MALDI-MSI method for visually analyzing distribution of active ingredients of rhizoma smilacis glabrae in tissues in rat body

Abstract

The invention discloses an MALDI-MSI method for visually analyzing distribution of active ingredients of rhizoma smilacis glabrae in tissues in a rat body. The space metabolism distribution characteristics of the rhizoma smilacis glabrae bioactive components in a rat body are analyzed by utilizing an MALDI-MSI mass spectrum imaging technology, 9-aminoacridine is creatively used as a matrix based on the characteristics of the rhizoma smilacis glabrae active components, and the concentration of the 9-aminoacridine matrix and MALDI mass spectrum parameters are optimized, so that the sensitivity of the rhizoma smilacis glabrae bioactive components is improved. The ion inhibition effect of the matrix on the glabrous greenbrier rhizome active component is eliminated, the signal response of the glabrous greenbrier rhizome active component is obviously enhanced, the mass spectrum imaging quality level is improved, and the in-situ visual detection of the glabrous greenbrier rhizome active component in different tissues and organs of rats is successfully realized.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component analysis, and particularly relates to a MALDI-MSI method for visually analyzing the tissue distribution of active ingredients of Smilax glabra in rats. Background Art

[0002] Smilax glabra (Smilax glabra Roxb.) is the dried rhizome of the perennial herbaceous plant Smilax glabra (Liliaceae), primarily grown in southern and south-central my country. It is a traditional Chinese medicinal herb with a cold nature, a sweet and bland flavor, and enters the liver and stomach meridians, boasting the effects of clearing heat and dampness, detoxifying, and reducing swelling. It is not only a low-toxic herb but also possesses excellent pharmacodynamics. Modern pharmacological research has demonstrated its anti-tumor, antiviral, antibacterial, anti-inflammatory, and hypoglycemic properties. It possesses a rich array of pharmacological activities, with its main chemical components including flavonoids, steroids, phenylpropanoids, organic acids, volatile oils, tannins, and inorganic elements. Research has shown that its primary active ingredients are flavonoids, such as astilbin, chrysanthemin, epicatechin, and resveratrol, which play a significant role in lowering uric acid. The different active chemical components of Smilax glabra often play distinct roles in improving and treating diseases.

[0003] Traditional LC-MS / MS methods for drug tissue distribution measurement require tissue homogenization to extract analytes, resulting in an inability to obtain information on the spatial distribution of drugs within organ substructures or specific regions, and instead only reflecting average tissue concentrations. Common mass spectrometry imaging techniques include MALDI, DESI, and SIMS. Matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI) has become the most widely used due to its high sensitivity, label-free detection, and broad molecular coverage (including lipids, metabolites, peptides, and proteins). MALDI-MSI offers high spatial resolution, reaching 10 μm or even lower. In recent years, the continuous development of MALDI time-of-flight (TOF) mass spectrometers and devices has enabled and enabled MALDI-MSI to acquire high-resolution data rapidly and with high throughput, enabling even higher sensitivity and the detection of a wider range of compounds. This technique offers numerous advantages, including label-free operation, high coverage, high sensitivity, and a wide detection range. These characteristics enable it to simultaneously image and analyze multiple known and unknown molecules, obtain the spatial distribution, relative content and structural information of different molecules, and realize qualitative, quantitative and positional analysis of their molecules. Currently, MALDI-MSI has been widely used to study the spatial distribution of small and large molecules in biological samples. However, in the process of using MALDI-MSI technology to image the active ingredients of Smilax glabra, common matrices have a relatively serious ion suppression effect on the active ingredients of Smilax glabra, which inhibits the signal response of the active ingredients of Smilax glabra, thereby affecting the quality level of its mass spectrometry imaging, and cannot effectively realize the in situ visualization detection of the active ingredients of Smilax glabra in different tissues and organs of rats. Summary of the Invention

[0004] In response to the above technical problems, the present invention develops a MALDI-MSI method for visually analyzing the tissue distribution of Smilax glabra active ingredients in rats, which is used to analyze the spatial distribution characteristics of the main bioactive ingredients of Smilax glabra (5-O-caffeoylshikimic acid, astilbin, chrysanthemin and quercetin) in rats. The present invention utilizes MALDI-MSI mass spectrometry imaging technology to analyze the spatial metabolic distribution characteristics of Smilax glabra bioactive ingredients in rats. Based on the characteristics of Smilax glabra active ingredients, 9-aminoacridine is creatively used as a matrix, and the 9-aminoacridine matrix concentration and MALDI mass spectrometry parameters are optimized to eliminate the ion suppression effect of the matrix on the active ingredients of Smilax glabra, significantly enhance the signal response of the active ingredients of Smilax glabra, improve the quality level of its mass spectrometry imaging, and successfully realize the in situ visual detection of the active ingredients of Smilax glabra in different tissues and organs of rats.

[0005] The purpose of the present invention can be achieved by the following solutions:

[0006] In a first aspect, the present invention provides a method for visually analyzing the tissue distribution of an active ingredient of Smilax glabra in rats, comprising the following steps:

[0007] S1. Spraying 9-aminoacridine matrix onto the tissue section to be tested containing the active ingredient of Smilax glabra;

[0008] S2. Perform MALDI mass spectrometry imaging analysis on the tissue slice to be tested after spraying in step S1.

[0009] As an embodiment of the present invention, in step S1, the method for preparing the tissue slice to be tested includes: embedding the tissue of the rat after gavage with a standard decoction of Smilax glabra, freezing the tissue, and performing frozen sectioning, and vacuum drying to obtain the tissue slice to be tested.

[0010] Furthermore, the tissues include liver, kidney, large intestine, small intestine, and stomach tissues.

[0011] Furthermore, the embedding agent used for embedding includes gelatin, and its mass percentage is 8-15%.

[0012] Furthermore, the freezing treatment temperature is -70 to -80°C for 3 to 4 hours; and the temperature of the frozen sections is -15 to -25°C.

[0013] Furthermore, the vacuum drying time is 15-30 minutes.

[0014] Furthermore, the thickness of the tissue slice to be tested is 15-25 μm. The present invention also includes quickly transferring the tissue slice to the conductive surface of an ITO conductive glass slide before spraying, and performing optical image acquisition under an optical microscope in order to determine the acquisition area.

[0015] As an embodiment of the present invention, in step S1, the concentration of 9-aminoacridine in the 9-aminoacridine matrix is 10-15 mg / mL.

[0016] As an embodiment of the present invention, in step S2, the mass spectrometry detection of the MALDI mass spectrometry imaging analysis is performed in a negative ion mode.

[0017] In one embodiment of the present invention, in step S2, the mass spectrometry analysis conditions for the MALDI mass spectrometry imaging analysis include: a mass-to-charge ratio range of 200-800 m / z, a detector voltage of 2.20-2.60 kV, a laser spot count of 100-120 shots, a laser intensity of 60-65, and a laser spot diameter of 1-2 μm. The present invention obtains the spatial distribution of bioactive components through MALDI mass spectrometry imaging analysis.

[0018] As one embodiment of the present invention, step S2 further includes analyzing the data obtained from the MALDI mass spectrometry imaging analysis, performing peak extraction using a list of related compound data, and then performing imaging analysis and visualization. In some embodiments, the data obtained from the MALDI mass spectrometry imaging analysis is analyzed using IMAGEREVEAL MS software.

[0019] Furthermore, the related compounds include 5-O-caffeoylshikimic acid, astilbin, chrysantheminin, and quercetin.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention uses MALDI-MSI mass spectrometry imaging technology to analyze the spatial metabolic distribution characteristics of the bioactive components of Smilax glabra in rats. Based on the characteristics of the active components of Smilax glabra, 9-aminoacridine is creatively used as a matrix to eliminate the ion suppression effect of the matrix on the active components of Smilax glabra, significantly enhancing the signal response of the active components of Smilax glabra, improving the quality level of its mass spectrometry imaging, and successfully realizing the in situ visualization detection of the active components of Smilax glabra in different tissues and organs of rats.

[0022] 2. By optimizing the 9-aminoacridine matrix concentration and MALDI mass spectrometry parameters, the present invention further eliminates the ion suppression effect of the matrix on the target compound, achieves precise localization of the distribution differences of the active ingredient in target organs such as the liver, kidney, and intestine, and reveals its tissue-specific metabolic patterns.

[0023] 3. The MALDI-MSI method of the present invention intuitively shows the distribution characteristics of the effective ingredients of Smilax glabra by constructing a visual atlas, which provides an important spatial metabolomics basis for explaining its pharmacodynamic material basis and guiding clinical precision medication. Compared with traditional pharmacokinetic methods, this technology can synchronously obtain the spatiotemporal distribution information of multiple active ingredients, which has innovative guiding value for optimizing Chinese medicine compatibility schemes and dosage control. In addition, the method of the present invention has a simple operating procedure, can not only quantitatively analyze the detected compounds, but also analyze the in situ distribution characteristics of the compounds, and provide a visual research platform for explaining the pharmacodynamic material basis of Smilax glabra and its metabolic mechanism in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0025] Figure 1 This is the MALDI imaging image of characteristic ions of the matrix screening optical image in Example 1;

[0026] Figure 2This is a MALDI imaging diagram of the tissue distribution of the effective ingredients of Smilax glabra in Example 1 in the liver, kidney, large intestine, small intestine, and stomach of rats;

[0027] Figure 3 is the ionic strength of the active ingredients of Smilax glabra in each tissue and organ in Example 1;

[0028] Figure 4 The spatial distribution of the four active ingredients of Smilax glabra in Example 1 in the kidney;

[0029] Figure 5 The results of 9-AA matrix concentration screening in Comparative Example 1 are as follows;

[0030] Figure 6 This is the laser intensity screening result in Comparative Example 2. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, provide detailed implementation methods and specific operating procedures, and will help those skilled in the art to further understand the present invention. It should be pointed out that the scope of protection of the present invention is not limited to the following embodiments, and a number of adjustments and improvements made under the premise of the concept of the present invention all fall within the scope of protection of the present invention.

[0032] Example 1

[0033] (1) Preparation of standard decoction: Weigh 100g of Smilax glabra slices and add 900mL of purified water to soak for 30min. Then, use high heat to decoct for 20min, and simmer for 50min to maintain a slight boil over low heat, for a total of 70min. Then, filter the residue with a sieve and collect the filtrate in a beaker. Add 800mL of water to the residue, simmer for 15min over high heat and 40min over low heat for a second decoction, and filter and collect the decoction again. Combine the two decoctions and place them in a clean container for later use. Concentrate the combined decoction under reduced pressure using a rotary evaporator, and the concentrated decoction is used for administration.

[0034] (2) Animal experiment: The experimental animals were kept in an environment with a temperature of about 25°C and a relative humidity of 50% ± 5%, and were provided with free access to food and water. All experimental animals were adaptively fed for one week before the start of the experiment. Six male SD rats were selected in the experiment and given a standard decoction of Smilax glabra (dose 6 g / kg) by gavage. One hour after administration, the liver, kidney, large intestine, small intestine and stomach tissues were quickly dissected and collected, and placed in a -80°C refrigerator for freezing to ensure the stability of the biological samples.

[0035] (3) Sample embedding: Each tissue was placed in a silicone mold (2×2×2 cm) and embedded with 10% gelatin solution, and then frozen at -80°C for 4 h.

[0036] (4) Matrix screening: Prepare a mixed standard solution of 5-O-caffeoylshikimic acid, astilbin, chrysanthemin, and quercetin with pure methanol at a concentration of 20 μg / mL. Then screen the matrices 1,5-DAN, 9-AA (9-aminoacridine), CHCA, and DHB, each with a concentration of 10 mg / mL. 1,5-DAN and DHB matrices use 70% methanol solution as solvent, and 9-AA and CHCA matrices use 80% methanol solution and 50% acetonitrile solution as solvent, respectively. Figure 1 As shown in the results, it was found that using 9-AA as a matrix can significantly enhance the signal response of the active ingredients of Smilax glabra, and its chromatographic peak intensity is significantly better than other matrices. At the same time, the mass spectrometry imaging quality reaches the optimal level. Therefore, 9-AA was finally selected as the matrix for mass spectrometry imaging analysis of Smilax glabra components entering the blood.

[0037] (5) Preparation of frozen sections: Use a cryostat to cut frozen tissues of various organs, with a thickness of 20 μm. The temperature of the cryostat is -20°C.

[0038] (6) Optical image acquisition: Transfer the tissue sections to an optical microscope for optical image acquisition. Transfer method: After the ITO slide is pre-cooled for 10 minutes on a cryostat, the intact tissue sections are quickly attached to the slide and dried in a vacuum dryer for 20 minutes.

[0039] (7) Matrix Deposition: After collecting optical images of tissue sections, a laser desorption / ionization matrix 9-AA was applied using a spray gun to ensure uniform distribution of the matrix on the tissue sections. The tissue sections were vacuum dried before sample loading. The matrix 9-AA concentration was 10 mg / mL.

[0040] (8) Mass spectrometry imaging analysis: Rat tissue sections coated with 9-AA matrix were subjected to mass spectrometry imaging analysis using a Shimadzu iMScope QT mass spectrometer in negative ion mode. The experimental parameters were set as follows: mass-to-charge ratio covering the 200-800 m / z range, detector voltage 2.20 kV, laser intensity 60, single-point excitation number 100 shots, and laser spot diameter precisely controlled to 1 μm. By comparing the mass spectrometry fragmentation patterns of the standard compound database and literature reports (Shi W, Jia M, Li X, Zhao X, Wang C, Fan G, Lou Y. Comprehensive characterization of the active ingredients of Smilax glabra Roxb based on chemical fingerprinting, metabolic fingerprinting and pharmacodynamic fingerprinting. Front Pharmacol. 2025Apr 23; 16: 1519054. doi: 10.3389 / fphar.2025.1519054), four characteristic active ingredients in Smilax glabra Roxb were successfully identified: 5-O-caffeoylshikimic acid, astilbin, quercitrin and engeletin. The detailed information of each compound is shown in Table 1. By extracting the characteristic ion intensity and combining it with the algorithm, the following was obtained: Figure 2 The high spatial resolution MALDI mass spectrometry imaging diagram shown. Figure 2 and Figure 3 It can be seen that there are obvious differences in the spatial distribution of the bioactive ingredients in Smilax glabra. The four active ingredients from Smilax glabra are widely distributed in the liver, kidney, stomach, large intestine and small intestine of rats. The overall distribution of 5-O-caffeoylshikimic acid is large intestine > small intestine > stomach > kidney > liver; the overall distribution of astilbin is: stomach > small intestine > large intestine > kidney > liver; the overall distribution of flavonoids glycosides is: stomach > large intestine > small intestine > kidney > liver; the overall distribution of quercetin is: stomach > small intestine > large intestine > kidney > liver. Then Figure 4 The distribution of these four chemical components in the kidney shows that 5-O-caffeoylshikimic acid and quercetin are primarily distributed in the rat renal medulla. Unlike the distribution patterns of 5-O-caffeoylshikimic acid and quercetin in the kidney, astilbin and cyperitoside are primarily distributed in the renal cortex. Overall, this study analyzed exogenous drugs in SD rat kidney sections using MALDI-MSI technology, obtaining information on the spatial distribution and abundance of these substances in the rat kidney.

[0041] Table 1 Detailed information of the four active ingredients of Smilax glabra

[0042]

[0043]

[0044] Comparative Example 1

[0045] The method of this comparative example is basically the same as that of Example 1, except that the concentration of substrate 9-AA in step (4) is adjusted to 1 mg / mL and 20 mg / mL for comparison with 10 mg / mL in Example 1. The results are as follows: Figure 5 The matrix 9-AA significantly enhanced the signal intensity of each compound, with 10 mg / mL achieving even greater signal enhancement compared to 1 mg / mL. This indicates that increasing matrix concentration generally improves the ionization efficiency and signal intensity of a molecule, thereby enhancing the sensitivity of mass spectrometry imaging. However, there was no significant difference in signal intensity between 10 mg / mL and 20 mg / mL concentrations, and excessively high matrix concentrations may cause matrix interference and reduce the detection sensitivity of the target molecule. Therefore, a matrix concentration of 10 mg / mL was selected for this experiment.

[0046] Comparative Example 2

[0047] The method of this comparative example is basically the same as that of Example 1, except that the laser intensity in step (8) is adjusted to 50 and 70 for comparison with 60 in Example 1. The results are as follows: Figure 6 The results show that the ionization efficiency and signal intensity of each compound are optimal at a laser intensity of 60, so the laser intensity was adjusted to 60 during detection. By optimizing the laser intensity, the intensity and signal-to-noise ratio of the mass spectrometry signal can be improved, thereby obtaining higher-quality mass spectrometry images.

[0048] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for visually analyzing the tissue distribution of the active ingredients of Smilax glabra in rats, characterized in that: The following steps are involved: S1. Spraying 9-aminoacridine matrix onto the tissue section to be tested containing the active ingredient of Smilax glabra; S2. Perform MALDI mass spectrometry imaging analysis on the tissue slice to be tested after spraying in step S1.

2. The method according to claim 1, characterized in that In step S1, the method for preparing the tissue slice to be tested includes: embedding the tissue of the rat after gavage with a standard decoction of Smilax glabra, freezing the tissue, performing frozen sectioning, and vacuum drying to obtain the tissue slice to be tested.

3. The method according to claim 2, characterized in that The tissues include liver, kidney, large intestine, small intestine, and stomach tissue.

4. The method according to claim 2, characterized in that The embedding agent used for embedding includes gelatin, and its mass percentage is 8-15%.

5. The method according to claim 2, characterized in that Also includes at least one of the following technical features: The freezing treatment temperature is -70 to -80°C and the time is 3 to 4 hours; The temperature of the frozen section is -15 to -25°C; The vacuum drying time is 15-30min; The thickness of the tissue section to be tested is 15-25 μm.

6. The method according to claim 1, wherein In step S1, the concentration of 9-aminoacridine in the 9-aminoacridine matrix is 10-15 mg / mL.

7. The method according to claim 1, characterized in that In step S2, the mass spectrometry detection of the MALDI mass spectrometry imaging analysis is performed in a negative ion mode.

8. The method according to claim 1, characterized in that In step S2, the mass spectrometry analysis conditions of the MALDI mass spectrometry imaging analysis include: mass-to-charge ratio range of 200-800 m / z, detector voltage of 2.20-2.60 KV, laser spot number of 100-120 Shots, laser intensity of 60-65, and laser spot diameter of 1-2 μm.

9. The method according to claim 1, characterized in that Step S2 also includes: analyzing the data obtained by the MALDI mass spectrometry imaging analysis, performing peak extraction using a list of relevant compound data, and then performing imaging analysis and visualization.

10. The method according to claim 9, characterized in that The related compounds include 5-O-caffeoylshikimic acid, astilbin, chrysantheminin and quercetin.

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