF) mass spectrometry imaging (MSI) in a single run covered not only the natural products that were commonly detected but also less reported molecules, illustrating the high throughput and high sensitivity of the method. It was also delighted to find that a couple of important bioactive molecules (references could be seen in Table 2) from Isatis indigotica, such as uracil, adenine, hypoxanthine, 4(3H)-quinazolinone, deoxyvasicinone, 2,4(1H,3H)-quinazolinedione, isalexin, guanine, indirubin, and indigotin, were identified in an untargeted, label-free, and multiplexed way without extraction or isolation. Among them, uracil, adenine, guanine, indirubin, and indigotin are often used as the chemical markers for authentication of the root of Isatis indigotica, and they were detected in all the investigated samples. Using mass spectrometry imaging, the herb could be identified in a rapid way with multiple indexes, comparing to routine TLC or HPLC methods.

As indicated in Figure 6, the optical microscope embedded in the iMScope made it possible to acquire optical images and ion distribution images in the same instrument. The convenient assessment allowed for an unprecedented visualization of the spatial distribution of phytochemicals in the dried root of Isatis indigotica. Consequently, the localization and spatial information of some molecules in the root tissue were elucidated, which were related to the botanical structure of the herb. In all, a majority of the phytochemicals were shown to be more abundant in phloem, the nutrition-storing tissue of Isatis indigotica, than in xylem, the principle water-conducting tissue. Nevertheless, the signals for some identified constituents were considerably lower, and their mass spectrometry imaging (MSI) localization was therefore not so distinctive. Comparing with the LC-MS analysis after laser microdissection, microscopic mass spectrometry imaging using matrix-assisted laser desorption can reveal the spatial distribution of compounds in a more precise and direct way. Besides, segmentation and dissection might bring uncontrollable pollution, compound migration, or denaturation. However, the differentiation of isomers and absolute quantitation were not available by MALDI-MSI currently.

FIGURE 6.

FIGURE 6

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Optical image (A), mass spectrometry image (B), and overlay image (C) of the dried root of Isatis indigotica.

Finally, based on the ion images, data were collected from the whole tissue and analyzed by partial least squares (PLSs), and the dried root of Isatis indigotica from four habitats was differentiated unambiguously. Spectra collected from the whole tissue, the outer part of the tissue (cork, cortex, and phloem), and the inner part of the tissue (cambium and xylem) were also inputted for principal component analysis (PCA). However, no distinct cluster was observed for samples from different habitats.

Among other possibilities, the results from this study can be applied to increase the extraction yield of a given active component in Isatis indigotica, which is promising in research fields, such as pharmaceutical applications and industrial production. Moreover, the location of specific metabolites is helpful to improve the understanding of the relationship between compound distribution and plant structure as well as function. Combining with the chemometric method, mass spectrometry imaging (MSI) provides a simple and rapid approach for distinguishing habitats of traditional Chinese medicine (TCM) and exploring the environmental effects of plant growth. Finally, similar studies on other traditional Chinese medicine (TCM) are underway in our laboratory.

Acknowledgments

The authors gratefully acknowledge the technical advice provided by Shuang-shuang Di and Honggang Nie from Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University.

Data Availability Statement

The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Author Contributions

LN designed and performed the experiments, analyzed the data, and wrote the manuscript. JD, LH, CL, and XQ assisted in performing the experiments. SK collected and authenticated the dried root of Isatis indigotica. ZD and SM revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 81303194).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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