A method for analyzing the spatial distribution of functional components in burdock roots

Abstract

The present invention discloses a method for analyzing the spatial distribution of functional components in burdock root, and relates to the field of analytical detection technology. The method comprises the following steps: first, embedding the burdock root with an embedding agent, slicing it after freezing treatment to obtain burdock root tissue slices, and performing a vacuum drying treatment; then spraying the burdock root tissue slices with a matrix; then performing MALDI mass spectrometry imaging analysis on the burdock root tissue slices sprayed with the matrix to obtain the spatial distribution of functional components in the burdock root; the matrix used is DHAP. The present invention optimizes the MALDI-MSI matrix and uses the MALDI-MSI analysis method in negative ion detection mode to establish a high-coverage, visualized mass spectrometry imaging method for chemical components in burdock root, thereby achieving visualized characterization of sugars, organic acids, and flavonoids in burdock root, providing new technical support for the development and comprehensive utilization of burdock components.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to a method for analyzing the spatial distribution of functional components in burdock roots. Background Art

[0002] Burdock (Arctium lappa L.), also known as "evil fruit," "rat sticky," and "powerful," is a biennial plant of the genus Arctium in the Asteraceae family. Its roots, stems, leaves, and fruits are all used medicinally. The "Ming Yi Bie Lu" (Famous Doctors' Prescriptions) states that burdock rhizomes can treat "typhoid fever, fever, and thirst, and long-term consumption can reduce weight and prevent aging." Modern research has revealed that burdock root is rich in various chemical components, including inulin, cellulose, polyphenols, flavonoids, and amino acids, exhibiting diverse pharmacological effects, including hypoglycemic, anti-aging, and constipation relief. It is also popular as a nutritional health food. Therefore, research on the chemical composition of burdock root is crucial for elucidating the mechanisms of action of its functional components.

[0003] Currently, researchers at home and abroad often use a variety of analytical techniques to characterize functional components, including liquid chromatography / gas chromatography-mass spectrometry, high-speed countercurrent chromatography, and ion chromatography. These techniques offer advantages such as high accuracy, good reproducibility, and a wide linear range. However, these techniques suffer from complex and time-consuming pretreatment processes, as well as the loss of spatial distribution information of important metabolites, making them incapable of in situ characterization of endogenous plant components. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for analyzing the spatial distribution of functional components in burdock roots to solve the problems existing in the above-mentioned prior art. The visual characterization of sugars, organic acids and flavonoids in burdock roots provides technical support for the development and comprehensive utilization of burdock components.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for analyzing the spatial distribution of functional components in burdock roots, comprising the following steps:

[0007] (1) Sample preparation: Burdock root was embedded in an embedding agent, sliced ​​after freezing, and the burdock root tissue slices were obtained, which were then vacuum dried;

[0008] (2) Matrix spraying: spraying the vacuum-dried burdock root tissue slices with the matrix to obtain burdock root tissue slices sprayed with the matrix;

[0009] (3) The burdock root tissue sections sprayed with the matrix were subjected to MALDI mass spectrometry imaging analysis to obtain the spatial distribution of functional components in the burdock root;

[0010] The matrix used for matrix spraying in step (2) is DHAP.

[0011] Furthermore, the embedding agent is an OCT embedding agent.

[0012] Furthermore, the freezing treatment in step (1) is carried out at a temperature of -20±2°C and for a time of 40±10 min, preferably at a temperature of -20°C and for 40 min.

[0013] Furthermore, the spraying density in step (2) is 0.0025±0.0005 mg / mm 2 The spraying cycle number is 18±4 times. The preferred spraying density is 0.0025mg / mm 2 ; The number of spraying cycles is 18 times.

[0014] Furthermore, the nozzle temperature of the matrix spraying is 60±2°C, preferably 60°C.

[0015] Furthermore, the mass-to-charge ratio detected by MALDI mass spectrometry imaging analysis is 100-1500.

[0016] Furthermore, the sample scanning resolution during MALDI mass spectrometry imaging analysis is 50 to 200 μm, preferably 100 μm.

[0017] Furthermore, the functional ingredients include sugars, organic acids and flavonoids.

[0018] The present invention discloses the following technical effects:

[0019] The present invention optimizes the MALDI-MSI matrix and utilizes the MALDI-MSI analysis method in negative ion detection mode to establish a high-coverage, visualized mass spectrometry imaging method for the chemical components in burdock root. This method achieves visualized characterization of sugars, organic acids, and flavonoids in burdock root, providing new technical support for the development and comprehensive utilization of burdock components. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 The mass spectra and ion intensities of caffeic acid and quercetin in different matrices are shown in Figure 1, where (a) is the mass spectrum of caffeic acid, (b) is the mass spectrum intensity of caffeic acid, (c) is the mass spectrum of quercetin, and (d) is the mass spectrum intensity of quercetin.

[0022] Figure 2For the organizational structure of burdock;

[0023] Figure 3 The mass spectrum and imaging diagram of burdock sugar; (a) is the mass spectrum of burdock sugar, and (b) is the imaging diagram;

[0024] Figure 4 This is the MALDI-MSI image of organic acids in burdock root;

[0025] Figure 5 This is the MALDI-MSI image of flavonoids in burdock root. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] With the development of medicinal plant metabolomics, the role of endogenous plant molecules in processes such as signal transduction and defense regulation has become widely recognized. Existing research has demonstrated that burdock root contains a variety of endogenous molecular components, including polysaccharides, organic acids, flavonoids, and alkaloids, which play a vital role in its growth and metabolism, as well as in the transport and storage of nutrients. This study employed MALDI-MSI analysis in negative ion detection mode using a DHAP matrix to analyze the spatial distribution of these substances in burdock root, aiming to provide a reference for research related to its chemical composition.

[0032] Example 1 Analysis of the spatial distribution of functional components in burdock

[0033] 1. Instruments and Reagents

[0034] Rapiflex MALDI Tissuetyper TM TOF / TOF mass spectrometer (Bruker, Germany, equipped with SmartBeam TM 3D 355nm laser and flexAnalysis data processing workstation), Thermo CryoStar NX50NOVPD microtome (Thermo Fisher Scientific), ITO conductive glass indium tin oxide (square resistance ≤ 6Ω, South China Xiangcheng Technology Co., Ltd.), HTX TM-Sprayer TM A matrix sprayer (HTX Technology, USA), a vacuum dryer (Shanghai Yueci Electronic Technology Co., Ltd.), and SCiLS Lab 2018b mass spectrometry imaging data analysis software (Germany GmbH) were used.

[0035] 2,5-Dihydroxybenzoic acid (DHB, purity >99.0%), α-cyano-4-hydroxycinnamic acid (HCCA, purity >99.0%), 2,5-dihydroxyacetophenone (DHAP, purity >99.0%), 9-aminoacridine (9-AA, purity >99.0%), N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC, purity ≥98.0%), and trifluoroacetic acid (TFA, purity >99.0%) were purchased from Sigma, USA. Caffeic acid and quercetin (purity >99.0%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Acetonitrile and methanol (mass spectrometry grade, Merck, Germany), and OCT embedding medium were purchased from Sakura, USA.

[0036] Fresh burdock root (white-skinned variety, also known as yellow burdock), Lanling, Cangshan, Shandong.

[0037] 2. Matrix Screening

[0038] DHAP, 9-AA, and NEDC matrices were precisely prepared at a concentration of 20 mg / mL in a 1:1 acetonitrile / water (containing 0.1% TFA, v / v) solvent. A mixed matrix of DHB and HCCA (w / w = 1:1) was precisely prepared at a concentration of 20 mg / mL in a 70% acetonitrile, 25% methanol, and 5% water (containing 0.1% TFA, v / v) solvent. 6 mmol / L standard solutions of caffeic acid and quercetin were prepared in methanol. First, 1 μL of caffeic acid (6 mM, methanol) and quercetin (6 mM, methanol) were added dropwise to a stainless steel target plate. The plate was then placed in a desiccator. After approximately 30 minutes, the solvent in the sample solution evaporated. The three matrices were then dripped onto the dried sample. A blank was also dripped with the DHB and HCCA matrix mixture as a standard (for instrument calibration). After complete drying, MALDI analysis was performed.

[0039] 3. MALDI-TOF Mass Spectrometry Analysis

[0040] Place the MALDI target plate containing the sample into the instrument and operate the instrument using the Flex-Control software. First, select the established MALDI method based on the properties of the target and the mass spectrometric signal, with the ion mode set to negative. Then, position the mouse over the standard sample and click Start. The mass spectrometric signal for the sample will appear on the interface. Next, click Calibration, Automatic Assign, and ensure that the Er (error) is no more than 5 ppm and the mass spectrometric peaks of at least five standards are aligned. Then, click Apply. Next, click Sample, select the minimum laser value suitable for sample detection, click File, Save Method. Then, position the mouse over the sample, click Start, and save the data file. Analyze the data using the Flex-Analysis software. Click File, Open Data File, Browse, Open, then Mass List, Find. Find the target peak in the Mass List on the right, record the peak intensity, and export the mass spectrum and ASSⅡ File (TXT file). The optimal matrix suitable for the detection of target components was screened based on ionic strength and background interference.

[0041] In the present invention, in negative ion mode, DHAP performed best and had the highest ionic strength when used as a matrix compared to 9-AA and NEDC. Therefore, DHAP was selected as a matrix to perform spatial distribution analysis of burdock components.

[0042] Figure 1The mass spectra and ion intensities of caffeic acid and quercetin in different matrices are shown in Figure 1. (a) is the mass spectrum of caffeic acid, (b) is the mass spectrum intensity of caffeic acid, (c) is the mass spectrum of quercetin, and (d) is the mass spectrum intensity of quercetin.

[0043] 4. Sample Preparation

[0044] Fresh burdock roots were cut into 0.5 cm segments. OCT embedding medium was added dropwise to the center of a specimen holder at room temperature until the thickness reached approximately 2 mm. A pre-prepared cross-section of the burdock segment was then pressed upward onto the OCT embedding medium. The OCT embedding medium was then added dropwise around the burdock segment to encapsulate the segment. The burdock segment was then placed in a cryostat and pre-frozen for 40 minutes (-20°C). Within 30 seconds of placement in the cryostat, the transparent OCT embedding medium surrounding the burdock root gradually transformed into an opaque white solid. The specimen holder was then mounted on the cryostat's target head and coarsely sliced ​​(50 μm). Once the cross-section was smooth, fine slices were then made, adjusting the thickness to 16 μm for the complete root tissue cross-section (including the pith, xylem, cambium, phloem, cortex, and epidermis). The sliced ​​tissue sections were then mounted on ITO conductive glass and evacuated in a vacuum desiccator for 30 minutes.

[0045] 5. Matrix spraying

[0046] The vacuum-dried burdock root tissue slices were fixed in a matrix sprayer, and then the spraying program was set: the nozzle temperature was 60°C, the matrix solution flow rate was 0.03 mL / min, the nozzle movement spacing was 3 mm, the nozzle movement speed was 1200 mm / min, and the number of spraying cycles was 18 times. The optimal matrix spray density on the slices was about 0.0025 mg / mm 2 Then, use a syringe to draw up the matrix spray volume calculated by the software and slowly inject it into the matrix sprayer. Click Start. After spraying is complete, vacuum dry. Next, add the DHB and HCCA standard mixture dropwise to the conductive glass and mark the position for molecular weight calibration in subsequent imaging analysis. Use correction fluid to mark the position on the conductive glass slide, then scan the slide area with a scanner for subsequent instrument positioning of the sample.

[0047] 6. MALDI mass spectrometry imaging analysis

[0048] The conductive glass sprayed with matrix was placed on the Rapiflex MALDI Tissuetyper TMPerform mass spectrometry imaging analysis using a TOF / TOF mass spectrometer. First, select the imaging method from the Flex-control software, locate the standard sample, and automatically calibrate the mass spectrometer signal. Set the laser energy to 70% and the mass-to-charge ratio (m / z) range to 100-1500. Click Save Method. Create a new file name using the Flex-imaging software, select the same method as the Flex-control, and click Preview to find the scanned image. Use the correction fluid marker to locate the sample. Click Finish, select the sample area, and circle the background signal peak where there is no sample or standard sample. Click Step 5. Scan the sample at a resolution of 100 μm. Once the sample scan is complete, click Step 6 (Load Result).

[0049] Example 2 Distribution characteristics of carbohydrates in burdock roots

[0050] In negative ion mode, sugar substances tend to show hydrogen reduction peaks, forming [MH] - In the present invention, it was found that there were a series of mass spectrum peaks with a difference of 162Da in the mass spectrum of burdock root tissue, and they had the same spatial distribution position and were widely distributed in the burdock root tissue (pith, secondary xylem, cambium, secondary phloem and cortex). It was further speculated that these mass spectrum peaks were the hexose residue signals of burdock root carbohydrate substances. Figure 2 As shown in the above, the present invention found that there were 6 carbohydrate mass spectrometry signals in burdock root tissue, including m / z 503.1 (DP=3), m / z 665.1 (DP=4), m / z 827.1 (DP=5), m / z 989.1 (DP=6), m / z 1151.1 (DP=7) and m / z 1313.1 (DP=8) ([MH] - ) mass spectrum peaks, the spatial distribution of burdock root carbohydrates is similar, widely distributed in the burdock root tissue, and the epidermis contains almost no burdock carbohydrate components.

[0051] Figure 2 For the organizational structure of burdock; Figure 3 The mass spectrum and imaging diagram of burdock sugar, where (a) is the mass spectrum of burdock sugar and (b) is the imaging diagram.

[0052] Example 3 Distribution characteristics of organic acids in burdock roots

[0053] Organic acids are an important type of acidic metabolite in plants and are currently the most isolated and identified compounds from burdock roots. Studies have found that organic acids can improve plant disease resistance and stress resistance, and help improve plant quality. Caffeic acid and quinic acid compounds in burdock roots have good free radical scavenging ability and have protective ability against oxidative damage. The present invention selected white burdock roots for research, and the mass spectrometry imaging results of representative organic acids are shown below. Figure 4 As shown. In addition to caffeic acid phenethyl ester ([MH] - ,m / z 283.3)( Figure 4 b) is mainly distributed outside the pith, while the other three organic acids are mainly distributed in the epidermis and cortex, such as caffeic acid ([MH] - ,m / z 179.1)( Figure 4 a) Mainly distributed in the cortex and rarely in the secondary phloem; D-(-)quinic acid ([MH] - ,m / z 191.1)( Figure 4 c) Mainly distributed in the cortex; dicaffeoylquinic acid ([M-2H] - ,m / z 514.5)( Figure 4 d) Mainly distributed in the epidermis.

[0054] Example 4 Tissue Distribution of Flavonoids in Burdock Root

[0055] Flavonoids are an important class of chemical compounds found primarily in higher plants and ferns. Within plants, flavonoids primarily exist as glycosides bound to sugars, while some exist in a free form. In plant tissues such as fruits, leaves, and flowers, flavonoids are primarily glycosides; in the hard tissues of the xylem, they exist as free aglycones. Literature reports indicate that burdock root contains multiple flavonoid components, including quercetin, luteolin, glycyrrhizin, and quercetin rhamnoside, which exhibit antioxidant and lipid-lowering activities.

[0056] Flavonoids are more easily detected by mass spectrometry in negative ion mode, among which representative flavonoids are Figure 5 As shown. Figure 5 a It can be seen that quercetin ([MH] - , 301.2) are distributed in all tissues; luteolin ([MH] - , 285.2) is abundant in the epidermis and pith of burdock root tissue ( Figure 5 b); Liquiritin ([MH] - , 417.4)( Figure 5 c) concentrated in the secondary phloem; quercetin glucoside ([MH] - , 609.5)( Figure 5 d) Mainly distributed in the cortex adjacent to the epidermis.

[0057] This study used white-muscle burdock as the research subject and used MALDI-MSI in negative ion detection mode to characterize the chemical components of burdock roots in situ. The results showed that the DHAP matrix was the most effective for detection, enabling visual characterization of sugars, organic acids, and flavonoids in the root. Furthermore, significant differences in the distribution of different compounds within burdock root tissue were observed. This study, the first to visualize the spatial distribution of different compounds within burdock roots, provides new technical support for research into burdock quality and comprehensive utilization, burdock metabolism, and bioremediation matrix studies.

[0058] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for analyzing the spatial distribution of functional components in burdock roots, characterized in that: The following steps are involved: (1) Sample preparation: Burdock root was embedded in an embedding agent, sliced ​​after freezing, and the burdock root tissue slices were obtained, which were then vacuum dried; (2) Matrix spraying: spraying the vacuum-dried burdock root tissue slices with the matrix to obtain burdock root tissue slices sprayed with the matrix; (3) The burdock root tissue sections sprayed with the matrix were subjected to MALDI mass spectrometry imaging analysis to obtain the spatial distribution of functional components in the burdock root; The matrix used for matrix spraying in step (2) is DHAP; The embedding medium is an OCT embedding medium; The freezing treatment temperature in step (1) is -20±2°C and the time is 40±10 min; The functional components include sugars, organic acids and flavonoids.

2. The method according to claim 1, characterized in that The spraying density in step (2) is 0.0025±0.0005mg / mm 2 ; The number of spraying cycles is 18±4 times.

3. The method according to claim 1, characterized in that The nozzle temperature for matrix spraying was 60±2°C.

4. The method according to claim 1, wherein The mass-to-charge ratio detected by MALDI mass spectrometry imaging analysis is 100-1500.

5. The method according to claim 1, characterized in that The sample scanning resolution during MALDI mass spectrometry imaging analysis is 50 to 200 μm.

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