Glehnia root slice sample preparation method and spatial distribution positioning method

Abstract

The invention provides a radix glehniae slice sample preparation method and a spatial distribution positioning method, and belongs to the technical field of traditional Chinese medicine analysis. In the preparation method of the slice sample, an adhesive tape transfer method is adopted to carry out slice pretreatment on the radix glehniae, so that the sample slice is complete and free of shrinkage, tissue breakage is prevented, meanwhile, the influence of an adhesive tape adhesive layer on a sample detection signal is eliminated, the sample breakage and shrinkage phenomena caused by direct slicing are avoided, and the distribution of active ingredients of traditional Chinese medicines is basically not influenced. According to the positioning method, an AFADESI-MSI technology is adopted to perform scanning analysis on tissue slices, through an optimized sample pretreatment method, a spray solvent and mass spectrum parameters, components in the radix glehniae can have a good imaging effect, spatial distribution information of chemical components in the radix glehniae can be accurately positioned, and the accuracy of the positioning of the radix glehniae is improved. The method is used for observing spatial distribution modes of different compounds in radix glehniae, and is beneficial to understanding ecological and physiological effects of different chemical components in plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine analysis, and relates to a method for preparing a Glehnia littoralis slice sample and a method for spatial distribution positioning. Background Art

[0002] Glehnia littoralis is the dried root of the Umbelliferae plant Glehnia littoralis Fr. Schmidt ex Miq., which has a rich and diverse chemical composition, mainly including polysaccharides, coumarins, polyacetylenes, lignans, phenolic acids and other substances. Glehnia littoralis In recent years, the research field of Glehnia littoralis has mainly focused on the characterization analysis of chemical components, the accurate determination of multi-component contents, and the exploration of differential markers. There is still an obvious lack in the research on the spatial distribution of Glehnia littoralis chemical components within the tissue. The methods for determining the active components of Glehnia littoralis mostly rely on LC-MS technology for analysis, and this analysis requires a complex sample preparation process. When imaging and analyzing Glehnia littoralis according to the traditional slicing method, due to the lack of targeted consideration and effective protection measures for the root tissue structure characteristics of plants, the Glehnia littoralis is extremely prone to fragmentation and shrinkage during the operation due to mechanical stress, damaging the sample integrity, interfering with the imaging effect, and reducing the imaging quality. To a certain extent, this hinders the construction of a comprehensive and systematic cognitive system for Glehnia littoralis.

[0003] In recent years, the research on Glehnia littoralis has mainly focused on the characterization analysis of chemical components, the accurate determination of multi-component contents, and the exploration of differential markers. There is still an obvious lack in the research on the spatial distribution of Glehnia littoralis chemical components within the tissue. The methods for determining the active components of Glehnia littoralis mostly rely on LC-MS technology for analysis, and this analysis requires a complex sample preparation process. When imaging and analyzing Glehnia littoralis according to the traditional slicing method, due to the lack of targeted consideration and effective protection measures for the root tissue structure characteristics of plants, the Glehnia littoralis is extremely prone to fragmentation and shrinkage during the operation due to mechanical stress, damaging the sample integrity, interfering with the imaging effect, and reducing the imaging quality. To a certain extent, this hinders the construction of a comprehensive and systematic cognitive system for Glehnia littoralis. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing a Glehnia littoralis slice sample and a method for spatial distribution positioning to solve the problem that the existing slice sample preparation method cannot ensure the sample integrity.

[0005] The fresh medicinal material of Glehnia littoralis is tough, and the plant tissue becomes brittle after freezing. When preparing tissue sections, shrinkage, partial tissue peeling, or fragmentation are likely to occur, all of which will affect the accurate characterization and analysis of the component spatial distribution.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions: The present application provides a method for preparing a Glehnia littoralis slice sample, and the method includes: S01: After embedding the Glehnia littoralis sample with an embedding agent, attach a transparent tape to the cut surface of the Glehnia littoralis sample, and press thermally for 15 - 20 s until there are no bubbles at the joint; slice uniformly to form Glehnia littoralis slices.

[0007] Pick, wash the fresh Glehnia littoralis sample and store it at -80 °C, and place it at -20 °C for overnight thawing one day before slicing. Before slicing, turn on the cryostat for pre-cooling so that the temperature of the cryostat chassis is -25 ~ -30 °C and the temperature of the sample head is -18 °C.

[0008] After the cryostat is pre-cooled, place the Glehnia littoralis sample in the cryostat cabinet for 30 - 40 minutes to balance the temperature of the Glehnia littoralis sample in the cryostat, so as to make the Glehnia littoralis sample reach an appropriate hardness, prevent liquefaction or shrinkage during sectioning, and ensure that the sections are flat and adhere to the Fisher Superfrost Plus positively charged anti - detachment glass slides. At the same time, place the Fisher Superfrost Plus positively charged anti - detachment glass slides in the cryostat for pre - cooling for 20 minutes.

[0009] Select a position with uniform thickness at the front end of the Glehnia littoralis sample, cut a Glehnia littoralis tissue segment about 2 cm in length, add embedding medium on the base tray and spread it flat as the bottom layer. Before the embedding medium completely hardens, place the tissue segment on the embedding medium and gently press to make the sample stand upright on the base tray. After the sample position is fixed, use the embedding medium to wrap the sample. The thickness of the embedding medium is about 2 - 3 mm to form a Glehnia littoralis sample.

[0010] In this application, sectioning the Glehnia littoralis tissue segment after embedding not only simplifies the operation process but also reduces the corner cutting tension, making the sections more complete and undamaged. To reduce the impact of the embedding medium on the instrument and sample detection, gelatin with a concentration of 10 - 20% is selected as the embedding medium in this application.

[0011] Cut a transparent tape that is 2 - 3 mm wider than the cross - section of the Glehnia littoralis sample and 2 - 4 cm long. Attach the transparent tape to the cut surface of the Glehnia littoralis sample from bottom to top and press it thermally for 15 - 20 s to ensure that there are no air bubbles at the joint between the transparent tape and the Glehnia littoralis sample. After setting the section thickness of the cryostat, slice at a constant speed to obtain Glehnia littoralis sections.

[0012] During sectioning, the Glehnia littoralis sample and anti - detachment glass slides are all placed in the cryostat throughout the process to ensure that they are always in a constant low - temperature environment during sectioning. The method of thermal pressing can effectively block the conduction of hand temperature to the Glehnia littoralis sample, thereby preventing the components of the Glehnia littoralis sample from dissolving or shifting and ensuring the accuracy and reliability of experimental data.

[0013] The thickness and dryness of the Glehnia littoralis sections have a significant impact on the integrity of the tissue structure, the retention amount of the analyte, and the overall ion response intensity in positive and negative ion modes. If the sections are too thin, it is easy to damage the pores of Glehnia littoralis, making the tissue structure incomplete, resulting in less retained analyte content and being unfavorable for detection. If the sections are too thick, it will inhibit the ionization of the Glehnia littoralis sample, resulting in reduced sensitivity. In this application, the section thickness is 10 - 15 μm, and 15 μm is preferably selected as the section thickness for Glehnia littoralis imaging.

[0014] S02: Stick the sliced Glehnia littoralis on a pre-cooled glass slide, and heat the back of the pre-cooled glass slide with the temperature of the hand until the sliced Glehnia littoralis and the embedding agent become translucent.

[0015] Based on the observation during the slicing process, when the sliced Glehnia littoralis cut off still adheres to the surface of the transparent tape, it needs to be transferred to a glass slide for subsequent operations and analysis. Therefore, the sliced Glehnia littoralis needs to be placed on a pre-cooled FisherSuperfrost Plus positively charged adhesive-free glass slide, and the sliced Glehnia littoralis is swept and pressed thermally insulated from bottom to top to make it completely adhere to the glass slide. Then, heat the back of the pre-cooled glass slide for 5 - 10 s with the temperature difference between the hand temperature and the pre-cooled glass slide, which can effectively promote the transfer of the sliced Glehnia littoralis from the transparent tape to the glass slide. Further optimization reveals that when the sliced Glehnia littoralis and the embedding agent are in a translucent state, the transfer efficiency is the highest, and the sample structure damage can be minimized.

[0016] S03: After quick-freezing for 2 - 3 min, tear off the transparent tape to obtain a sliced Glehnia littoralis sample.

[0017] If the transparent tape is peeled off immediately after the sliced Glehnia littoralis is transferred from the transparent tape to the glass slide, it will cause damage to the structural integrity of the sample, and at the same time, the adhesive of the transparent tape will remain on the surface of the sliced Glehnia littoralis. Based on this, in this application, the sliced Glehnia littoralis and the glass slide are quickly frozen for 2 - 3 min to make the sliced Glehnia littoralis present as a white solid state. At this time, the operation of peeling off the transparent tape can be carried out to improve the complete transfer rate of the sliced Glehnia littoralis, so that the tissue morphological structure of the sliced Glehnia littoralis remains intact, and a wrinkle-free and complete sliced Glehnia littoralis sample is obtained.

[0018] On the other hand, this application also provides a method for spatially distributing and positioning components in a Glehnia littoralis sample, and this method includes: S01: Place the sliced Glehnia littoralis sample prepared by the above-mentioned sliced Glehnia littoralis sample preparation method in a pre-cooled dryer for 1 - 2 h, slowly return to room temperature, and then vacuum dry for 10 min.

[0019] Since the sliced Glehnia littoralis sample will be affected by factors such as temperature, humidity, and oxidation when placed for too long, which will cause physical or chemical changes in the measured substances in the sample, and is not conducive to the accuracy and repeatability of the active ingredient localization detection. If the sliced Glehnia littoralis sample is over-dried, its brittleness will increase significantly, which makes the sliced sample extremely easy to break during the imaging process, and thus it is difficult to obtain a complete mass spectrometry image. If the drying time of the sliced Glehnia littoralis sample is insufficient, its residual water content still remains at a relatively high level, resulting in regional diffusion of the analyte, an increase in the coefficient of variation of the mass spectrometry signal intensity, and a significant reduction in the experimental repeatability.

[0020] Based on this, to ensure the integrity of the Glehnia littoralis slice samples and the consistency of the experiments, the prepared Glehnia littoralis slice samples are stored in a low-temperature manner. Before performing the spatial distribution localization analysis of the components, the Glehnia littoralis slice samples are placed in a pre-cooled desiccator for 1 - 2 h to slowly recover to room temperature. The process of taking and transporting should be rapid to avoid the formation of water mist on the Glehnia littoralis slice samples, which may affect the imaging quality. After recovering to room temperature, the Glehnia littoralis slice samples are dried under vacuum to remove the possible water mist on the samples.

[0021] The length of the vacuum drying time will affect the imaging quality. When the vacuum drying time is relatively long, the brittleness of the Glehnia littoralis slice samples increases significantly, resulting in problems such as an increase in the slice breakage rate, loss of tissue integrity, and interruption of the mass spectrometry signal continuity during the subsequent mass spectrometry imaging process. When the vacuum drying time is relatively short, the residual water content in the Glehnia littoralis slice samples remains at a relatively high level, which will lead to regional diffusion of the analytes, an increase in the coefficient of variation of the mass spectrometry signal intensity, and a decrease in the experimental repeatability. Therefore, through experimental verification in this application, when the vacuum drying time is 10 min, the above situations can be avoided, ensuring the accuracy and repeatability of the analysis.

[0022] S02: Place the dried Glehnia littoralis slice samples on the imaging platform, and use AFADESI - MSI to collect and analyze data to obtain the spatial distribution information of different active components in the Glehnia littoralis in the Glehnia littoralis slice samples.

[0023] Place the dried Glehnia littoralis slice samples on the imaging Like a platform , the mass spectrometry system is a Thermo Scientific Q Exactive Plus mass spectrometer, and the imaging system is an aerodynamic assisted desorption electrospray ionization mass spectrometry imaging (AFADESI - MSI) with high - resolution mass spectrometry. Data collection and analysis are performed on the Glehnia littoralis slice samples to obtain the spatial distribution information of different active components in the Glehnia littoralis in the Glehnia littoralis slice samples. Among them, the data processing software includes MassImager, MarkerView, Matlab, and SIMCA. The mass spectrometry imaging conditions are as follows: the spray solvent for detecting components is 85% acetonitrile, and the flow rate is set at 5 μl / min; the spatial resolution is 100 μm, the scanning speed Vx is 0.2 mm / s, and Dy is 0.1 mm; in the negative ion mode, the capillary voltage is - 4500 V, the spray pressure is 0.61 MPa, the capillary temperature is 100 °C, the spray voltage is 3.5 kV, the sheath gas flow rate is 30, and the auxiliary gas flow rate is 6; in the positive ion mode, the capillary voltage is 4500 V, the spray pressure is 0.61 MPa, the capillary temperature is 320 °C, the spray voltage is 3.8 kV, the sheath gas flow rate is 5, and the auxiliary gas flow rate is 1.

[0024] In this application, the AFADESI-MSI technology uses aerodynamic-assisted desorption electrospray ionization. During the electrospray process, high-speed airflow is used to transport charged droplets to the surface of the Glehnia littoralis Fr. Schmidt ex Miq. slice sample, ionizing the analytes in the Glehnia littoralis Fr. Schmidt ex Miq. slice sample. The ionized analytes enter the mass spectrometer under the action of an electric field, are separated and detected according to their mass-to-charge ratios, and then the detected ion signals are converted into images through data processing software, so as to accurately locate the spatial distribution information of different components in Glehnia littoralis Fr. Schmidt ex Miq.. The AFADESI-MSI technology has the characteristics of high sensitivity, wide dynamic range, large imaging space and operation flexibility. It can directly ionize the sample tissue without complex sample pretreatment, and basically does not change the spatial distribution information of the components in the sample to be measured, which is conducive to studying the distribution of various active components in Glehnia littoralis Fr. Schmidt ex Miq., helps to explore the physiological and ecological functions of the active components of Glehnia littoralis Fr. Schmidt ex Miq., and at the same time helps to extract target components from specific parts and improve the utilization rate of Glehnia littoralis Fr. Schmidt ex Miq..

[0025] After the imaging analysis by AFADESI-MSI, most of the active components in Glehnia littoralis Fr. Schmidt ex Miq. exist in the forms of M-H, M+H, M+K, and M+Na, and the spatial distribution information of different active components in Glehnia littoralis Fr. Schmidt ex Miq. is as follows: psoralen and kaempferol are distributed in the phloem, cambium and epidermis of Glehnia littoralis Fr. Schmidt ex Miq.; umbelliferone is distributed in the epidermis and phloem of Glehnia littoralis Fr. Schmidt ex Miq.; bergapten, falcarindiol, phellopterin / isophellopterin, imperatorin / isoimperatorin, caffeic acid, and ferulic acid are distributed in the epidermis of Glehnia littoralis Fr. Schmidt ex Miq.; chlorogenic acid, mannose / glucose, and galacturonic acid are distributed in the epidermis and xylem of Glehnia littoralis Fr. Schmidt ex Miq.; vanillic acid is distributed in the xylem of Glehnia littoralis Fr. Schmidt ex Miq.; isoimpinellin is distributed in the phloem of Glehnia littoralis Fr. Schmidt ex Miq..

[0026] The present invention has the following beneficial effects: (1) In this application, the tape transfer method is used to simply and quickly pretreat the Glehnia littoralis Fr. Schmidt ex Miq. before slicing, ensuring that the sample slices are complete and non-shrunken, preventing tissue fragmentation, and at the same time eliminating the influence of the adhesive layer of the tape on the sample detection signal, avoiding the sample fragmentation and shrinkage phenomenon caused by direct slicing, and basically not affecting the spatial distribution of the active components.

[0027] (2) The AFADESI-MSI technology is used to scan and analyze the tissue slices. Through optimized sample pretreatment methods, spray solvents and mass spectrometry parameters, the components in Glehnia littoralis Fr. Schmidt ex Miq. can have good imaging effects, accurately locate the spatial distribution information of the chemical components in Glehnia littoralis Fr. Schmidt ex Miq., and are used to observe the spatial distribution patterns of different compounds in Glehnia littoralis Fr. Schmidt ex Miq., which helps to understand the ecological and physiological functions of different chemical components in plants.

[0028] (3) Through data import, processing and analysis, and matching and identification in the database, the separation and extraction rate of the active components in Glehnia littoralis Fr. Schmidt ex Miq. is improved, and the utilization rate of Glehnia littoralis Fr. Schmidt ex Miq. is optimized.

[0029] (4) This method is applicable to the preparation of all ginseng slice tissues and can be widely used in multiple fields such as plant science research, industrial production process optimization, and drug R & D. Description of the Drawings

[0030] Figure 1 It is a slice image of the Glehnia littoralis slices prepared in Example 1 of this application; Figure 2 It is the mass spectrometry imaging map of the Glehnia littoralis slices in positive and negative ion modes in Example 3 of this application; Figure 3 It is the mass spectrometry imaging map of the Glehnia littoralis slices prepared by the direct slicing method in positive and negative ion modes; Figure 4 It is the mass spectrometry imaging map of the Glehnia littoralis samples in positive and negative ion modes under OCT embedding agent; Figure 5 It is the mass spectrometry imaging map of the Glehnia littoralis samples in positive and negative ion modes when the slice thickness is 20μm; Figure 6 It is the mass spectrometry imaging map of the Glehnia littoralis samples in positive ion mode under different vacuum drying times; Figure 7 It is the mass spectrometry imaging map of the Glehnia littoralis samples in positive ion mode under different spraying solvents. Detailed Embodiments

[0031] The technical solutions of the present invention will be further explained and illustrated through specific embodiments below.

[0032] Example 1 The example of this application provides a method for preparing Glehnia littoralis slice samples, and this method includes: S101: Pick and wash the fresh Glehnia littoralis samples and store them at -80°C. Place them at -20°C for overnight thawing one day before slicing. Turn on the cryostat for pre-cooling before slicing so that the temperature of the cryostat cabinet is -25°C and the temperature of the sample head is -18°C.

[0033] After the pre-cooling of the cryostat is completed, place the Glehnia littoralis samples in the cryostat cabinet for 40 minutes. At the same time, place the Fisher Superfrost Plus positively charged anti - detachment glass slides in the cryostat for pre-cooling for 20 minutes. Select a position with uniform thickness at the front end of the Glehnia littoralis samples, cut a Glehnia littoralis tissue segment with a length of about 2 cm, add gelatin with a concentration of 10% on the base and spread it flat as the bottom layer. Place the tissue segment on the gelatin before the gelatin completely hardens, gently press it to make the sample stand upright on the base. After the sample position is fixed, wrap the sample with gelatin, and the thickness of the gelatin is about 3 mm to form Glehnia littoralis samples.

[0034] Cut a transparent tape that is 3 mm wider than the cross-section of the Glehnia littoralis sample and 4 cm long. Attach the transparent tape to the cut surface of the Glehnia littoralis sample from bottom to top, and thermally insulate and press for 20 s until there are no air bubbles at the joint between the transparent tape and the Glehnia littoralis sample.

[0035] When the transparent tape is completely attached to the Glehnia littoralis sample, after setting the section thickness of the cryostat to 15 μm, section at a constant speed to obtain Glehnia littoralis sections.

[0036] S102: Attach the Glehnia littoralis sections to a pre-cooled glass slide, and heat the back of the pre-cooled glass slide with body temperature for 10 s until the Glehnia littoralis sections and the gelatin become translucent.

[0037] S103: Rapidly freeze the Glehnia littoralis sections and the glass slide for 3 min until the Glehnia littoralis sections become white and solid. Slowly tear off the transparent tape from the lower end at a constant speed to obtain a non-shrunken and complete Glehnia littoralis section sample. Place the glass slide with the Glehnia littoralis section sample in a pre-cooled glass slide box, cover the outer layer with plastic wrap and put it into a self-sealing bag, and then quickly transfer it to a -80 °C refrigerator for storage.

[0038] Example 2 The embodiment of the present application provides a method for preparing a Glehnia littoralis section sample, and this method includes: S201: Pick and wash a fresh Glehnia littoralis sample and store it at -80 °C. Place it at -20 °C for overnight thawing one day before sectioning. Turn on the cryostat for pre-cooling before sectioning so that the temperature of the cryostat cabinet is -30 °C and the temperature of the sample head is -18 °C.

[0039] After the pre-cooling of the cryostat is completed, place the Glehnia littoralis sample in the cryostat cabinet for 40 min. At the same time, place Fisher Superfrost Plus positively charged anti - detachment glass slides in the cryostat for pre-cooling for 20 min. Select a position with uniform thickness at the front end of the Glehnia littoralis sample, cut a Glehnia littoralis tissue segment with a length of about 2 cm, add gelatin with a concentration of 20% on the base tray, and spread it flat as the bottom layer. Place the tissue segment on the gelatin before the gelatin completely hardens, and gently press to make the sample stand upright on the base tray. After the sample position is fixed, use gelatin to wrap the sample, and the thickness of the gelatin is about 2 mm to form a Glehnia littoralis sample.

[0040] Cut a transparent tape that is 2 mm wider than the cross-section of the Glehnia littoralis sample and 2 cm long. Attach the transparent tape to the cut surface of the Glehnia littoralis sample from bottom to top, and thermally insulate and press for 15 s until there are no air bubbles at the joint between the transparent tape and the Glehnia littoralis sample.

[0041] When the transparent tape is completely attached to the Glehnia littoralis sample, after setting the section thickness of the cryostat to 10 μm, section at a constant speed to obtain Glehnia littoralis sections.

[0042] S202: Place the Glehnia littoralis slices on a pre-cooled glass slide, and heat the back of the pre-cooled glass slide with the temperature of the hand for 5 s until the Glehnia littoralis slices and the gelatin become translucent.

[0043] S203: Quickly freeze the Glehnia littoralis slices and the glass slide for 2 min until the Glehnia littoralis slices become white and solid. Tear off the transparent tape to obtain a non-shrunken and complete Glehnia littoralis slice sample. Place the glass slide with the Glehnia littoralis slice sample in a pre-cooled glass slide box, cover the outer layer with plastic wrap and put it into a self-sealing bag, and then quickly transfer it to a -80 °C refrigerator for storage.

[0044] Comparative Example 1 The comparative example of this application provides a method for preparing a Glehnia littoralis slice sample. This method is the same as that of Example 1, except that after the Glehnia littoralis sample is prepared, it is sliced uniformly by the direct slicing method to obtain Glehnia littoralis slices with a slice thickness of 15 μm. Then, place the Glehnia littoralis slices on the knife table and gently flatten the Glehnia littoralis slices with a fine brush. Invert the front side of a FisherSuperfrost Plus positively charged anti- detachment glass slide at room temperature on the Glehnia littoralis slices. At this time, the Glehnia littoralis slices and the gelatin will be in a translucent state, indicating that the Glehnia littoralis slices have been successfully transferred to the glass slide, and it is necessary to ensure that they are fully attached to each other.

[0045] Comparative Example 2 The comparative example of this application provides a method for preparing a Glehnia littoralis slice sample. This method is the same as that of Example 1, except that the Glehnia littoralis tissue segments are embedded with OCT embedding medium.

[0046] Comparative Example 3 The comparative example of this application provides a method for preparing a Glehnia littoralis slice sample. This method is the same as that of Example 1, except that the thickness of the Glehnia littoralis slices is 20 μm.

[0047] Example 3 The example of this application provides a method for spatially distributing and positioning components in a Glehnia littoralis sample. This method includes: S301: Transfer the Glehnia littoralis slice sample prepared in Example 1 to a -20 °C refrigerator overnight to reach an equilibrium state, and then place it in a pre-cooled desiccator for 2 h to slowly return to room temperature. After returning to room temperature, dry the Glehnia littoralis slice sample under vacuum for 10 min.

[0048] S302: Place the dried sliced Glehnia littoralis samples on the imaging platform. The imaging system is AFADESI-MSI. Conduct data acquisition and analysis on the sliced Glehnia littoralis samples to obtain the spatial distribution information of different active ingredients in the sliced Glehnia littoralis samples within Glehnia littoralis. Among them, the mass spectrometry imaging conditions are as follows: The spray solvent for detecting ingredients is 85% acetonitrile, and the flow rate is set at 5 μl / min; Spatial resolution: 100 μm, scanning speed Vx: 0.2 mm / s, Dy: 0.1 mm; In the negative ion mode, capillary voltage: -4500 V, spray pressure: 0.61 MPa, capillary temperature: 100 °C, spray voltage: 3.5 kV, sheath gas flow rate: 30, auxiliary gas flow rate: 6; In the positive ion mode, capillary voltage: 4500 V, spray pressure: 0.61 MPa, capillary temperature: 320 °C, spray voltage: 3.8 kV, sheath gas flow rate: 5, auxiliary gas flow rate: 1.

[0049] Example 4 The embodiment of the present application provides a method for spatially distributing and positioning components in a Glehnia littoralis sample, and this method includes: S401: Transfer the sliced Glehnia littoralis samples prepared in Example 2 to a refrigerator at -20 °C overnight to reach an equilibrium state, and then place them in a pre-cooled dryer for 1 h to slowly return to room temperature. After returning to room temperature, dry the sliced Glehnia littoralis samples under vacuum for 10 min.

[0050] S402: Place the dried sliced Glehnia littoralis samples on the imaging flat Platform , The imaging system is AFADESI-MSI. Conduct data acquisition and analysis on the sliced Glehnia littoralis samples to obtain the spatial distribution information of different active ingredients in the sliced Glehnia littoralis samples within Glehnia littoralis. Among them, the mass spectrometry imaging conditions are as follows: The spray solvent for detecting ingredients is 85% acetonitrile, and the flow rate is set at 5 μl / min; Spatial resolution: 100 μm, scanning speed Vx: 0.2 mm / s, Dy: 0.1 mm; In the negative ion mode, capillary voltage: -4500 V, spray pressure: 0.61 MPa, capillary temperature: 100 °C, spray voltage: 3.5 kV, sheath gas flow rate: 30, auxiliary gas flow rate: 6; In the positive ion mode, capillary voltage: 4500 V, spray pressure: 0.61 MPa, capillary temperature: 320 °C, spray voltage: 3.8 kV, sheath gas flow rate: 5, auxiliary gas flow rate: 1.

[0051] The raw data in the.raw format was converted to the.cdf format using Xcalibur software, and the converted.cdf format data was imported into the MassImager imaging processing software to obtain the imaging contour map of the Glehnia littoralis tissue section. The peaks of ions in the Glehnia littoralis data were aligned, normalized, and zero-valued preprocessed using MarkerView and Matlab software. Multivariate statistical analysis was performed on the data using SIMCA software. Potential biomarkers were identified and attributed with reference to relevant literature and databases (such as the HMDB database).

[0052] Comparative Example 4 The comparative example of this application provides a method for spatially distributing and positioning the components in a Glehnia littoralis sample. This method is the same as that in Example 3, except that the Glehnia littoralis section sample was dried under vacuum for 15 minutes.

[0053] Comparative Example 5 The comparative example of this application provides a method for spatially distributing and positioning the components in a Glehnia littoralis sample. This method is the same as that in Example 3, except that the Glehnia littoralis section sample was dried under vacuum for 20 minutes.

[0054] Comparative Example 6 The comparative example of this application provides a method for spatially distributing and positioning the components in a Glehnia littoralis sample. This method is the same as that in Example 3, except that the spray solvent in the mass spectrometry imaging conditions was 70% acetonitrile.

[0055] Comparative Example 7 The comparative example of this application provides a method for spatially distributing and positioning the components in a Glehnia littoralis sample. This method is the same as that in Example 3, except that the spray solvent in the mass spectrometry imaging conditions was 85% methanol.

[0056] The Glehnia littoralis section sample prepared in Example 1 was photographed to obtain the attached Figure 1 . From the attached Figure 1 It can be seen that there are a large number of holes in the cross-section of the Glehnia littoralis section sample prepared in Example 1, and the tissue structure is complete, with no glue, no shrinkage, and no breakage on the surface, and the structural properties of Glehnia littoralis are well retained.

[0057] Attached Figure 2 shows the imaging maps of the spatial distribution positions of Glehnia littoralis compounds obtained in the positive and negative ion modes for the Glehnia littoralis sample based on the AFADESI-MSI technology in Example 3 of this application. Among them, most of the compounds in the Glehnia littoralis sample exist in the forms of M-H, M+H, M+K, and M+NA. The specific compound information is shown in Table 1.

[0058] Table 1: Compound information of Glehnia littoralis detected by the AFADESI-MSI mass spectrometry imaging technology From the attachedFigure 2 It can be seen that psoralen m / z 186.0317 ([C 11 H6O3]), kaempferol m / z 286.0477 ([C 15 H 10 O6]) are distributed in the phloem, cambium and epidermis of Glehnia littoralis; umbelliferone m / z 162.0317 ([C9H6O3]) is mainly distributed in the epidermis and phloem, with a higher abundance in the epidermis; bergapten m / z 338.1518 ([C 21 H 22 O4]), falcarindiol m / z260.1776 ([C 17 H 24 O2]), phellopterin / isophellopterin m / z 270.0892 ([C 16 H 14 O4]), imperatorin / isoirnperatorin m / z 270.0892 ([C 16 H 14 O4]), caffeic acid m / z 180.0423 ([C9H8O4]), ferulic acid m / z 194.0579 ([C 10 H 10 O4]) have a higher abundance in the epidermis of Glehnia littoralis; chlorogenic acid m / z 354.0951 ([C 16 H 18 O9]), mannose / glucose m / z180.0634 ([C6H 12 O6]), galacturonic acid m / z 194.0427 ([C6H 10 O7]) have a higher abundance in the epidermis and xylem; vanillic acid m / z 168.0423 ([C8H8O4]) has a higher abundance in the xylem; isoimpinellin m / z 246.0528 ([C 13 H 10 O5]) is mainly distributed in the phloem.

[0059] According to the spatial distribution positioning method in Example 3, the AFADESI-MSI detection was performed on the Glehnia littoralis slice samples prepared in Comparative Examples 1-3 respectively. Taking 14 different compounds in Glehnia littoralis as reference ions, the effects of different slicing methods, different embedding agents, and different slice thicknesses on the component imaging effect were observed, and the attached Figures 3 - 5 .

[0060] From the attached Figure 3It can be seen that the prepared Glehnia littoralis slices by the direct slicing method are extremely prone to shrinkage and fragmentation. During the operation of attaching to the glass slide, it is difficult for such slices to adhere to the surface of the glass slide completely and tightly. The above defects result in differences in the distribution of compounds on the slice surface, and further cause the lack of imaging of some compounds during the imaging process, seriously affecting the integrity and accuracy of the imaging results.

[0061] From the attachment Figure 4 It can be seen that the OCT embedding medium has a significant impact on the imaging response of compounds in Glehnia littoralis, specifically manifested as a decrease in the abundance of compounds, affecting the accuracy of imaging. In addition, the OCT embedding medium also has an adverse effect on the edge area of imaging, resulting in a blurred edge interface and reducing the resolution and clarity of imaging.

[0062] From the attachment Figure 5 It can be seen that when the slice thickness increases, the imaging signal of compounds in Glehnia littoralis changes, resulting in a decrease in imaging resolution, and problems such as blurred images and loss of details appear. At the same time, the uneven distribution of compounds in different layers causes signal overlap, resulting in impaired imaging quality and unable to accurately reflect the true distribution characteristics of Glehnia littoralis compounds.

[0063] Taking the compounds in Glehnia littoralis as reference ions, the attachment Figure 6 shows the mass spectrometry imaging maps of Glehnia littoralis samples under different vacuum drying times in the positive ion mode based on the AFADESI-MSI technology in Comparative Examples 4 and 5 of the present application; the attachment Figure 7 shows the mass spectrometry imaging maps of Glehnia littoralis samples under different spray solvents in the positive ion mode based on the AFADESI-MSI technology in Comparative Examples 6 and 7 of the present application.

[0064] From the attachment Figure 6 It can be seen that as the drying time continues to extend, a large amount of moisture in the Glehnia littoralis samples is lost, resulting in fragmentation of its tissue structure, and the longer the drying time, the more serious the damage degree of the Glehnia littoralis samples. The fragmentation of the Glehnia littoralis samples changes the spatial distribution of compounds, interferes with the normal acquisition of imaging signals, and significantly reduces the imaging quality.

[0065] From the attachment Figure 7 It can be seen that when 70% acetonitrile and 85% methanol sprays rapidly volatilize on the surface of Glehnia littoralis samples, some compounds in Glehnia littoralis are detached from the imaging surface or unevenly distributed, and then the signal attenuation of compounds occurs during the imaging process, resulting in the lack of compound imaging information, and some compounds cannot even be detected by the imaging system, affecting the comprehensive analysis of the components of Glehnia littoralis.

[0066] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A method for preparing a Glehnia littoralis slice sample, characterized in that, Comprising: After embedding the Glehnia littoralis samples with an embedding agent, attach a transparent tape to the cut surface of the Glehnia littoralis samples, and thermally press for 15 - 20 s until there are no air bubbles at the joint; slice at a uniform speed to form Glehnia littoralis slices; Attach the Glehnia littoralis slices to a pre-cooled glass slide, and heat the back of the pre-cooled glass slide using the body temperature of the hand until the Glehnia littoralis slices and the embedding agent become semi-transparent; After quick-freezing for 2 - 3 min, tear off the transparent tape to obtain Glehnia littoralis slice samples.

2. The method for preparing the Glehnia littoralis slice sample according to claim 1, wherein, The fresh Glehnia littoralis samples are washed and stored at -80 °C, and placed at -20 °C for overnight thawing one day before slicing.

3. The method for preparing the Glehnia littoralis slice sample according to claim 1, wherein, The embedding agent is gelatin with a concentration of 10 - 20%.

4. The method for preparing the Glehnia littoralis slice sample according to claim 1, wherein Before slicing, place the Glehnia littoralis samples in the casing of the slicing machine for 30 - 40 min.

5. The method for preparing the Glehnia littoralis slice sample according to claim 1, wherein During slicing, the temperature of the casing of the slicing machine is -25~-30 °C, and the temperature of the sample head is -18 °C.

6. The method for preparing the Glehnia littoralis slice sample according to claim 1, wherein The slice thickness is 10 - 15 μm.

7. A method for spatially distributing and positioning components in a Glehnia littoralis sample, characterized in that, Comprising: Place the Glehnia littoralis slice samples prepared by the method for preparing Glehnia littoralis slice samples according to any one of claims 1 - 6 in a pre-cooled desiccator for 1 - 2 h, slowly return to room temperature, and then vacuum dry for 10 min; Place the dried Glehnia littoralis slice samples on an imaging platform, collect and analyze data using AFADESI-MSI to obtain the spatial distribution information of different active ingredients in the Glehnia littoralis in the Glehnia littoralis slice samples.

8. The method for localizing the spatial distribution of components in the Glehnia littoralis sample according to claim 7, wherein The mass spectrometry imaging conditions are as follows: the spray solvent for detecting components is 85% acetonitrile, and the flow rate is set at 5 μl / min; spatial resolution: 100 μm, scanning speed Vx: 0.2 mm / s, Dy: 0.1 mm; in the negative ion mode, capillary voltage: -4500 V, spray pressure: 0.61 MPa, capillary temperature: 100 °C, spray voltage: 3.5 kV, sheath gas flow rate: 30, auxiliary gas flow rate: 6; in the positive ion mode, capillary voltage: 4500 V, spray pressure: 0.61 MPa, capillary temperature: 320 °C, spray voltage: 3.8 kV, sheath gas flow rate: 5, auxiliary gas flow rate:

1.

9. The method for locating the spatial distribution of components in the Glehnia littoralis sample according to claim 7, characterized in that, The spatial distribution information of the different active ingredients in the Glehnia littoralis is as follows: Psoralen and kaempferol are distributed in the phloem, cambium and epidermis of Glehnia littoralis; Umbelliferone is distributed in the epidermis and phloem of Glehnia littoralis; Bergapten, falcarindiol, phellopterin / isophellopterin, imperatorin / isoimperatorin, caffeic acid, ferulic acid are distributed in the epidermis of Glehnia littoralis; Chlorogenic acid, mannose / glucose, galacturonic acid are distributed in the epidermis and xylem of Glehnia littoralis; Vanillic acid is distributed in the xylem of Glehnia littoralis; Isoimpinellin is distributed in the phloem of Glehnia littoralis.

Images