Mass spectrum imaging method for visually analyzing terpenoid and coumarin components in ferula asafetida stems

Abstract

The invention provides a mass spectrum imaging method for visually analyzing terpenoid and coumarin components in ferula asafetida stems, and belongs to the technical field of molecular imaging. The method comprises the following steps: embedding a to-be-detected sample of a ferula ferulaeoides stem, freezing and slicing the to-be-detected sample with the thickness of 30 microns, performing matrix-assisted laser desorption ionization mass spectrometry imaging on the obtained slice in a positive ion mode, and obtaining the spatial distribution information of terpenoids and coumarins in the to-be-detected tissue sample in situ according to the accurate mass-to-charge ratio of ions in an imaging picture. The slice under specific conditions has a relatively complete tissue structure, the established mass spectrum imaging method does not need complex sample pretreatment, basically does not change the spatial distribution information of active components in a sample to be detected, and can accurately position the spatial distribution information of a plurality of compounds at the same time; therefore, the visualization of the internal active ingredients of the ferula ferulaeoides stems is realized. The invention provides a new technical support for researching the anabolic pathway and physiological function of terpenoid and coumarin compounds in ferula plants in the future, and has important significance in improving the separation and extraction rate of active ingredients in ferula and the utilization rate of ferula stem tissues.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular imaging, and in particular relates to a mass spectrometry imaging method for in-situ identification of terpenoid and coumarin components in ferula stems. Background Art

[0002] Ferula multiflora Ferula feruloides (Steud.) Korovin is a perennial once-fruiting herbaceous plant of the Apiaceae family and the genus Ferula. Its medicinal parts are oleoresin and roots. Ferula multicolumbula is widely distributed in the arid desert area on the edge of the Junggar Basin in my country. It has the effects of strengthening the stomach, eliminating accumulation, dispelling cold and relieving pain. It is an important medicine for the treatment of gastric ulcers and gastritis. Ferula multicolumbula contains rich characteristic components such as volatile oils, sesquiterpenes and coumarins. Among them, terpenoids, such as D-limonene and guaiacol, are one of the active ingredients of Ferula multicolumbula, and have pharmacological effects such as anti-tumor and insecticide. Coumarin, as another major active ingredient of Ferula multicolumbula, has pharmacological effects such as anti-neuroinflammatory and antibacterial. In recent years, research on Ferula multicolumbula has mainly focused on the characterization of multiple chemical components, the determination of multiple component contents and pharmacological activities. However, the spatial distribution information of active ingredients such as terpenes and coumarins in Ferula multicolumbula is still unclear.

[0003] Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is a new analytical technique for studying the types and distribution of molecules or ions at the tissue cell or subcellular level. It can achieve in-situ qualitative and quantitative analysis of multiple substances on the sample surface without complex pre-treatment. The sample molecules to be tested are desorbed and ionized with the assistance of the matrix to obtain a set of mass spectra associated with the spatial position of the sample. The target mass-to-charge ratio ( m / z ) tissue distribution images.

[0004] The secondary metabolite pathways in medicinal plants are complex, multi-layered, and have obvious spatial characteristics. The structure and physiological activity of metabolites are closely related to their location in plant organs or tissues. Therefore, using MALDI-MSI to study the distribution of various types of bioactive components (such as terpenes and coumarins) in the stems of Ferula is of great significance for the physiological functions, growth and development metabolism, and extraction and separation of active ingredients of Ferula multicolum. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a mass spectrometry imaging method for visually analyzing terpenoids and coumarin components in the stems of Ferula asafoetida. Based on the combined technology of cryosectioning and MALDI-MSI, the method can clearly perform in situ analysis of terpenoids and coumarin compounds in the stems of Ferula asafoetida, and can be used to observe the distribution patterns of different compounds in the stems of Ferula asafoetida.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a mass spectrometry imaging method for visually analyzing terpenoids and coumarin components in Ferula foetida stems, wherein the mass spectrometry imaging method specifically comprises the following steps: S1, embedding the sample to be tested of the stem of Ferula multicolum with an embedding agent and then performing cryosectioning; S2, placing the slice obtained in S1 under an optical microscope for observation to obtain an optical image of the tissue slice; S3, using an automatic matrix sprayer to deposit the matrix DHB ionized by laser desorption assisted deposition on the slice obtained in S1 to obtain a tissue slice coated with the matrix; S4, using matrix-assisted laser desorption ionization mass spectrometry imaging to collect and analyze data on the tissue slices obtained in S3, and obtain the spatial distribution of terpenes and coumarin components therein. Terpenes include D-limonene ( m / z 137.1323) and guaiacol ( m / z 245.1890); coumarin ingredients include 5,7-dihydroxycoumarin ( m / z 179.0337), umbelliferone ( m / z 200.9944) and Angelica dahurica alcohol ( m / z 343.0579).

[0007] The slices of the present invention under specific conditions have a relatively complete tissue structure, and the MALDI-MSI technology is used without complex sample pretreatment, and the spatial distribution information of the active components in the sample to be tested is basically not changed, and the spatial distribution information of multiple compounds can be accurately located at the same time, so as to obtain the visualization of the active components inside the Ferula stem, and provide new ideas, methods and data support for the physiological functions, growth and development metabolism, and active component extraction and separation of the Ferula multi-umbrella plant.

[0008] In view of the plant characteristics of the stem of Ferula multicolumbella, the present invention selects embedding first and then freezing and slicing, which can obtain more complete tissue slices with clearer contours, so as to obtain an ideal in situ imaging effect.

[0009] In combination with the first aspect, the stem of Ferula multiflora in S1 is freshly harvested stem of Ferula multiflora.

[0010] In combination with the first aspect, the embedding agent used in S1 is 5% CMC (carboxymethyl cellulose) embedding agent, which has a better embedding effect on the tissue and can obtain a higher quality sectioning effect.

[0011] In combination with the first aspect, the chamber temperature and specimen head temperature of the microtome in S1 were set to −20 °C.

[0012] Preferably, the thickness of the tissue slice in S1 is 30 μm to ensure that a relatively complete tissue slice is obtained, and the slice surface does not form ripples and can be flatly attached to the slide, thereby maximizing the efficiency of extracting the target compound from the tissue slice.

[0013] In combination with the first aspect, the optical image in S2 is acquired at an acquisition magnification of 10 times.

[0014] Preferably, an automatic matrix sprayer is used in S3 to deposit the DHB matrix, with 70% methanol aqueous solution as the solvent, the DHB matrix solution concentration is 30 mg / mL, the matrix flow rate is 20 μL / min, the nitrogen flow rate is 5 L / min, the nozzle movement speed is 8 mm / s, and the spraying time is 50 min / glass slide.

[0015] Combined with the first aspect, the ablation spot diameter caused by the minimum laser beam focusing in S4 is 5 μm, and the laser step length is set at 15 μm. Set the mass spectrometry detection parameters: scanning range, m / z =70–1050 Da; spray voltage is +4 kV; capillary temperature is 250 °C; acquisition speed is 1.5 piex / s; mass resolution is 70,000, m / z is 200 Da, and the mass spectrometer is operated in positive ion mode. The mass accuracy provided in the entire measurement is better than 2 ppm RMS error.

[0016] The invention provides application of the method in in-situ visual analysis of terpenoid and coumarin components and distribution in ferula stems.

[0017] The beneficial effects of the present invention are as follows: the present invention utilizes matrix-assisted laser desorption ionization mass spectrometry imaging technology, optimizes the sample pretreatment method, the matrix and the parameters of the automatic matrix sprayer, and simultaneously determines a suitable mode for observing the distribution of active components of the genus Ferula, i.e., MALDI-MSI in the positive ion mode, and obtains the spatial distribution information of terpenes and coumarins in the stem of Ferula multi-umbrella in situ. The slices of the present invention under specific conditions have a relatively complete tissue structure, and the mass spectrometry imaging method established does not require complex sample pretreatment, and basically does not change the spatial distribution information of the active components in the sample to be tested, and can accurately locate the spatial distribution information of multiple compounds at the same time, thereby obtaining the visualization of the active components inside the stem of Ferula multi-umbrella. The present invention provides new technical support for the future study of the synthetic metabolic pathways and physiological functions of terpenes and coumarins in Ferula plants, and is of great significance in improving the separation and extraction rate of active components in Ferula and the utilization rate of Ferula stem tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0019] Figure 1This is a microstructural analysis of the stem tissue of Ferula multicolum.

[0020] Figure 2 These are optical images of Ferula multiflora stem tissue sections at different slice thicknesses.

[0021] Figure 3 The mass spectra of the reference solution in DHB matrix-assisted positive and negative ion modes.

[0022] Figure 4 These are matrix-assisted laser desorption ionization mass spectrometry images and optical images of terpenoids and coumarins in tissue sections of Ferula multiflora stems. Specific implementation methods The following is a detailed description of a mass spectrometry imaging method for visually analyzing terpenes and coumarins in Ferula stems provided by the present invention in conjunction with the accompanying drawings and examples.

[0024] Example 1 Optimization of frozen sectioning process of Ferula multicolum stem

[0025] Frozen sections of Ferula multiumbellata stems: Wash and dry fresh Ferula multiumbellata stems, cut tissue segments with a thickness of about 2 cm and place them in embedding boxes, embed them with 5% CMC solution, wait until the embedding agent is completely solidified, take out the embedding box, drip 5% CMC embedding agent on the sample holder, quickly stick the embedding block to the sample holder and freeze it for 5 min, fix the sample to the blade, adjust the blade travel to 10–100 μm, set the box temperature and sample head temperature of the freezing microtome to −20 ℃, set the section thickness to 20, 25, and 30 μm, and attach tissue sections of different thicknesses to clean slides.

[0026] Optical tissue slice images were obtained using an optical microscope for tissue slices of different thicknesses. The structural integrity was observed through the slice optical images ( Figure 1 ), thereby determining the slicing conditions. Figure 2 It can be seen that according to the wrinkles and cracks in the optical images of each group of slices, the condition when the slice thickness is 30 μm is better.

[0027] Example 2 Determination of the Mass Spectrometry Imaging Matrix of Ferula multiflora

[0028] Using 70% methanol aqueous solution as solvent, DHB, CHCA and 9AA were prepared into matrix solutions: CHCA and 9AA concentrations were 10 mg / mL, and DHB concentration was 30 mg / mL; 7-hydroxycoumarin reference substance was prepared into a 1 mg / mL reference substance mixed solution, and D-limonene was used as the stock solution. Take 10 μL of the mixed reference solution (standard stock solution), 10 μL of the matrix solution, and 10 μL of the ultrapure solution, and take 3 μL of the mixed solution and drop it on a glass slide, dry it at room temperature for 24 h, and then analyze it with MALDI in positive and negative ion modes respectively.

[0029] The ion peak of d-limonene was not detected in CHCA and 9AA matrices. In addition, in positive ion mode, both compounds were detected using DHB matrix solution, and the relative peak intensity of the compound ion peak was higher than that in negative ion mode ( Figure 3 Therefore, the MALDI mass spectrometry imaging of the active components in the stems of Ferula multiflora was performed in the positive ion mode using DHB matrix.

[0030] Example 3 A method for in situ visualization analysis of terpenoids and coumarins and their spatial distribution in the stems of Ferula multiflora using MALDI-MSI technology, comprising the following steps:

[0031] (1) Cryosectioning of Ferula multiumbellata stems: Wash and dry fresh Ferula multiumbellata stems, cut tissue segments with a thickness of about 2 cm and place them in an embedding box. Embed them with 5% CMC solution. After the embedding agent is completely solidified, take out the embedding box, drip 5% CMC embedding agent on the sample holder, quickly stick the embedding block on the sample holder and freeze-fix for 5 min. Fix the sample to the blade head, adjust the blade head travel to 10–100 μm, set the chamber temperature and sample head temperature of the cryostat to −20 °C, set the tissue section thickness to 30 μm, and attach it to a clean slide.

[0032] (2) Preparation of matrix solution: Use 70% methanol aqueous solution as solvent to prepare DHB matrix solution with a concentration of 30 mg / mL.

[0033] (3) Matrix spraying: Use the German TransMIT SMALDIPrep Matrix Sprayer automatic matrix spraying device to spray the tissue sections placed on the sample stage with matrix. Before use, open the pressure reducing valve to control the pressure between 0.2–0.3 MPa, and check whether there is any leakage on the gas line interface and the inside of the metal plate; before the first spraying of the matrix, clean it (the spraying pipeline and the front end pipeline of the injection needle are cleaned twice in total), and the cleaning parameters are as follows: the cleaning liquid is methanol-water solution (methanol: water = 7:3), the cleaning liquid flow rate is 30 μL / min, the nitrogen flow rate is 5 L / min, and the cleaning time is 5 min / time; after cleaning, set the spraying parameters: the matrix flow rate is 20 μL / min, the nitrogen flow rate is 5 L / min, the nozzle movement speed is 8 mm / s, and the spraying time is 50 min / slide.

[0034] (4) Perform mass spectrometry imaging analysis in MALDI-MSI positive ion mode: Use the German TransMITAP-SMALDI10 atmospheric pressure scanning matrix-assisted laser desorption ionization ion source equipped with the Thermo Scientific QExactiveTM mass spectrometer to perform mass spectrometry imaging on the sample to be tested. First, remove the tissue section from the sample stage, then click Calibrate stage for calibration, and place the sample after calibration; control the movement and focus of the objective lens: first move the Z-axis focus to see the sample clearly, then move the X and Y axes to circle the sample area to be tested, and finally fine-tune the Z axis to minimize the laser spot and maximize the signal of mass spectrometry detection, that is, the ablation spot diameter caused by the minimum laser beam focus is 5 μm, and the laser step length is set to 15 μm; set the mass spectrometry detection parameters: scanning range, m / z =70–1050 Da; spray voltage is +4 kV; capillary temperature is 250 °C; acquisition speed is 1.5 piex / s; mass resolution is 70,000, m / z The mass spectrometer was operated in positive ion mode and the mass accuracy was better than 2 ppm RMS error throughout the measurement.

[0035] (5) Accurate mass-to-charge ratio of ions in the image m / z The specific material structure is obtained by comparing the value information with the relevant standard database. Using Mirion software, the compound is subjected to mass spectrometry imaging data extraction to obtain its ion intensity and high-resolution dimensional mass spectrometry imaging diagram. The compound spatial distribution results can be displayed in different colors, and the color depth represents the different amounts of the analyte. Figure 4 As shown in the figure, they are D-limonene in the stem of Ferula multiflora ( m / z 137.1323), guaiacol ( m / z 245.1890), 5,7-dihydroxycoumarin ( m / z 179.0337), umbelliferone ( m / z 200.9944) and Angelica dahurica alcohol ( m / z 343.0579) of MALDI imaging. Among them, D-limonene ( m / z 137.1323) is mainly distributed in the epidermis, cortex and resin ducts; guaiacol ( m / z 245.1890) has sporadic deposition in the epidermis and cortex; 5,7-dihydroxycoumarin ( m / z 179.0337) are mainly distributed in the resin ducts; umbelliferone ( m / z 200.9944) are scattered in the epidermis and vascular bundles; Angelica dahurica alcohol ( m / z343.0579) are mainly distributed in the resin canals and parenchyma. In summary, the distribution of these terpenes and coumarins in the stems of Ferula multicolum has some similarities, mainly distributed in the epidermis and resin canals. In addition, D-limonene ( m / z 137.1323), 5,7-dihydroxycoumarin ( m / z 179.0337) and Angelica dahurica alcohol ( m / z 343.0579) with higher signal intensity in the stems of Ferula multicolum.

Claims

1. A mass spectrometry imaging method for visual analysis of terpenoids and coumarins in Ferula stems, characterized in that: The mass spectrometry imaging method comprises the following steps: S1, embedding the sample to be tested of the stem of Ferula multicolum with an embedding agent and then performing cryosectioning; S2, placing the slice obtained in S1 under an optical microscope for observation to obtain an optical image of the tissue slice; S3, using an automatic matrix sprayer to deposit the matrix DHB ionized by laser desorption assisted deposition on the slice obtained in S1 to obtain a tissue slice coated with the matrix; S4. The slices obtained in S3 are used for data collection and analysis using matrix-assisted laser desorption ionization mass spectrometry imaging to obtain the spatial distribution of terpenoids and coumarin components therein.

2. The mass spectrometry imaging method for visually analyzing terpenoids and coumarins in Ferula stems according to claim 1, characterized in that: The asafoetida stems are freshly collected asafoetida stems.

3. The mass spectrometry imaging method for visually analyzing terpenes and coumarins in stems of Ferula foetida according to claim 1, characterized in that: The embedding medium used in S1 was 5% CMC embedding medium.

4. The mass spectrometry imaging method for visually analyzing terpenes and coumarins in stems of Ferula foetida according to claim 1, characterized in that: The chamber temperature and specimen head temperature of the microtome in S1 were set to −20 °C.

5. The mass spectrometry imaging method for visually analyzing terpenes and coumarins in Ferula stems according to claim 1, characterized in that: The thickness of tissue sections in S1 was 30 μm.

6. The mass spectrometry imaging method for visually analyzing terpenes and coumarins in Ferula stems according to claim 1, characterized in that: The optical image in S2 was acquired at a magnification of 10 times.

7. The mass spectrometry imaging method for visually analyzing terpenes and coumarins in Ferula stems according to claim 1, characterized in that: In S3, an automatic matrix sprayer was used to deposit the DHB matrix, with a matrix concentration of 30 mg / mL, a matrix flow rate of 20 μL / min, a nitrogen flow rate of 5 L / min, a nozzle movement speed of 8 mm / s, and a spraying time of 50 min / glass slide.

8. The mass spectrometry imaging method for visually analyzing terpenes and coumarins in Ferula stems according to claim 1, characterized in that: In S4, the laser step size was set at 15 μm, and the mass spectrometer was operated in positive ion mode.

9. The application of the mass spectrometry imaging method for visually analyzing terpenes and coumarins in stems of Ferula asafoetida according to any one of claims 1 to 8, comprising any one of the following applications: (1) Application in determining the spatial distribution of terpenoid components in the stems of Ferula; (2) Its application in determining the spatial distribution of coumarin components in the stems of Ferula.

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