Visual detection method for chemical components in radix ophiopogonis

Abstract

The invention belongs to the technical field of analysis and detection, and provides a visual detection method for chemical components in radix ophiopogonis. The method comprises the following steps: embedding a radix ophiopogonis root sample to be detected, and slicing to obtain a sliced sample; carrying out matrix spraying on the slice sample to obtain a sprayed slice; carrying out matrix-assisted laser desorption ionization mass spectrometry imaging on the sprayed slice to obtain a visual result; the matrix sprayed by the matrix is a 2, 5-dihydroxy benzoic acid solution, and the concentration of the 2, 5-dihydroxy benzoic acid is 20-40 mg / mL; the 2, 5-dihydroxybenzoic acid solution also contains trifluoroacetic acid, and the volume concentration of the trifluoroacetic acid is 0.05-0.15%; the solvent is an acetonitrile aqueous solution (the volume ratio of acetonitrile to water is (6-8): (4-2)). According to the method, clear in-situ analysis is carried out on the chemical components in the radix ophiopogonis by utilizing matrix-assisted laser desorption ionization mass spectrometry imaging, so that the spatial distribution and content difference of the components in the radix ophiopogonis are obtained, and the result is more visual due to spatial presentation.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine analysis and detection, and in particular to a visual detection method for chemical components in Ophiopogon japonicus. Background Art

[0002] Ophiopogon japonicus is the dried root of Ophiopogon japonicus, a plant of the genus Ophiopogon in the Liliaceae family. It was first recorded in Shennong's Herbal Classic and listed as a top-grade product. Its main effects are nourishing yin, promoting body fluid, moistening the lungs and clearing the heart. Ophiopogon japonicus is widely used in clinical diseases such as myocardial ischemia, thrombosis, diabetes, and neurodegeneration. There are more than 130 prescriptions containing Ophiopogon japonicus in the pharmacopoeia, accounting for about 17% of Chinese medicine preparations for the treatment of cardiovascular and cerebrovascular diseases, such as Shenmai Injection and Shensong Yangxin Capsules. Ophiopogon japonicus is a commonly used bulk Chinese medicinal material with a long history of use. Its traditional main production areas are Sichuan and Zhejiang provinces, and are called Sichuan Ophiopogon japonicus and Zhejiang Ophiopogon japonicus, respectively.

[0003] At present, the main method for detecting Ophiopogon japonicus is liquid chromatography-mass spectrometry, which is: soaking Ophiopogon japonicus and detecting the obtained extract by liquid chromatography-mass spectrometry. However, this detection method can only obtain the content of related substances in Ophiopogon japonicus, and the distribution of related substances is unknown. That is, there is an urgent need for an accurate and visual detection method. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a visual detection method for the chemical components in Ophiopogon japonicus. The detection method provided by the present invention can realize spatial presentation of the material components in Ophiopogon japonicus, and the result is more intuitive.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for visualizing the chemical components in Ophiopogon japonicus, comprising the following steps: After embedding the sample to be tested of the root of Ophiopogon japonicus, slicing is performed to obtain a slice sample; Performing matrix spraying on the sliced ​​sample to obtain sprayed slices; Performing matrix-assisted laser desorption ionization mass spectrometry imaging on the sprayed slices to obtain a visualized result; The concentration of 2,5-dihydroxybenzoic acid in the 2,5-dihydroxybenzoic acid solution is 20-40 mg / mL; the 2,5-dihydroxybenzoic acid solution also contains trifluoroacetic acid, and the volume concentration of trifluoroacetic acid is 0.05-0.15%; the solvent of the 2,5-dihydroxybenzoic acid solution is an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 6-8:4-2.

[0006] Preferably, in the 2,5-dihydroxybenzoic acid solution, the volume ratio of acetonitrile to water is 7:3, and the volume concentration of trifluoroacetic acid is 0.1%.

[0007] Preferably, the parameters of the matrix spraying include: the flow rate of the sprayer at 60° C. is 0.04-0.06 mL / min, the track speed is 800-1000 mm / min, the distance of the nozzle movement is 0.5-1.5 mm, and the number of spraying cycles is 7-8 times.

[0008] Preferably, the method further comprises: washing and freezing the root sample of Ophiopogon japonicus before embedding, wherein the freezing temperature is -90 to -70°C.

[0009] Preferably, the embedding agent is an aqueous solution containing hydroxypropyl methylcellulose and polyvinyl pyrrolidone, wherein the mass concentration of hydroxypropyl methylcellulose is 7-8%, and the mass concentration of polyvinyl pyrrolidone is 2-3%.

[0010] Preferably, the slices are fixed in an axial direction, and the temperature of the slices is -30 to -10°C.

[0011] Preferably, after the slicing, the method further comprises: transferring the obtained thin slices to the surface of a glass sheet and drying them to obtain slice samples.

[0012] Preferably, the thickness of the sliced ​​sample is 40-60 μm.

[0013] Preferably, the parameters of the matrix-assisted laser desorption ionization mass spectrometry imaging include: positive ion mode scanning, the scanning range is 150~1250m / z; the spatial resolution is 40~60μm; the spray voltage is 3000~5000V; the ion source temperature is 200~300℃; and the laser attenuation is 27~29°.

[0014] Preferably, after the matrix-assisted laser desorption ionization mass spectrometry imaging, the obtained mass spectrum data is converted into a Udp file and then analyzed by Mirion data software to obtain a visualization result.

[0015] The invention provides a visual detection method for chemical components in Ophiopogon japonicus.

[0016] The present invention uses matrix-assisted laser desorption ionization mass spectrometry imaging to detect Ophiopogon japonicus, which can realize spatial presentation of the material components in Ophiopogon japonicus, and the results are more intuitive. In particular, for Ophiopogon japonicus with a long growth period, the visualization detection method of the present invention can intuitively obtain the changes in the position and content of related substances with different years. At the same time, the selection of the matrix can accurately and efficiently realize the detection of the chemical components in Ophiopogon japonicus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1~Figure 5 This is a visualization analysis result diagram of different substances in Ophiopogon japonicus of different growth years; Figure 6 This is the chromatogram of components of Ophiopogon japonicus in different growth years. DETAILED DESCRIPTION

[0018] The present invention provides a method for visualizing the chemical components in Ophiopogon japonicus, comprising the following steps: After embedding the sample to be tested of the root of Ophiopogon japonicus, slicing is performed to obtain a slice sample; Performing matrix spraying on the sliced ​​sample to obtain sprayed slices; Performing matrix-assisted laser desorption ionization mass spectrometry imaging on the sprayed slices to obtain a visualized result; The concentration of 2,5-dihydroxybenzoic acid in the 2,5-dihydroxybenzoic acid solution is 20-40 mg / mL; the 2,5-dihydroxybenzoic acid solution also contains trifluoroacetic acid, and the volume concentration of trifluoroacetic acid is 0.05-0.15%; the solvent of the 2,5-dihydroxybenzoic acid solution is an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 6-8:4-2.

[0019] Unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0020] The invention embeds the sample to be tested in the root of Ophiopogon japonicus and then slices it to obtain the slice sample.

[0021] In the present invention, the root sample of Ophiopogon japonicus is preferably washed and frozen before embedding. In the present invention, the washing reagent is preferably water. In the present invention, the freezing temperature is preferably -90 to -70°C, and more preferably -80°C.

[0022] In the present invention, the embedding agent is preferably an aqueous solution containing hydroxypropyl methylcellulose and polyvinyl pyrrolidone, in which the mass concentration of hydroxypropyl methylcellulose is preferably 7-8%, more preferably 7.5%; the mass concentration of polyvinyl pyrrolidone is preferably 2-3%, more preferably 2.5%. In the present invention, the embedding method preferably includes: placing the Ophiopogon japonicus root sample to be tested in the embedding agent.

[0023] In the present invention, the slice is preferably fixed in the axial direction. In the present invention, the temperature of the slice is preferably -30 to -10°C, more preferably -20°C.

[0024] After the slicing, the present invention preferably further comprises: transferring the obtained thin slices to the surface of a glass sheet and drying them to obtain slice samples.

[0025] In the present invention, the drying is preferably carried out in a drying oven. The present invention does not impose any specific limitation on the drying time, as long as the water vapor on the glass sheet can be completely evaporated.

[0026] In the present invention, the thickness of the sliced ​​sample is preferably 40-60 μm, and specifically preferably 40 μm, 50 μm or 60 μm.

[0027] After obtaining the sliced ​​sample, the present invention performs matrix spraying on the sliced ​​sample to obtain sprayed slices.

[0028] In the present invention, the substrate of the substrate spraying is 2,5-dihydroxybenzoic acid solution. In the present invention, the concentration of 2,5-dihydroxybenzoic acid in the 2,5-dihydroxybenzoic acid solution is 20~40mg / mL, preferably 30mg / mL, specifically 20mg / mL, 25mg / mL, 30mg / mL, 35mg / mL or 40mg / mL. In the present invention, the 2,5-dihydroxybenzoic acid solution also contains trifluoroacetic acid, and the volume concentration of trifluoroacetic acid is 0.05~0.15%, preferably 0.1%, specifically 0.05%, 0.1% or 0.15%. In the present invention, the solvent of the 2,5-dihydroxybenzoic acid solution is acetonitrile aqueous solution, and the volume ratio of acetonitrile and water in the acetonitrile aqueous solution is 6~8:4~2, preferably 7:3, specifically 6:4, 7:3 or 8:2.

[0029] In the present invention, the parameters of the substrate spraying include: the flow rate of the sprayer at 60°C is preferably 0.04~0.06mL / min, specifically 0.04mL / min, 0.05mL / min or 0.06mL / min; the track speed is preferably 800~1000mm / min, specifically 800mm / min, 900mm / min or 1000mm / min; the distance moved by the nozzle is preferably 0.5~1.5mm, specifically 0.5mm, 1mm or 1.5mm; the number of spraying cycles is preferably 7~9 times, specifically 7 times, 8 times or 9 times.

[0030] After the sprayed slices are obtained, the present invention performs matrix-assisted laser desorption ionization mass spectrometry imaging on the sprayed slices to obtain visualized results.

[0031] In the present invention, the parameters of the matrix-assisted laser desorption ionization mass spectrometry imaging include: positive ion mode scanning, the scanning range is preferably 150~1250m / z; the spatial resolution is preferably 40~60μm, specifically 40μm, 45μm, 50μm, 55μm or 60μm; the spray voltage is preferably 3000~5000V, specifically 3000V, 3500V, 4000V, 4500V or 5000V; the ion source temperature is preferably 200~300℃, specifically 200℃, 220℃, 250℃, 270℃ or 300℃; the laser attenuation is preferably 27~29°, specifically 27°, 28° or 29°.

[0032] After the matrix-assisted laser desorption ionization mass spectrometry imaging, the present invention preferably further comprises converting the obtained mass spectrum data into a Udp file and obtaining a visualization result through Mirion data analysis software.

[0033] The visual detection method for chemical components in Ophiopogon japonicus provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] The experimental materials involved in the following examples are shown in Table 1, and the experimental instruments are shown in Table 2.

[0035] Table 1 Experimental materials

[0036] Table 2 Experimental instruments

[0037] Example 1 The tubers were collected from fresh Ophiopogon japonicus of one-year-old, two-year-old and three-year-old in Deqing, Zhejiang Province, with three plants collected from each growth year. The collected Ophiopogon japonicus was briefly washed and quickly frozen at -80°C. Then, the sample was fixed axially on the sample holder of the freezing microtome using an embedding medium (7.5% hydroxypropyl methylcellulose (Hypromellose, HMPC) + 2.5% polyvinylpyrrolidone (Polyvinylpyrrolidone, PVP)) and cut into 50μm thick tissue sections at -20°C. The cut slices were quickly transferred to the surface of the glass slide and melted and adhered at the same time.

[0038] After taking out the slices of Ophiopogon japonicus, open them and place them in a dryer to dry for 15 minutes. After the water vapor on the glass slide is completely dried, place the glass slide in the matrix sprayer and select a matrix containing 30 mg / mL of DHB (2,5-dihydroxybenzoic acid) solution with a volume concentration of 0.1% trifluoroacetic acid (acetonitrile: water = 7 / 3, v / v) for matrix spraying. The matrix spraying parameters are set as follows: the flow rate of the sprayer at 60°C is set to 0.05 mL / min, the track speed is set to 900 mm / min, the nozzle movement distance is 1 mm, and the number of spraying cycles is 8 times.

[0039] After taking out the slices of Ophiopogon japonicus sprayed with the matrix, the scanning parameters were set through the mass spectrometry imaging platform control program software: positive ion mode scanning, scanning range of 150~1250m / z; spatial resolution of 50μm; spray voltage of 4000V; ion source temperature of 250℃; laser attenuation of 28°.

[0040] After the original mass spectrometry data is converted into a Udp file, the data is visualized using Mirion data analysis software. The results are as follows: Figure 1~Figure 5 shown.

[0041] from Figure 1~Figure 5It can be seen that the 25 ingredients are 5,7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chromone-4-one (5,7-dihydroxy-6-methyl-3-hydroxybenzyl flavonoids), 5-hydroxy-3-(3-hydroxy-4-methoxybenzyl)-7-methoxy-6-methyl-4H-chromen-4-one (5-hydroxy-3-(3-hydroxy-4-methoxybenzyl)-7-methoxy-6-methyl-tetrahydroflavonoids), Np-trans-Coumaroyltyramine (N-trans-p-coumaryltyramine), N-trans-Feruloyltyramine (N-trans-feruloyltyramine), Ophiopogonanone B (Ophiopogon japonicus flavonoids B), Borneol 7-O-[beta-D-apiofuranosyl-(1->6)]-beta-D-glucopyranoside-2 (Borneoside), Ophiopogonone A (Ophiopogon japonicus flavonoids A), Methylophiopogonanone B (Methylophiopogon japonicus dihydroisoflavonoids B), Ophiop acidamide (Ophiopogone), 6-aldehydo-isoophipogonone B (6-aldehyde iso-Ophiopogon japonicus flavonoids B), Methylophiopogonanone A (Methylophiopogon japonicus dihydroisoflavonoids A), 5,7,4'-trihydroxy,3'-methoxyl-6,8-dimethyl-homisoflavanone (5,7,4'-trihydroxy-3'-methoxy-6,8-dimethylhomisoflavanone), 6-aldehydo-isoophipogonone A (6-aldehyde isophiopogon japonicus flavonoids A), 5-Hydroxy-7-methoxy-3',4'-methoxy-6,8-dimethylhomoisoflavanone (5-hydroxy-7-methoxy-3',4'-methyleneoxy-6,8-dimethylhomoisoflavanone), 6-Formyl-isoophiopogonanone A (6-aldehyde isophiopogon japonicus flavonoids A), Ophiopogonanone C (Ophiopogon japonicus flavonoids C), 2-HydroxymethylMethylophiopogonanone A (2-hydroxymethylmethylophiopogon dihydroisoflavonoids A), 5,7,2'-Trihydroxy-8,4'-dimethoxy-6-methylhomoisoflavone (5,7,2'-trihydroxy-8,4'-dimethoxy-6-methyl homoisoflavone), Ophiopogonanone E (Ophiopogon japonicus flavonoids E), Ophiopogonanone D (Ophiopogon japonicus flavonoids D), Stigmasterol (Stigmasterol), Ruscogenin (Ruscogenin), Borneol 7-O-[beta-D-apiofuranosyl-(1->6)]-beta-D-glucopyranoside (Borneoside), Ophiofurospiside B (Ophiopogonin B), OphiopogoninRa (Ophiopogonin Ra) in Ophiopogon japonicus have significant changes in distribution and content with the change of growth time.

[0042] Verification Example The samples of Ophiopogon japonicus with different growth years in Example 1 were analyzed by LC-MS. The chromatograms of the components of Ophiopogon japonicus with different growth years are as follows: Figure 6 The information of compounds detected in the chromatogram is shown in Table 3.

[0043] Table 3 Information on compounds in extracts of Ophiopogon japonicus from different growth years

[0044] The variation trend of the components obtained by the above LC-MS results is the same as the trend of the component content 1 obtained in Example 1, indicating that the results of the detection method provided by the present invention are credible.

[0045] Although traditional LC-MS analysis can also screen out the different components of Ophiopogon japonicus in different growth years, compared with mass spectrometry imaging analysis, it lacks spatial presentation and is not intuitive enough.

[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for visualizing the chemical components in Radix Ophiopogonis, comprising the following steps: After embedding the root sample of Ophiopogon japonicus to be tested, slicing is performed to obtain a slice sample; Performing matrix spraying on the sliced ​​sample to obtain sprayed slices; Performing matrix-assisted laser desorption ionization mass spectrometry imaging on the sprayed slices to obtain a visual result; The matrix of the matrix spraying is a 2,5-dihydroxybenzoic acid solution, the concentration of 2,5-dihydroxybenzoic acid in the 2,5-dihydroxybenzoic acid solution is 20-40 mg / mL; the 2,5-dihydroxybenzoic acid solution also contains trifluoroacetic acid, and the volume concentration of trifluoroacetic acid is 0.05-0.15%; the solvent of the 2,5-dihydroxybenzoic acid solution is an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 6-8:4-2.

2. The visual detection method according to claim 1, characterized in that: In the 2,5-dihydroxybenzoic acid solution, the volume ratio of acetonitrile to water is 7:3, and the volume concentration of trifluoroacetic acid is 0.1%.

3. The visual detection method according to claim 1, characterized in that: The parameters of the matrix spraying include: the flow rate of the sprayer at 60° C. is 0.04-0.06 mL / min, the track speed is 800-1000 mm / min, the distance of the nozzle movement is 0.5-1.5 mm, and the number of spraying cycles is 7-8 times.

4. The visual detection method according to claim 1, characterized in that: The method also includes: washing and freezing before embedding the Ophiopogon japonicus root sample to be tested, wherein the freezing temperature is -90 to -70°C.

5. The visual detection method according to claim 1, characterized in that: The embedding agent is an aqueous solution containing hydroxypropyl methylcellulose and polyvinyl pyrrolidone, wherein the mass concentration of hydroxypropyl methylcellulose is 7-8%, and the mass concentration of polyvinyl pyrrolidone is 2-3%.

6. The visual detection method according to claim 1, characterized in that: The slices are fixed in an axial direction, and the temperature of the slices is -30 to -10°C.

7. The visual detection method according to claim 1, characterized in that: After the slicing, the method further comprises: transferring the obtained thin slices to the surface of a glass sheet and drying them to obtain slice samples.

8. The visual detection method according to claim 1, characterized in that: The thickness of the sliced ​​sample is 40-60 μm.

9. The visual detection method according to claim 1, characterized in that: The parameters of the matrix-assisted laser desorption ionization mass spectrometry imaging include: positive ion mode scanning, a scanning range of 150-1250 m / z; a spatial resolution of 40-60 μm; a spray voltage of 3000-5000 V; an ion source temperature of 200-300° C.; and a laser attenuation of 27-29°.

10. The visual detection method according to claim 1, characterized in that: After the matrix-assisted laser desorption ionization mass spectrometry imaging, the obtained mass spectrum data is converted into a Udp file and then analyzed by Mirion data analysis software to obtain a visualization result.

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