Method for detecting spatial distribution of active ingredients in garlic

Abstract

The present application relates to the technical field of substance detection, in particular to a method for detecting spatial distribution of active ingredients in garlic. The method for detecting spatial distribution of active ingredients in garlic comprises the following steps: freezing and slicing the embedded garlic to form garlic slices, then precipitating a matrix on the surface of the garlic slices by sublimation, and finally analyzing by MALDI-MSI. The method can visually detect sulfur-containing compounds, free amino acids, phenolic compounds and other active ingredients in garlic, which is conducive to researchers to prepare single active ingredients with higher purity according to the detection results, and is also conducive to further research and utilization of garlic.

Description

Technical Field

[0001] This invention relates to the field of substance detection technology, and more specifically, to a method for detecting the spatial distribution of active ingredients in garlic. Background Technology

[0002] Garlic (Allium sativum L.) has a unique pungent flavor and is known worldwide as a spice that enhances the taste and flavor of food. Garlic contains a wealth of active ingredients, including sulfur compounds, free amino acids, and phenolic compounds, which can exert important physiological functions such as lowering high blood pressure, lowering cholesterol, preventing cancer, and providing antioxidant effects. Understanding the tissue distribution of these active ingredients in garlic can reveal their metabolic pathways and mechanisms. Therefore, exploring the spatial distribution variations of active ingredients in garlic not only helps to elucidate their metabolic pathways and physiological functions but also provides valuable guidance for the processing of functional foods.

[0003] Methods for detecting active ingredients in garlic include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). However, these techniques involve cumbersome sample preparation and complex data processing. For example, LC-MS and GC-MS require sample extraction, purification, and enrichment, and derivatization may be necessary during detection. Most importantly, these methods cannot display the spatial distribution of multiple target compounds in tissue samples in real time and in situ; the spatial distribution information of these active ingredients in garlic remains unclear. Therefore, an effective method for detecting the spatial distribution of active ingredients in garlic is urgently needed.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting the spatial distribution of active ingredients in garlic. The detection method provided in this invention can accurately analyze the spatial distribution of active ingredients in garlic, which is beneficial for analyzing the synthetic metabolism and physiological functions of active ingredients based on the detected spatial distribution results, and scientifically guiding garlic breeding and processing.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a method for detecting the spatial distribution of active ingredients in garlic, comprising: freezing and slicing embedded garlic to form garlic slices, then precipitating a matrix onto the surface of the garlic slices by sublimation, and then analyzing it using MALDI-MSI.

[0008] In an optional embodiment, the freezing conditions include: a freezing temperature of -15°C to -25°C, preferably -20°C; and a freezing time of 20-25 minutes, preferably 20 minutes.

[0009] In an optional embodiment, the garlic slices are 25-35 micrometers thick, preferably 30 micrometers.

[0010] In an optional embodiment, the active ingredient is selected from at least one of sulfur-containing compounds, free amino acids, and phenolic compounds.

[0011] In an optional embodiment, the embedding medium is any one of gelatin, water, paraffin and carboxymethyl cellulose, preferably gelatin.

[0012] In an optional embodiment, the matrix is ​​selected from any one of α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), CHCA containing 0.1% trifluoroacetic acid (TFA), and DHB containing 0.1% TFA, preferably DHB matrix.

[0013] In an optional embodiment, the sublimation precipitation step includes: sublimating the matrix and then spraying it onto the surface of the garlic slices.

[0014] In an optional implementation, the m / z range is 100-1000 in positive / negative ionization mode during MALDI-MSI analysis.

[0015] In an optional implementation, the conditions for MALDI-MSI analysis include a voltage of 3.2-3.7 kV, preferably 3.5 kV.

[0016] In an optional implementation, the conditions for MALDI-MSI analysis include a repetition frequency of 950-1050 Hz, preferably 1000 Hz.

[0017] The present invention has the following beneficial effects: The detection method provided by the embodiments of the present invention can visualize the detection of various active ingredients in garlic, such as sulfur-containing compounds, free amino acids and phenolic compounds. This is beneficial for researchers to analyze the synthesis, metabolism and physiological functions of active ingredients based on the detection results, and to scientifically guide garlic breeding and processing. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a cross-sectional view of garlic provided in an embodiment of the present invention;

[0020] Figure 2 Mass spectrometry image of sulfur-containing compounds in garlic bulbs provided in an embodiment of the present invention;

[0021] Figure 3 This is a mass spectrometry image of free amino acids in garlic bulbs provided in an embodiment of the present invention;

[0022] Figure 4 This is a mass spectrometry image of phenolic compounds in garlic bulbs provided in an embodiment of the present invention;

[0023] Figure 5 Optical microscope images and spatial mass spectrometry images of slices frozen at different times provided in embodiments of the present invention;

[0024] Figure 6 Optical microscope images and spatial mass spectrometry images of frozen sections of different thicknesses provided in embodiments of the present invention;

[0025] Figure 7 The analysis heatmap shows the ion signals of 64 active ingredient standards in four different matrices. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] Mass spectrometry imaging (MSI) is a powerful label-free analytical technique that uses mass spectrometers to identify the molecular composition of biological samples and display the distribution of compounds in tissues as images, providing a completely new perspective for biological research in recent years. Various mass spectrometry imaging techniques have been developed, among which MALDI-MSI (matrix-assisted laser desorption / ionization mass spectrometry) is the most widely used commercial tissue imaging technique and has been applied as an important research tool in multiple research fields. MALDI-MSI is capable of simultaneously detecting thousands of ions in a single data acquisition, including multiple components such as proteins, polysaccharides, lipids, and polyphenols. Compared with traditional methods for analyzing sample extracts, direct analysis of sample tissues is more advantageous. Therefore, MALDI-MSI can serve as a valuable technique to reveal the distribution of food factors and improve the reliability of component analysis for quality control and food safety.

[0028] Furthermore, embodiments of the present invention provide a method for detecting the spatial distribution of active ingredients in garlic, comprising:

[0029] Fresh garlic is used as the sample to be tested. The garlic is cut into three parts: top, middle, and bottom. Each part is placed in a reagent embedding cassette (e.g., 17×17×5mm). The embedding medium is poured into the cassette until it completely covers the sample. The embedding medium provided in this embodiment can be any one of gelatin, water, paraffin, and carboxymethyl cellulose, such as a 10% aqueous gelatin solution.

[0030] Then, freeze at -15°C to -25°C for 20-25 minutes. Specifically, the freezing temperature includes, but is not limited to, any value or a range between -15°C and -25°C, such as -15°C, -20°C, and -25°C, with -20°C being preferred. The freezing time is any value or a range between 20 and 25 minutes, such as 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, and 25 minutes, with 20 minutes being preferred.

[0031] After freezing, the garlic is sliced ​​to form garlic slices. The thickness of the garlic slices is 25-35 micrometers, for example, any value between 25-35 micrometers or any range between any two values, such as 25 micrometers, 26 micrometers, 27 micrometers, 28 micrometers, 29 micrometers, 30 micrometers, 31 micrometers, 32 micrometers, 33 micrometers, 34 micrometers, and 35 micrometers. For example, 30 micrometers is preferred.

[0032] After slicing, the slices were transferred to indium tin oxide-plated glass slides and frozen slices were kept in a cryostat, held in place until they appeared completely dry for further MALDI matrix deposition. The matrix was then evenly applied to the frozen garlic slices using a Shimadzu vacuum matrix sprayer (IMLayer).

[0033] The matrix is ​​selected from any one of CHCA, DHB, CHCA containing 0.1% TFA and DHB containing 0.1% TFA, preferably DHB.

[0034] Analysis was performed using MALDI-MSI. Specifically, MALDI imaging data of tissue sections were acquired on an iMScopeTrio spectrometer (Shimadzu Corporation, Kyoto, Japan). Internal quality calibration was first performed using the m / z values ​​of the DHB matrix cluster to ensure the reliability and accuracy of the imaging analysis. The laser diameter and laser intensity were set to 2 and 65 (arbitrary units in iMScope), respectively, and ions in the m / z range of 100-1000 were measured in positive / negative ion mode. Other parameter settings were as follows: sample voltage 3.2-3.7 kV, e.g., 3.50 kV; repetition frequency 950-1050 Hz, e.g., 1000 Hz; and 200 laser irradiations per pixel under optimized parameters.

[0035] All collected data were analyzed using Imaging MS Solution software (Shimadzu, Kyoto, Japan). The same software was used for visualization and relative quantitative analysis.

[0036] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0037] Example 1

[0038] This embodiment provides a method for detecting the spatial distribution of active ingredients in garlic, including:

[0039] Fresh garlic (see diagram of fresh garlic cross-section) Figure 1 As the sample to be tested, fresh garlic is cut into three parts: top, middle and bottom. Each part is placed in a reagent embedding box (e.g., 17×17×5mm). A 10% gelatin aqueous solution is poured into the mold until the embedding medium completely covers the sample.

[0040] The sections were then frozen at -20°C for 20 minutes, and 30 μm thick sections were prepared using a cryostat and transferred to indium tin oxide-coated glass slides. The frozen sections were kept in a cryostat and held in place until they appeared completely dry for further MALDI matrix deposition. The DHB matrix was uniformly sublimated onto the frozen garlic sections using a Shimadzu IMLayer vacuum matrix sprayer.

[0041] DHB solution: Weigh 70 mg of DHB and dissolve it in a mixture of 7 mL of acetonitrile and 3 mL of purified water. Mix well and store in the dark.

[0042] Gelatin solution: Weigh 1g of gelatin and dissolve it in 10mL of purified water. Dissolve the gelatin in a water bath at 45℃.

[0043] MALDI imaging data of tissue sections were acquired using an iMScope Trio spectrometer (Shimadzu Corporation, Kyoto, Japan). Internal quality calibration was first performed using the m / z values ​​of the DHB matrix cluster to ensure the reliability and accuracy of the imaging analysis. The laser diameter and intensity were set to 2 and 65 (arbitrary units in iMScope), respectively, and ions in the m / z range of 100-1000 were measured in positive / negative ion mode. Other parameter settings were as follows: sample voltage 3.50 kV, repetition rate 1000 Hz, and 200 laser irradiations per pixel under optimized parameters.

[0044] See results Figures 2-4 ,in, Figure 2 Optical microscope images and spatial mass spectrometry images of sulfur-containing compounds; Figure 3 Optical microscope image and spatial mass spectrometry image of free amino acids; Figure 4 These are optical microscope images and spatial mass spectrometry images of phenolic compounds.

[0045] Results analysis (1):

[0046] according to Figure 2 It was found that six sulfur-containing compounds were detected in the garlic sample, namely the ions [M+H]. + At m / z 162.06, S-allyl-L-cysteine / S-(trans-1-propenyl)-L-cysteine ​​(SAC / SPC) ion [M+H] + Allicin and [M+H] ions at m / z 178.05 + γ-L-glutamyl-S-methyl-L-cysteine ​​(GSMC) and the ion [M+Na] at m / z 265.08 + The γ-L-glutamyl-S-allyl-L-cysteine / γ-L-glutamyl-S-(trans-1-propenyl)-L-cysteine ​​(GSAC / GSPC) was detected at m / z 312.14. SAC / SPC was distributed in the sprouting leaves and true leaves of garlic, with a small, uneven distribution in the storage leaves. The ion signal intensity decreased from the top to the bottom of the garlic plant. Alliin had a stronger ion signal intensity than the other four sulfur-containing compounds. Its distribution in garlic was similar to SAC / SPC, mainly in the sprouting leaves and true leaves, with a small, uneven distribution in the storage leaves. The ion signal intensity decreased from the top to the bottom. Unlike the other sulfur-containing compounds, GSMC and GSAC / GSPC were mainly distributed in the storage leaves of garlic, and the ion signal intensity increased from the top to the bottom, which was opposite to the vertical distribution of the other detected sulfur-containing compounds.

[0047] Results analysis (2):

[0048] Amino acids are essential substances for maintaining human life, playing vital physiological roles and possessing unique flavor characteristics. Amino acids are amphoteric compounds, and due to their low ionization efficiency, they are difficult to detect by MALDI-MSI. However, the detection method provided in this invention can detect five free amino acids, namely the [M+H] ions. + At m / z 104.10, γ-aminobutyric acid (GABA) ions [M+H] are present. + Glutamine and [M+H] ions at m / z 147.08 + Lysine and [M+H] ions at m / z 147.11 + Cysteine ​​and the [M+H] ion at m / z 160.11 + Arginine at m / z 175.12.

[0049] GABA possesses antihypertensive and anti-anxiety effects and participates in the biosynthesis of glutamate family amino acids. GABA is evenly distributed in garlic, and among the detected free amino acids, its ion signal intensity is second only to arginine. Glutamine and lysine are mainly distributed in storage leaves, with slightly weaker ion signals in sprouting leaves and true leaves. Cysteine ​​showed a low ion signal intensity in garlic, mainly distributed in storage leaves, and was not detected in the epidermis, sprouting leaves, or true leaves. Arginine is the most abundant free amino acid in garlic, accounting for more than half of the total amino acids; therefore, its ion signal intensity is higher than the other four free amino acids, and it is mainly distributed in the storage leaves of garlic.

[0050] The intensity of free amino acids increases sequentially from the top to the bottom of the garlic plant. Therefore, it is speculated that the synthesis of free amino acids in garlic is concentrated in the bottom part of the plant. Further research is needed to investigate the synthesis pathway of free amino acids.

[0051] During garlic growth, cysteine ​​and glutamate first react to form GSMC, GSAC, and GSPC. Subsequently, under the action of γ-glutamyl transferase, SMC, SAC, and SPC are generated. Then, through the action of oxidase, the sulfhydryl groups are oxidized to sulfoxide groups, producing methylalliin and alliin. Figure 2 and Figure 3 It can be seen that cysteine, GSMC, GSAC, and GSPC are mainly distributed in storage leaves, while SAC, SPC, methyl allicin, and allicin are mainly distributed in sprouting leaves and true leaves. Therefore, it is reasonable to infer that the above reactions proceed from storage leaves to sprouting leaves and true leaves.

[0052] Results analysis (3):

[0053] Phenolic compounds are mostly in the form of [M+Na] + [M+K]+ It is presented in the form of Na, because phenolic compounds contain substances that readily react with Na. + or K + The binding sites include oxygen on the carbonyl group and oxygen on the ether bond. Studies have shown a strong positive correlation between potassium ion content and total phenol content, suggesting that potassium ions may facilitate the transport of phenolic compounds.

[0054] Four phenolic compounds were detected in garlic, namely the ions [M+H]. + Resveratrol and [M+K] ions at m / z 229.94 + Citric acid and its ions [M+K] at m / z 230.95 + Citric acid and its ion [M+H] at m / z 311.00 + Hyperoside at m / z 465.02.

[0055] Resveratrol, citric acid, naringenin, and hyperoside exhibit relatively similar distributions in garlic, being evenly distributed across the epidermis, storage leaves, sprouting leaves, and true leaves. Naringenin shows a stronger ionic signal intensity than the other three phenolic compounds detected in garlic. This invention provides the first report on the detailed tissue-specific distribution and ionic signal intensity of the phenolic compounds resveratrol, citric acid, naringenin, and hyperoside in garlic, offering new insights into a more comprehensive understanding of the physiological activities of garlic.

[0056] Matrix optimization

[0057] 0.5 μL of each of the 64 active ingredient standard solutions was dropped onto a stainless steel plate, and four matrices were applied to each target compound standard solution before detection.

[0058] The detection conditions are as follows: laser diameter and laser intensity are set to 2 and 65 respectively (any unit in iMScope), and ions in the m / z range of 100-1000 are measured in positive / negative ion mode. The voltage is 1.75 kV, the repetition frequency is 1000 Hz, and each pixel is irradiated 200 times under optimized parameters.

[0059] The standard solutions of 64 active ingredients are prepared as follows:

[0060] (1) If the target compound is a liquid

[0061] Liquid standards are available in 100 μg / mL or 1000 μg / mL specifications. Accurately pipette a certain amount of standard solution, dilute it with acetonitrile or methanol to prepare a standard working solution of appropriate concentration (1 μg / mL), and store it in a brown glass bottle at -18℃ or below, protected from light.

[0062] (2) If the target compound is a solid

[0063] Accurately weigh an appropriate amount of the standard substance (accurate to 0.1 mg), dilute to 10 mL with acetonitrile or methanol, prepare a standard stock solution of a certain concentration, and store it below -18℃ protected from light.

[0064] The four matrices are as follows: CHCA, CHCA containing 0.1% TFA, DHB, and DHB containing 0.1% TFA.

[0065] See results Figure 7 .

[0066] according to Figure 7 As shown in Table 1, when using DHB containing 0.1% TFA as the matrix, the active ingredient with the lowest ionic intensity detectable in positive ion mass spectrometry is a sulfur-containing compound. Therefore, three matrices were initially selected: CHCA, CHCA containing 0.1% TFA, and DHB, for detection in positive ion mass spectrometry.

[0067] Table 1. List of Standard Compounds for 64 Active Ingredients

[0068]

[0069]

[0070]

[0071] Subsequently, matrix optimization was further performed using actual garlic samples.

[0072] Table 2. Detectable compounds in each matrix in actual garlic samples.

[0073]

[0074] The results are shown in Table 2. When using DHB as the matrix, 15 active substances were detected, making it the matrix with the highest number of detected compounds among the three matrices. In MALDI-MSI analysis, DHB was more suitable for detecting garlic samples than the other three matrices. Therefore, DHB was selected as the matrix used in this experiment, and detection was performed in positive ionization mode of mass spectrometry.

[0075] Optimization of freezing time

[0076] Garlic slices were frozen for 10–40 minutes. The freezing time of garlic slices was optimized by selecting the mass spectrometry responses of three representative active ingredients: arginine, allicin, and naringenin. Optical microscope images and spatial mass spectrometry images are shown below. Figure 5 As shown.

[0077] according to Figure 5It was found that a freezing time of 10 min was too short and affected the ion signal intensity, with allicin exhibiting a weaker ion signal intensity compared to other freezing times. From a freezing time of 20 min onwards, there was no significant difference in the ion signal intensity of the active ingredients, and the optical image showed good adhesion between the gelatin embedding medium and the sample. However, when the freezing time exceeded 30 min, the extended freezing time did not further enhance the signal intensity of the active ingredients in the MALDI-MSI analysis; instead, it led to freeze crystallization, affecting the structural morphology of garlic. Therefore, 20 min was selected as the optimal freezing time for garlic samples.

[0078] Slice thickness optimization

[0079] The structure of garlic makes it difficult to obtain thin, intact slices without affecting its morphology. Therefore, slice thickness is another important factor to consider when slicing garlic. Garlic slices with a thickness of 10–50 μm were prepared, and the mass spectrometry response of alliin was used to optimize the slice thickness. Optical microscope images and spatial mass spectrometry images are shown below. Figure 6 As shown.

[0080] according to Figure 6 It was found that thinner tissue sections (10, 20 μm) tend to shrink, leaving more voids on the tissue surface and disrupting the integrity of the garlic sample shape, resulting in lower ionic signal intensity of the active ingredient. When the garlic slice thickness reaches 30 μm, the thicker tissue section can better maintain the integrity of the garlic slice and obtain a stronger ionic signal intensity. However, when the slice thickness exceeds 40 μm, the excessively thick tissue section leads to incomplete ionization, isolating the target ion and failing to further enhance the signal intensity of the active ingredient in MALDI-MSI analysis. Therefore, 30 μm was chosen as the slice thickness for subsequent experiments.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting spatial distribution of active ingredients in garlic, characterized by, Comprising: frozen sectioning of the embedded garlic to form a garlic section, and then precipitating the matrix onto the surface of the garlic section by sublimation, and then analysing using MALDI-MSI; the freezing conditions include a freezing temperature of -20°C and a freezing time of 20 minutes; the active ingredient is selected from the group consisting of S-(trans-1-propenyl)-L-cysteine, alliin, γ-L-glutamyl-S-(trans-1-propenyl)-L-cysteine, γ-aminobutyric acid, glutamine, cysteine, resveratrol, citric acid, naringenin and hyperoside.

2. The method for detecting the spatial distribution of active ingredients in garlic according to claim 1, characterized by, the thickness of the garlic section is 25-35 microns.

3. The method for detecting the spatial distribution of active ingredients in garlic according to claim 2, characterized by, the thickness of the garlic section is 30 microns.

4. The method for detecting spatial distribution of active ingredients in garlic according to claim 1, characterized in that, the embedding medium used for embedding is any one of gelatin, water, paraffin and carboxymethyl cellulose.

5. The method for detecting spatial distribution of active ingredients in garlic according to claim 1, characterized in that, the matrix is selected from the group consisting of α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid containing 0.1% trifluoroacetic acid and 2,5-dihydroxybenzoic acid containing 0.1% trifluoroacetic acid.

6. The method for detecting spatial distribution of active ingredients in garlic according to claim 1, characterized in that, the step of precipitating by sublimation includes sublimating the matrix and then spraying it onto the surface of the garlic section.

7. The method for detecting spatial distribution of active ingredients in garlic according to claim 1, characterized in that, MALDI-MSI analysis in positive / negative ionization mode m / z ranging from 100 to 1000.

8. The method for detecting spatial distribution of active ingredients in garlic according to claim 1, characterized in that, the conditions during analysis using MALDI-MSI include a voltage of 3.2-3.7 kilovolts.

9. The method for detecting the spatial distribution of active ingredients in garlic according to claim 8, characterized by, the conditions during analysis using MALDI-MSI include a voltage of 3.5 kilovolts.

10. The method for detecting the spatial distribution of active ingredients in garlic according to claim 1, characterized by, the conditions during analysis using MALDI-MSI include a repetition frequency of 950-1050 Hz.

11. The method for detecting the spatial distribution of active ingredients in garlic according to claim 10, characterized by, the conditions during analysis using MALDI-MSI include a repetition frequency of 1000 Hz.

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