Mass spectrum imaging method for visually analyzing spatial distribution of bioactive components in phyllanthus emblica and olive

Abstract

The invention provides a mass spectrum imaging method for visually analyzing in-vivo and in-vitro spatial distribution of bioactive components in phyllanthus emblica and olive by optimizing instrument parameters and screening a matrix to avoid interference of the matrix to a target compound. According to the established mass spectrum imaging method, on the basis of obtaining a high-resolution morphological image, the in-vitro spatial distribution difference of 36 components in fresh phyllanthus emblica and olives is efficiently and visually analyzed, and the main components comprise phenolic acids, tannins, flavonoids, carboxylic acids, saccharides, terpenoids, sterols and the like. Meanwhile, spatial distribution differences of the 36 components, including heart, liver, spleen, lung and kidney, in various organs of a mouse body are analyzed. The difference of the same bioactive substances in the phyllanthus emblica and the olive is researched from a brand new perspective, the difference distribution of the presented same compounds verifies that the combined action of the same bioactive substances from different sources in vivo can show different pharmacological meanings, and an innovative thought is provided for distinguishing the phyllanthus emblica and the olive in clinical medication.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component analysis, and particularly relates to a mass spectrometry imaging method for visually analyzing the spatial distribution of bioactive components in emblica and olive. Background Art

[0002] Phyllanthus emblica L. is the mature fruit of the Euphorbiaceae family Phyllanthus emblica. It is also known as Yunnan olive, olive, oil emblica, Ammole, Mihan, Wangguo, Mubo, and emblica fruit. It is mainly distributed in the Philippines, Malaysia, Guizhou, Yunnan, Guangdong, Fujian, Taiwan and other regions in my country. Emblica emblica is a traditional Tibetan medicinal material. It belongs to the lung and spleen meridians. It tastes bitter, sour and astringent at first, but soon turns sweet, hence the name "emblica". It can be used to clear heat and relieve sore throat, moisten the lungs and resolve phlegm, and treat respiratory diseases such as cough and sore throat. According to modern scientific research, it has anti-inflammatory, antioxidant, anti-aging, and liver protection effects, and may also have certain therapeutic effects on cardiovascular diseases.

[0003] Olive (Canarium album Rauesch.), also known as yellow olive, green seed, green fruit, mountain olive, white olive, red olive, is the mature fruit of the genus Olive of the Oleaceae family. It is mainly distributed in Fujian, Yunnan, Taiwan and other places in my country. In addition to being native to southern China, Longnan City, Gansu Province in the north is also one of the best suitable areas for olive cultivation in China. At the same time, it is also cultivated in Japan and the Malay Peninsula. Olives are sweet, sour and astringent, and enter the lung and stomach meridians. The Compendium of Materia Medica records that they can produce body fluids, relieve thirst, treat sore throats, and detoxify fish and crabs. Modern scientific research has found that olives also have antibacterial, anti-inflammatory and analgesic effects, anti-hepatitis B virus, and liver protection. The common names of Phyllanthus emblica are Yunnan olives and olives, and the fruit morphology and bioactive ingredients are roughly the same as those of olives, and the efficacy is similar. Therefore, analyzing the differences between Phyllanthus emblica and olives is of great significance for clinical guidance of medication.

[0004] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) is a new soft ionization bioimaging technology. This method combines the advantages of matrix-assisted laser desorption ionization (MALDI) and time-of-flight mass spectrometry (TOF-MS) and is widely used in the qualitative or semi-quantitative detection of samples in the fields of proteomics, metabolomics, and drug development. This technology has high throughput, high sensitivity, high resolution, and strong molecular specificity. It can locate multiple biomolecules at a single time without the need for complex probe synthesis or labeling, and provides a molecular map of the abundance and spatial information of biomolecules in tissue sections. Nowadays, with the continuous development of MALDI-TOF-MSI, the development of new matrices, and the improvement of ionization technology, this technology has been transferred from biological macromolecules to the study of small molecule compounds such as drugs and metabolites, and the analysis objects have also expanded from animal tissue samples to plant and Chinese herbal medicine samples. Compared with traditional methods, the advantage of using MALDI-TOF-MSI technology to analyze emblica and olive, which have the same common name, fruit morphology, and similar bioactive ingredients, is that it can intuitively distinguish the spatial distribution information of their endogenous molecules in vivo and in vitro, and visualize the differences between emblica and olive, which is of great significance for clinical guidance of medication. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a mass spectrometry imaging method for visually analyzing the spatial distribution of bioactive components in emblica and olives. By screening and determining relevant instrument parameters and matrix types, the interference of the matrix on the target compounds is avoided, and the main representative bioactive components such as phenolic acids, tannins, flavonoids, carboxylic acids, sugars, terpenes and sterols can be visualized.

[0006] The mass spectrometry imaging method for visually analyzing the spatial distribution of bioactive components in emblica and olive provided by the present invention comprises the following steps: (1) Sample tissue preparation: Fresh emblica and olive were sawed open to present their cross-sections and embedded in embedding agents. They were placed in a freezing microtome and then sliced. The tissue slices were transferred to ITO-conductive glass slides. The heart, liver, spleen, lung, and kidney organs of mice were taken after oral administration of emblica and olive extracts. The organs did not need to be embedded. They were frozen and then sliced. The tissue slices were transferred to ITO-conductive glass slides. The embedding medium is gelatin; the freezing treatment temperature is -80±10℃, and the freezing section temperature is -20±5℃; the thickness of the Emblica emblica and olive tissue sections is 20μm; the thickness of the mouse organ tissue sections is 15μm; before transferring the tissue sections, the ITO-conductive slide is precooled in a freezing microtome for 5 to 10 minutes; Emblica emblica and olive are both fresh fruits.

[0007] (2) Optical image acquisition: The optical images of the embryonic phlox, olive, and mouse organ tissue sections on the ITO-conductive slide in step (1) were acquired under an optical microscope; the optical image acquisition magnification was 1.25 times.

[0008] (3) Matrix deposition: Handheld spray gun deposition assisted laser desorption ionization matrix DAN (1,5-diaminonaphthalene) was used to obtain tissue sections of Phyllanthus emblica, olive, and mouse organs after matrix coating; the matrix DAN concentration was 10 mg / ml.

[0009] (4) Mass spectrometry imaging analysis: MALDI mass spectrometry imaging analysis of tissue sections coated with the matrix can obtain the spatial distribution of 36 bioactive components; the main representative bioactive components include phenolic acids, tannins, flavonoids, carboxylic acids, sugars, terpenes, and sterols.

[0010] The mass spectrometry analysis of MALDI mass spectrometry imaging was performed in negative ion mode, and the mass spectrometry analysis conditions were as follows: mass-to-charge ratio range of 100-1000, detector voltage of 1.89±0.5KV, sample voltage of 3.0KV, imaging step length of 45±10μm, laser intensity ≥55, and laser spot diameter of 20±10μm.

[0011] Beneficial effects of the present invention: The present invention provides a mass spectrometry imaging method for visually analyzing the spatial distribution of bioactive components in emblica and olives. By screening and determining relevant instrument parameters and matrix types, the interference of the matrix on the target compound is avoided. Visual imaging of 36 endogenous molecules in emblica and olives in vivo and in vitro is achieved. The present invention studies the differences in the same bioactive substances in emblica and olives from a new perspective. The differential distribution of the same compounds presented verifies that the combined action of the same bioactive substances from different sources in vivo can show different pharmacological significances, providing an innovative idea for distinguishing emblica and olives in clinical medication. The present invention can intuitively distinguish the spatial distribution information of endogenous molecules in vivo and in vitro for emblica and olives with similar fruit morphology and bioactive components, and visualize the differences in the similarities between emblica and olives, which is of great significance for clinical guidance of medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Optical images and structural analysis of Phyllanthus emblica and olive tissue sections; Figure 2 Matrix screening optical images and characteristic ion MALDI imaging images; Figure 3 Optical images and characteristic ion MALDI imaging of Phyllanthus emblica and olive tissue sections (the upper part is olive tissue, the lower part is Phyllanthus emblica tissue); Figure 4Optical images of mouse heart tissue sections and characteristic ion MALDI imaging (the upper part is the heart of a mouse gavaged with Phyllanthus emblica extract, and the lower part is the heart of a mouse gavaged with Olive extract); Figure 5 Optical images of mouse liver tissue sections and characteristic ion MALDI imaging (the upper part shows the liver of mice gavaged with Phyllanthus emblica extract, and the lower part shows the liver of mice gavaged with Olive extract); Figure 6 Optical images of mouse spleen tissue sections and characteristic ion MALDI imaging (the upper part shows the spleen of mice gavaged with Phyllanthus emblica extract, and the lower part shows the spleen of mice gavaged with Olive extract); Figure 7 Optical images of mouse lung tissue sections and characteristic ion MALDI imaging (the upper part shows the lungs of mice gavaged with Phyllanthus emblica extract, and the lower part shows the lungs of mice gavaged with Olive extract); Figure 8 Optical images of mouse kidney tissue sections and characteristic ion MALDI imaging (the upper part is the kidney of the mouse gavaged with Phyllanthus emblica extract, and the lower part is the kidney of the mouse gavaged with Olive extract). DETAILED DESCRIPTION

[0013] The mass spectrometry imaging method for visually analyzing the spatial distribution of bioactive components in emblica and olive in vitro and in vivo provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples. Example

[0014] (1) Sample pretreatment: Select fresh, undamaged, and appropriately sized emblica and olives, wash them three times with deionized water, wipe them dry, and store them in a sealed container in a refrigerator at 4°C. Fast healthy male KM mice for 1 day and administer the drug by gavage (1.0 g of emblica and olive extracts dissolved in 1 ml of water, and the dose is calculated based on 40 g / kg of the original medicinal material). Remove the heart, liver, spleen, lung, and kidney and freeze them at -80±10°C.

[0015] (2) Sample embedding: Take out the emblica buds and olives stored in a sealed container at 4°C, saw them open to reveal their cross-sections, quickly place them in a silicone mold (5×5×2 cm) and embed them with gelatin, place them in a freezing microtome at -20°C for 15 min, and then freeze them at -80°C for 4 hours.

[0016] (3) Matrix screening: Prepare mixed standard solution: Prepare mixed solution of 7 standards including gallic acid (m / z 169.01), shikimic acid (m / z173.04), ellagic acid (m / z 301.00), chebulic acid (m / z 355.03), corilagin (m / z 633.07), chebulic acid (m / z 953.08) and chebulic acid (m / z 955.11) to screen matrix. DAN matrix deposition was carried out by manual spray gun method with matrix concentration of 10 mg / ml. 9-AA, CHCA and DHB matrix deposition was carried out by Shimadzu iMLayer matrix sublimator with matrix concentration of 10 mg / ml. Specific deposition conditions: 9-AA and CHCA matrix 300 mg, deposited at 250°C with a thickness of 0.5 μm, DHB matrix 300 mg, deposited at 180°C with a thickness of 1.5 μm. Figure 2 As shown in the figure, in the negative ion mode, when DAN matrix deposition was used, the seven standards were able to form clear and bright co-crystallization images with DAN, while the co-crystallization detection effects with 9-AA, CHCA and DHB were poor, so it was determined that DAN matrix deposition was used.

[0017] (4) Preparation of frozen sections: Use a cryostat to cut frozen Phyllanthus emblica and olive tissues, with a slice thickness of 20 μm. Cut frozen mouse organ tissues, with a slice thickness of 15 μm. The temperature of the cryostat is -20°C. Figure 1 , based on the optical images of Phyllanthus emblica and olive tissues and related literature, their microstructures were analyzed.

[0018] (5) Optical image acquisition: The optical image of the tissue section transferred to the ITO-conductive glass slide was acquired under an optical microscope, and the image acquisition magnification was 1.25 times. Transfer method: After the ITO-conductive glass slide was precooled for 5 to 10 minutes in a freezing microtome, the complete tissue section was tightly attached to the ITO-conductive glass slide.

[0019] (6) Matrix deposition: Handheld spray gun deposition assisted laser desorption ionization of matrix DNA to obtain matrix-coated tissue sections with a matrix DNA concentration of 10 mg / ml.

[0020] (7) Mass spectrometry imaging analysis: iMScopeTRIO mass spectrometer was used to scan and ionize each tissue section coated with DAN matrix in negative ion mode. The mass spectrometry instrument parameters were as follows: mass-to-charge ratio range of 100-1000, detector voltage of 1.89±0.5KV, sample voltage of 3.0KV, imaging step length of 45±10μm, laser intensity ≥55, and laser spot diameter of 20±10μm. The mass spectrometry information of the above bioactive ingredients was compared with the literature and database to determine their specific material structure. The ion intensity and visualization image of the active ingredient were extracted, as shown in Figure 2. Figure 3The figure shows the MALDI imaging of 36 endogenous molecules in Phyllanthus emblica and olive (Table 1 shows the specific information of 36 endogenous molecules in Phyllanthus emblica and olive). As can be seen from the figure, there are obvious differences in the spatial distribution of bioactive components in Phyllanthus emblica and olive. In olive tissue, only a few components, such as tannin compound ellagic acid (m / z301.00) and flavonoid compound quercetin (m / z 301.03), have a high relative abundance in the mesocarp, while other components are distributed more evenly in various tissues. However, the spatial distribution of Phyllanthus emblica components is completely opposite to this, and each type of component shows a unique distribution characteristic. Among the phenolic acid compounds, only a few, such as mucic acid-2-O-gallate (m / z 361.04), are distributed in the exocarp and mesocarp, and the rest are mainly concentrated in the endocarp. The distribution of carboxylic acid and tannin compounds is relatively uniform, but malic acid (m / z 133.01) and ellagic acid (m / z 301.00) are mainly found in the endocarp and seeds. The distribution characteristics of flavonoids are more significant, and their chemical components are mainly concentrated in the endocarp, seeds and seed coat. Figures 4 to 8 It showed that 36 components from emblica and olive were widely distributed in mice. Among them, phenolic acids were mainly distributed in the heart, lungs, and spleen, and the overall distribution was: lungs> spleen> heart> liver> kidney. Carboxylic acids were particularly significantly distributed in the heart and lungs, and the overall distribution was: lungs> heart> spleen> liver> kidney. Tannins were mainly distributed in the lungs, and the overall distribution was: lungs> spleen> heart> liver> kidney. Flavonoids were mainly distributed in the lungs and spleen, and the overall distribution was: lungs> spleen> liver> heart> kidney. Sugars were mainly distributed in the lungs and heart, and the overall distribution was: lungs> heart> spleen> kidney> liver. Terpenes and sterols were mainly distributed in the heart and lungs, and the overall distribution was: lungs> heart> spleen> liver> kidney. It is worth noting that the distribution of these components in the lungs is very significant, and this finding provides an important research basis for further exploring their pharmacological effects.

Claims

1. A mass spectrometry imaging method for visually analyzing the spatial distribution of bioactive components in Phyllanthus emblica and olive, the method comprising the following steps: (1) Sample tissue preparation: Fresh emblica and olive fruits were sawed open to present their cross-sections and embedded in embedding agents. They were placed in a freezing microtome and then sliced. The tissue slices were transferred to ITO-conductive glass slides. The heart, liver, spleen, lung, and kidney organs of mice gavaged with emblica and olive extracts were obtained. The organs did not need to be embedded. They were frozen and then sliced. The tissue slices were transferred to ITO-conductive glass slides. (2) Optical image acquisition: optical images of the embryonic physalis, olive, and mouse organ tissue sections on the ITO-conductive slide in step (1) are acquired under an optical microscope; (3) Matrix deposition: Deposition-assisted laser desorption ionization of matrix DAN was performed by handheld spray gun method to obtain matrix-coated Phyllanthus emblica, olive, and mouse organ tissue sections; (4) Mass spectrometry imaging analysis: MALDI mass spectrometry imaging analysis of tissue sections coated with the matrix to obtain the spatial distribution of bioactive components; the mass spectrometry of this imaging analysis was performed in negative ion mode; mass spectrometry analysis conditions: mass-to-charge ratio range of 100-1000, detector voltage of 1.89±0.5KV, sample voltage of 3.0KV, imaging step length of 45±10μm, laser intensity ≥55, and laser spot diameter of 20±10μm.

2. The mass spectrometry imaging method for analyzing the spatial distribution of bioactive components in emblica and olive according to claim 1, characterized in that: The embedding medium is gelatin.

3. The mass spectrometry imaging method for analyzing the spatial distribution of bioactive components in emblica and olive according to claim 1, characterized in that: The freezing treatment temperature in step (1) is -80±10°C, and the freezing section temperature is -20±5°C.

4. The mass spectrometry imaging method for analyzing the spatial distribution of bioactive components in Phyllanthus emblica and olive according to claim 1, characterized in that: In step (1), the thickness of the Emblica officinalis and Olive tissue sections is 20 μm, and the thickness of the mouse organ tissue sections is 15 μm.

5. The mass spectrometry imaging method for analyzing the spatial distribution of bioactive components in emblica and olive according to claim 1, characterized in that: Step (1) The ITO-conductive glass slide is precooled in a freezing microtome for 5 to 10 minutes before transferring the tissue sections.

6. The mass spectrometry imaging method for analyzing the spatial distribution of bioactive components in Phyllanthus emblica and olive according to claim 1, characterized in that: The acquisition magnification of the optical image in step (2) is 1.25 times.

7. The mass spectrometry imaging method for analyzing the spatial distribution of bioactive components in Phyllanthus emblica and olive according to claim 1, characterized in that: The matrix DNA concentration in step (3) is 10 mg / ml.

Images