Method for identifying different processed radix astragali products based on GC-IMS analysis of characteristic odor substances

Abstract

The invention belongs to the technical field of analysis and identification of traditional Chinese medicine decoction pieces, and relates to a method for identifying different processed products of radix astragali based on GC-IMS analysis of characteristic odor substances, and the method comprises the following steps: 1) preparing different processed products of radix astragali as samples to be detected; 2) carrying out GC-IMS analysis to obtain a GC-IMS two-dimensional spectrogram corresponding to the sample to be detected; 3) respectively carrying out three-dimensional spectrogram conversion, direct comparison diagram conversion, difference comparison diagram conversion and fingerprint spectrum analysis on the GC-IMS two-dimensional spectrogram, and 4) carrying out clustering heat map analysis, and distinguishing different radix astragali processed products according to the difference of volatile substances contained in the sample to be detected. According to the method, different radix astragali processed products are identified by analyzing substances with different smells, and a new thought is provided for establishing a quality control method for different radix astragali processed products.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and identification of traditional Chinese medicine decoction pieces, and relates to a method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances. Background Art

[0002] Astragalus membranaceus is the dried root of the leguminous plant Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. It was first recorded in "Shennong Ben Cao Jing" and was listed as a top-grade medicine. Since ancient times, there have been many processing methods for Astragalus membranaceus. Its earliest record was in the Han Dynasty, such as the method of removing the rhizome; in the Liu Song Dynasty of the Southern and Northern Dynasties, there was steaming; since the Song Dynasty, there have been processing methods such as honey frying, roasting with salt solution, stir-frying, honey steaming, honey frying, moist steaming with salt water, frying with crisp, boiling with wine, and salt frying; in the Yuan Dynasty, there was frying with salt and honey water; in the Ming Dynasty, processing techniques such as stir-frying with wine mixing, ginger juice frying, and stir-frying with rice washing water were added; in the Qing Dynasty, new processing methods such as processing with human milk, baking, vinegar frying, and nine-processed Astragalus membranaceus were created. With the development of society, some processing techniques have gradually been eliminated, and only Astragalus membranaceus and stir-fried Astragalus membranaceus with honey are included in the current edition of the pharmacopoeia.

[0003] Odor is one of the criteria for identifying traditional Chinese medicine decoction pieces. For Astragalus membranaceus, "the stronger the bean smell, the better". The bean smell comes from the enzymatic reaction products of polyunsaturated fatty acids in it. Under the action of lipoxygenase (LOX) and hydroperoxide lyase (HPL), polyunsaturated fatty acids containing cis,cis-1,4-pentadiene structure generate fatty acid hydroperoxides, and then further produce volatile substances such as small molecule alcohols, ketones, and aldehydes, making the beans produce a bean smell. After Astragalus membranaceus is processed by different methods, the types and contents of its chemical components will change, and the types and contents of chemical components will also affect the pharmacological effects of Astragalus membranaceus. Modern research on the chemical components of stir-fried Astragalus membranaceus with rice mainly focuses on carbohydrates and flavonoids, followed by amino acids and trace elements. Whether different processing methods have an impact on the bean smell of Astragalus membranaceus remains to be systematically studied.

[0004] Gas chromatography-ion migration spectrometry (GC-IMS) combines the advantages of high separation of gas chromatography and high sensitivity of ion migration spectrometry, and is a rapid and reliable analysis method for trace volatile organic gases. In recent years, GC-IMS technology has been gradually widely used in the detection of food, traditional Chinese medicine, and air pollutants. In this study, GC-IMS technology was used to analyze and compare the differential odor components of Astragalus membranaceus before and after stir-frying with rice, in order to provide a reference method for quickly and accurately identifying raw and stir-fried Astragalus membranaceus decoction pieces. Summary of the Invention

[0005] The object of the present invention is to provide a method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances. By analyzing the odor difference substances, different processed products of Astragalus membranaceus are identified, providing new ideas for establishing a quality control method for different processed products of Astragalus membranaceus.

[0006] In order to achieve the above object, the steps adopted by the present invention are as follows:

[0007] A method for identifying different processed products of Astragalus membranaceus based on GC IMS analysis of characteristic odor substances, comprising the following steps:

[0008] (1) Prepare different processed products of Astragalus membranaceus as test samples. The different processed products of Astragalus membranaceus are Astragalus membranaceus or stir-fried Astragalus membranaceus with rice.

[0009] (2) Use GC IMS analysis to obtain the corresponding GC-IMS two-dimensional spectra of the test samples respectively.

[0010] (3) Perform three-dimensional spectrum conversion, direct comparison graph conversion, difference comparison graph conversion, fingerprint spectrum analysis and cluster analysis on the GC IMS two-dimensional spectra in step (2) respectively. By the differences in the volatile substances contained in the test samples, different processed products of Astragalus membranaceus are distinguished.

[0011] (4) Intuitively analyze through the cluster heat map to obtain the volatile components with large differences between Astragalus membranaceus and its processed products.

[0012] In the method for identifying different processed products of Astragalus membranaceus based on GC IMS analysis of characteristic odor substances, in step (2), the determination method of the GC IMS spectrum is as follows: Weigh 0.5 g of Astragalus membranaceus or stir-fried Astragalus membranaceus with rice samples and place them in a 20 mL headspace vial. Incubate at 80 °C and 500 r·min-1 for 15 min, then inject 200 μL. The injection needle temperature is 85 °C, and 3 parallel groups are measured for each sample; GC-IMS separation conditions: The column temperature is 60 °C, the running time is 40 min, the carrier gas / drift gas is N2, the purity is ≥99.999%, the carrier gas flow rate: the initial flow rate is 2.0 mL·min-1 and is maintained for 2 min, linearly increased to 10.0 mL·min-1 within 8 min, linearly increased to 100.0 mL·min-1 within 10 min, and maintained for 20 min. The injection port temperature is 80 °C; the IMS migration tube temperature is 45 °C; the drift gas flow rate is 75.0 mL·min-1.

[0013] In the method for identifying different processed products of Astragalus membranaceus based on GC IMS analysis of characteristic odor substances, in step (3), each peak on the GC IMS two-dimensional spectrum is qualitatively analyzed through the NIST database and the IMS database to determine the volatile substances corresponding to each peak on the GC IMS two-dimensional spectrum.

[0014] The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances. In step (3), the volatile substances in the test sample are esters, alcohols, ketones, aldehydes, terpenes, pyrazines, sulfides, furans, thiazoles, organic acids, and benzene compounds.

[0015] The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances. Among the volatile substances in step (3), the volatile substances exist in two forms: monomers and dimers. The volatile substances are methyl acetate, propionic acid, acetic acid, 3-hydroxy-2-propanone, 2-heptanone, 1,8-cineole, (E)-2-hexenal, hexenal, diallyl sulfide, hexanenitrile, n-hexanol, n-pentanol, isopentanol, n-butanol, sec-pentanol, isobutanol, and n-propanol.

[0016] The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances. In step (3), the fingerprint pattern analysis automatically generates a fingerprint pattern from all the peaks to be analyzed in the GC-IMS two-dimensional spectrum. From the fingerprint pattern, the complete volatile substance information of different processed products of Astragalus membranaceus and the differences in the volatile substances contained in different processed products of Astragalus membranaceus can be seen. In step (4), the cluster heat map analysis determines the differences in the volatile substances contained in different processed products by the shades of color between different volatile components with significant differences in the same group.

[0017] The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances. The cluster analysis includes dynamic principal component analysis.

[0018] The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances. Both step (2) and step (3) are completed by VOCal software. Among them, the principal component analysis in step (3) is completed by SIMCA-14 software, and step (4) is completed by using the microsignal platform http: / / www.bioinformatics.com.cn / after differential compound analysis.

[0019] The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances. The different processed products of Astragalus membranaceus are raw Astragalus membranaceus or processed products of Astragalus membranaceus. Among Astragalus membranaceus and its processed products, Astragalus membranaceus is raw Astragalus membranaceus, and the processed product of Astragalus membranaceus is stir-fried Astragalus membranaceus with rice. The preparation method of stir-fried Astragalus membranaceus with rice is as follows: Take raw Astragalus membranaceus, that is, the cleaned Astragalus membranaceus slices. Place the rice in a preheated frying pan, set the temperature to 240 °C, put the rice in and stir-fry for 7 minutes until the rice turns slightly yellow, then put in the cleaned Astragalus membranaceus, stir-fry the rice and the medicine together, stir-fry for 10 minutes until the rice turns yellow, the color of the Astragalus membranaceus slices turns darker, and there is an aroma, then take out, sieve out the rice, and let it cool, then it is obtained.

[0020] The method for identifying different processed products of Astragalus membranaceus based on the analysis of characteristic odor substances by GC-IMS shows that the contents of methyl propionaldehyde, isovaleraldehyde, and 5-methyl-2-acetylfuran compounds in raw Astragalus membranaceus are significantly higher than those in stir-fried Astragalus membranaceus with rice; the content of (E,E)-2,4-hexadienal compound in stir-fried Astragalus membranaceus with rice is significantly higher than that in raw Astragalus membranaceus.

[0021] Odor is one of the most important quality indicators of traditional Chinese medicine. Its odor comes from the volatile components in traditional Chinese medicine. After traditional Chinese medicine is processed, its odor changes, and the volatile components in the decoction pieces also change accordingly. Therefore, the decoction pieces of traditional Chinese medicine can be identified and the degree of processing of the decoction pieces can be determined by odor. In this study, GC-IMS technology was used for the first time to analyze the changes in the content of volatile components during the frying process of Astragalus membranaceus, and the chemical composition differences before and after stir-frying Astragalus membranaceus with rice were obvious. This study provides data support for the research on the processing mechanism of Astragalus membranaceus and also provides a method reference for exploring the chemical composition differences of other traditional Chinese medicines before and after processing. Description of the Drawings

[0022] Figure 1 Three-dimensional chromatogram of gas chromatography-ion mobility spectrometry (GC-IMS) of volatile substances in Astragalus membranaceus and its processed products

[0023] Figure 2 Direct comparison chart of volatile components in different processed products of Astragalus membranaceus

[0024] Figure 3 Comparison chart of differences in volatile substances of stir-fried Astragalus membranaceus with rice with Astragalus membranaceus as a reference for deduction

[0025] Figure 4 Comparison of fingerprint spectra of volatile substances in different processed products of Astragalus membranaceus

[0026] Figure 5 Principal component analysis chart of Astragalus membranaceus and its processed products

[0027] Figure 6 OPLS-DA (A) and model cross-validation results (B) of Astragalus membranaceus and its processed products

[0028] Figure 7 Cluster heat map of volatile components of Astragalus membranaceus and its processed products Specific implementation method

[0030] The present invention will be described in detail below in conjunction with the drawings and embodiments.

[0031] Example 1:

[0032] 1 Experimental equipment and materials

[0033] Gas phase ion mobility spectrometry (G.A.S. company); BCE224-1CCN type ten-thousandth electronic analytical balance (Sartorius Scientific Instruments Co., Ltd.); CTC-PAL3 static headspace automatic sampler (CTC Analytics AG company); 20 mL headspace vial (Shandong Haineng Scientific Instruments Co., Ltd.); CY30 type electromagnetic medicine frying machine (Beijing Huoshi Machinery Manufacturing Co., Ltd.).

[0034] Astragalus membranaceus produced in Gansu (batch number 210506, Ma'anshan Jingquan Chinese Herbal Pieces Co., Ltd.), identified by Chief Pharmacist Zhu Yufeng of the Affiliated Hospital of Nanjing University of Chinese Medicine, as the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao of the Leguminosae family. Rice (Fengyang Banqiao Yixing Agricultural Development Co., Ltd.) was purchased from the market.

[0035] 2 Methods

[0036] 2.1 Samples

[0037] The processing of stir-frying Astragalus membranaceus with rice was from the previous research of the research group. Take 10 kg of purified Astragalus membranaceus herbal pieces, put 2.5 kg of rice into the preheated frying pan, set the temperature to 240 °C, put in the rice and stir-fry for 7 min first, stir-fry the rice until slightly yellow, then put in the purified Astragalus membranaceus, stir-fry the rice and the medicine together, stir-fry for 10 min until the rice turns yellow, the color of Astragalus membranaceus slices turns darker, and there is an aroma, then take out, sieve out the rice, spread it out to cool, and make samples separately for each batch.

[0038] 2.2 Experimental conditions

[0039] Headspace injection conditions: Weigh 0.5 g of Astragalus membranaceus or stir-fried Astragalus membranaceus with rice samples and place them in 20 mL headspace vials respectively. Incubate at 80 °C and 500 r·min -1 for 15 min and then inject 200 μL. The injection needle temperature is 85 °C, and 3 parallel groups are measured for each sample. GC-IMS separation conditions: MXT-WAX capillary chromatographic column (15 m × 0.53 mm, 1.0 μm, Restek company, USA), column temperature 60 °C, running time 40 min, carrier gas / drift gas N2 (purity ≥ 99.999%), carrier gas flow rate: initial flow rate 2.0 mL·min -1 Keep for 2 min, linearly increase to 10.0 mL·min within 8 min -1 and linearly increase to 100.0 mL·min within 10 min -1 and keep for 20 min. The injection port temperature is 80 °C. The IMS migration tube temperature is 45 °C; the drift gas flow rate is 75.0 mL·min -1 .

[0040] 2.3 Data processing

[0041] The retention indices of volatile compounds were calculated and used for qualitative analysis of the volatile components in Astragalus membranaceus and stir-fried Astragalus membranaceus with rice by comparing with the GC retention index NIST database and migration time IMS database built into the VOCal software. The Reporter plug-in in the VOCal data processing software was used to generate three-dimensional spectra, two-dimensional spectra, and differential spectra of volatile components respectively. The Gallery Plot plug-in was used to generate their fingerprint spectra, and the Dynamic PCA plug-in was used to generate principal component analysis diagrams for the comparison of volatile organic compounds between samples.

[0042] Principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and VIP analysis were performed using SIMCA-14.0 software to screen for odor-differentiating components in different processed Astragalus membranaceus, and a cluster heat map analysis of differential volatile compounds was carried out using the microsignal platform http: / / www.bioinformatics.com.cn / .

[0043] 3 Results and Analysis

[0044] 3.1 GC-IMS Spectral Analysis of Different Processed Products of Astragalus membranaceus

[0045] The Reporter plug-in of the VOCal software was used to directly compare the spectral differences between samples, and the characteristic volatile substances of different processed products of Astragalus membranaceus could be directly compared. The X-axis represents the ion migration time (normalized), the Y-axis represents the retention time of gas chromatography (s), and the Z-axis represents the signal peak intensity (V) (as Figure 1 ). It can be directly seen from the figure the differences in volatile organic compounds in different processed products of Astragalus membranaceus. For easy observation, a top view was taken for comparison.

[0046] In the direct comparison diagram of volatile components in different processed products of Astragalus membranaceus, the ordinate represents the retention time of gas chromatography (s), and the abscissa represents the ion migration time (normalized). The background of the whole figure is blue, and the red vertical line at 1.0 on the abscissa is the RIP peak (reaction ion peak, normalized). Each point on both sides of the RIP peak represents one kind of volatile substance. The color represents the concentration of the substance, white indicates a lower concentration, red indicates a higher concentration, and the darker the color, the greater the concentration ( Figure 2 ). It can be seen from the figure that there are differences in volatile substances before and after the processing of Astragalus membranaceus.

[0047] To further visually compare the differences in volatile components between different processed products of Astragalus membranaceus, the spectrum of raw Astragalus membranaceus slices was selected as a reference, and the spectrum of stir-fried Astragalus membranaceus with rice was subtracted from the reference to obtain a differential comparison diagram of different processed products of Astragalus membranaceus, as Figure 3As shown. If the volatile organic matter content of the two is the same, the background after deduction is white, while red represents that the concentration of the substance is higher in the target sample than in the reference, and blue represents that the concentration of the substance is lower than the reference ( Figure 3 ). Through the difference comparison chart, it can be more clearly seen that there are differences in the volatile substances in Astragalus membranaceus before and after processing, and there are also certain similarities in the volatile substances contained in Astragalus membranaceus processed by the two processes.

[0048] 3.2 Comparative analysis of the fingerprint spectra of volatile components of different processed products of Astragalus membranaceus

[0049] In order to clarify which specific substances in different processed products of Astragalus membranaceus are different, the GalleryPlot plug-in of VOCal software was used to select 2 different processed products of Astragalus membranaceus, and each sample was repeated 3 times. The fingerprint spectra were automatically generated for all the peaks to be analyzed in the obtained GC-IMS two-dimensional spectra. The results are shown in Figure 4 . Each row in the figure represents all the signal peaks selected in one Astragalus membranaceus sample, and each column in the figure represents the signal peaks of the same volatile substance in different Astragalus membranaceus samples. It can be seen from the figure the complete volatile substance information of each Astragalus membranaceus sample and the differences in the volatile substances contained in different Astragalus membranaceus samples.

[0050] Through preliminary comparison Figure 4 of the fingerprint spectra, it can be seen that different processed products of Astragalus membranaceus contain both common regions and their respective characteristic peak regions of volatile substances. Regions A and B in the figure show the volatile components with higher contents in Astragalus membranaceus, and regions C, D, and E show the parts with higher contents of volatile components in stir-fried Astragalus membranaceus with rice.

[0051] The results of the fingerprint spectra show that the contents of 45 substances such as butyric acid, propionic acid, borneol, α-terpineol, linalool, 1-octen-3-ol, 1-hexanol, Z-2-penten-1-ol, 1-pentanol, 3-methyl-1-butanol, 1-penten-3-ol, 1-butanol, 2-pentanol, 2-methyl-1-propanol, 1-propanol, ethanol, E-2-nonenal, E-2-heptenal, E-2-hexenal, 3-methyl-2-butenal, E-2-pentenal, acetal, octanal, hexanal, pentanal, butanal, propanal, 2-decanone, 6-methyl-5-hepten-2-one, 2-octanone, 1-penten-3-one, hexyl propionate, hexyl acetate, ethyl hexanoate, hexyl hexanoate, amyl acetate, butyl acetate, propyl acetate, ethyl acetate, methyl acetate, 2-pentylfuran, 1,8-cineole, p-xylene, diallyl sulfide, dimethyl disulfide, etc. in Astragalus membranaceus are relatively high.

[0052] The contents of 36 substances such as 2-methylpropanoic acid, acetic acid, benzaldehyde, citronellal, (E,E)-2,4-heptadienal, 2,4-hexadienal, Z-4-heptenal, nonanal, heptanal, 3-methylbutanal, 2-propenal, 2-methylpropanal, 2-furfuryl mercaptan, 2-heptanol, 3-methyl-1-pentanol, 5-methyl-2(3H)-furanone, 1-hydroxy-2-propanone, 3-hydroxy-2-butanone, 2-heptanone, 3-penten-2-one, 2-pentanone, 2-butanone, acetone, γ-butyrolactone, 2-acetyl-5-methylfuran, 2-acetyl-2-thiazoline, 2-acetylthiazole, 4,5-dimethylthiazole, 2,5-dimethylpyrazine, 2-methylpyrazine, terpinolene, limonene, 3-carene, hexenenitrile, dimethyl trisulfide, dimethyl sulfide, etc. are relatively high in stir-fried Astragalus membranaceus with rice.

[0053] 3.3 Qualitative analysis of the volatile component spectra in the samples

[0054] A total of 117 volatile components were obtained by gas-phase ion mobility spectrometry analysis, and 81 volatile components were identified. However, due to limitations in the NIST and IMS databases, only 100 components were identified, and 17 components remain to be confirmed. These include 12 classes of compounds such as esters, alcohols, ketones, aldehydes, terpenoids, pyrazines, sulfides, furans, thiazoles, organic acids, benzenes, etc. Some compounds exist as monomers (M), dimers (D), and trimers (T), as shown in Table 1. A total of 100 volatile organic compounds were detected in Astragalus membranaceus samples with different processing degrees, among which alcohols accounted for 25%; aldehydes accounted for 23%; ketones accounted for 15%; esters accounted for 12%; organic acids accounted for 6%; sulfides accounted for 5%; nitriles accounted for 2%; furans accounted for 2%; pyrazines accounted for 2%; benzenes accounted for 1%; thiazoles accounted for 1%. The results show that alcohols, aldehydes, ketones, and ester compounds are the main volatile components during the processing of Astragalus membranaceus.

[0055] Table 1 Volatile components in Astragalus membranaceus and its processed products

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Note: M - monomer; D - dimer; T - trimer; * - unclassified. M - monomer; D - dimer; T - trimer; * - unclassified. 3.4 Principal Component Analysis

[0064] PCA (Principal Component Analysis) is a commonly used statistical method for reducing the dimensionality of high - dimensional data through linear transformation while retaining the main variation patterns in the data. Now, the data of raw and processed Astragalus membranaceus are subjected to principal component analysis, as shown in Figure 5 . The contribution rates of PC1 and PC2 are 92.7% and 3.9% respectively. The cumulative contribution rate is 96.6%, indicating that PC1 and PC2 can better reflect the information of the main compounds in Astragalus membranaceus and its processed products, with good reliability. The aggregation degree among samples is relatively high, and the samples of Astragalus membranaceus with different processing methods are clustered into one category respectively, indicating that there are significant differences in chemical components before and after processing of Astragalus membranaceus, which can be effectively distinguished, and different processing methods have different effects on Astragalus membranaceus.

[0065] 3.5 Orthogonal Partial Least Squares - Discriminant Analysis

[0066] OPLS - DA (Orthogonal Parital Least Squares Discriminant Analysis) is a statistical method for classification and difference analysis, which is widely used in fields such as metabolomics, bioinformatics, and food science. By visualizing the differences between samples of different groups and identifying the variables that contribute most to the differences between groups. In the present invention, the selected compounds are used as the dependent variables, and different processed products are used as the independent variables. Through the OPLS - DA diagram, effective differentiation of Astragalus membranaceus and its processed varieties can be achieved. The fitting index (R x 2 ) of the independent variable in this analysis is 0.963, the fitting index (R y 2 ) of the dependent variable is 1, and the model prediction index (Q 2 ) is 0.998. Samples of Astragalus membranaceus and stir - fried Astragalus membranaceus with rice are clustered in their respective regions and show obvious two categories (as shown in Figure 6 (A)), indicating that significant changes have occurred in the volatile components of Astragalus membranaceus before and after processing. After 200 permutation tests, a permutation test plot is obtained, as shown in Figure 6 (B), to verify the reliability and statistical significance of the model, where Q 2= -0.662, indicating that the model does not have overfitting and has statistical significance. Taking FC > 1.5, VIP > 1 and P < 0.05 as indicators to screen the differential components of Astragalus membranaceus and its processed products, a total of 32 differential volatile components were screened out. Among them, 24 known components are (E,E)-2,4-heptadienal, 3-hydroxy-2-propanone-D, 3-hydroxy-2-propanone-M, (E,E)-2,4-hexadienal, 5-methyl-2-acetylfuran, 2-butanone, furfuryl mercaptan, 2-heptanol, 2-heptanone-D, 2-methylpropanal, 2-methylpyrazine, 2-propenal, 3-carene, 3-hydroxy-2-butanone, 3-methyl-pentanol, isovaleraldehyde, 3-penten-2-one, 4,5-dimethylthiazole, angelica lactone, heptanal, hexanenitrile-D, limonene, nonanal, terpinolene.

[0067] 3.6 Cluster heat map analysis of Astragalus membranaceus and its processed products

[0068] To more intuitively distinguish Astragalus membranaceus and its processed products, using the 32 differential components screened out as variables, the cluster heat map analysis of Astragalus membranaceus and stir-fried Astragalus membranaceus with rice samples was carried out on the microsignal platform (http: / / www.bioinformatics.com.cn / ), and the results are shown in Figure 7 . Among them, the abscissa represents different samples, the ordinate represents volatile flavor components, red indicates a significant up-regulation of relative content, and blue indicates a significant down-regulation of relative content. It can be seen from the figure that the contents of 2-methylpropanal, isovaleraldehyde, and 5-methyl-2-acetylfuran compounds in the raw Astragalus membranaceus are significantly higher than those of other samples; the content of (E,E)-2,4-hexadienal compound in stir-fried Astragalus membranaceus with rice is significantly higher than that of other samples.

[0069] 4 Discussion

[0070] Odor is one of the most important quality markers of traditional Chinese medicine. Different traditional Chinese medicines have their unique odors, which come from the volatile components of traditional Chinese medicine. During the processing of traditional Chinese medicine, unique odor changes may occur, which is also an important indicator for judging the processing degree of decoction pieces. Chinese herbal medicines can only be used as medicine after being processed into decoction pieces. Under the action of heating and auxiliary materials, the components of Chinese medicine decoction pieces will change, resulting in different odors. Therefore, odor is one of the important indicators for identifying Chinese medicine decoction pieces and judging the processing degree of decoction pieces. At present, sensor-type electronic noses or ultra-fast gas-phase electronic noses have been widely used in the research on characterizing the odor changes of traditional Chinese medicine during processing. Through this technology, the volatile components in traditional Chinese medicine can be preliminarily qualitatively analyzed. Compared with them, GC-IMS can not only detect trace volatile components, but also obtain analysis results in a shorter time, and the two-dimensional spectra, differential spectra and fingerprint spectra results can intuitively show the differences in volatile components. Through the GC-IMS technology, qualitative and quantitative analysis of Chinese medicine decoction pieces and their processed products can be carried out quickly, so it has broad application prospects.

[0071] In this study, the GC-IMS technique was used for the first time to analyze the changes in the content of volatile components during the stir-frying process of Astragalus membranaceus. A total of 81 volatile components, 100 peaks, and 17 unknown substances were identified. The FC values (fold change) of the two were calculated. With FC > 1.5, P < 0.05, and VIP > 1 as the screening criteria, a total of 32 components were up-regulated in stir-fried Astragalus membranaceus with rice compared to Astragalus membranaceus. The up-regulated components were mainly aldehydes and ketones, with flavors such as nuts, coffee, melon, wine, green fragrance, fat, chocolate, rose, citrus, lemon, woody fragrance, pear, butter, fruit, and herbs. By presenting the differential compounds in the form of a clustering heat map, it was found that the contents of 2-methylpropanal, isovaleraldehyde, and 5-methyl-2-acetylfuran were relatively high in raw Astragalus membranaceus, and the content of (E,E)-2,4-hexadienal was significantly up-regulated in stir-fried Astragalus membranaceus with rice. Through visualization means, the material connotation of the odor change in stir-fried Astragalus membranaceus with rice was intuitively presented, and the composition and change rules of volatile components during the stir-frying process of Astragalus membranaceus with rice were preliminarily revealed.

[0072] There are obvious differences in the chemical components of Astragalus membranaceus before and after stir-frying with rice. The effects of the type, dosage, and processing temperature of rice on the stir-frying process of Astragalus membranaceus with rice need to be further explored to establish the quality standard of the auxiliary material rice. This study provides data support for the research on the processing mechanism of Astragalus membranaceus and also provides a method reference for exploring the chemical component differences of other traditional Chinese medicines before and after processing.

[0073] The present invention uses the GC-IMS combination method to find a new method and idea for quickly judging different processed products of traditional Chinese medicine decoction pieces, and the results are accurate and reliable.

Claims

1. A method for identifying different processed products of Astragalus membranaceus by analyzing characteristic odor substances based on GC-IMS, which is characterized in that, The following steps are involved: (1) preparing different processed Astragalus as samples to be tested, wherein the different processed Astragalus is Astragalus or rice-fried Astragalus; (2) using GC-IMS analysis to obtain GC-IMS two-dimensional spectra corresponding to the samples to be tested; (3) performing three-dimensional spectrum conversion, direct comparison map conversion, difference comparison map conversion, fingerprint analysis and cluster analysis on the GC-IMS two-dimensional spectrum of step (2), and distinguishing different processed Astragalus membranaceus products based on the differences in volatile substances contained in the samples to be tested; (4) The volatile components of Astragalus and its processed products were obtained through intuitive analysis of cluster heat maps.

2. The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances according to claim 1, wherein, In the step (2), the determination method of the GC-IMS spectrum is as follows: Weigh 0.5 g of the astragalus or stir-fried astragalus with rice samples respectively and place them in a 20 mL headspace bottle. Incubate at 80 °C and 500 r·min -1 for 15 min, then inject 200 μL. The injection needle temperature is 85 °C, and three parallel determinations are made for each sample. GC-IMS separation conditions: The column temperature is 60 °C, the running time is 40 min, the carrier gas / drift gas is N2, the purity is ≥99.999%, and the carrier gas flow rate: the initial flow rate is 2.0 mL·min -1 is maintained for 2 min and linearly increased to 10.0 mL·min within 8 min -1 and linearly increased to 100.0 mL·min within 10 min -1 and maintained for 20 min. The inlet temperature is 80 °C; the IMS migration tube temperature is 45 °C; the drift gas flow rate is 75.0 mL·min -1 .

3. The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances according to claim 1, wherein In the step (3), each peak on the GC-IMS two-dimensional spectrum is qualitatively characterized by using the NIST database and the IMS database to determine the volatile substance corresponding to each peak on the GC-IMS two-dimensional spectrum.

4. The method for identifying different processed products of Astragalus membranaceus based on the analysis of characteristic odor substances by GC-IMS according to claim 1, wherein In the step (3), the volatile substances in the sample to be tested are esters, alcohols, ketones, aldehydes, terpenes, pyrazines, sulfides, furans, thiazoles, organic acids, and benzene compounds.

5. The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances according to claim 4, wherein, The volatile substances in the step (3) exist in two forms: monomer and dimer. The volatile substances are methyl acetate, propionic acid, acetic acid, 3-hydroxy-2-propanone, 2-heptanone, 1,8-cineole, (E)-2-vinylaldehyde, hexenal, diallyl sulfide, capronitrile, n-hexanol, n-pentanol, isopentanol, n-butanol, sec-pentanol, isobutanol, and n-propanol.

6. The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances according to claim 1, characterized in that, In the step (3), the fingerprint analysis is to automatically generate a fingerprint spectrum for all the peaks to be analyzed in the GC-IMS two-dimensional spectrum, and the complete volatile substance information of different processed Astragalus membranaceus products and the differences in volatile substances contained in different processed Astragalus membranaceus products can be seen from the fingerprint spectrum. In the step (4), the cluster heat map analysis is to judge the differences in volatile substances contained in different processed products through the depth of color between different volatile components with significant differences in the same group.

7. The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances according to claim 6, wherein The cluster analysis includes dynamic principal component analysis.

8. The method for identifying different processed products of Astragalus membranaceus based on the analysis of characteristic odor substances by GC-IMS according to claim 1, wherein, The steps (2) and (3) are both completed by VOCal software, wherein the principal component analysis in step (3) is completed by SIMCA-14 software, and step (4) is completed by differential compound analysis using the microbial information platform http: / / www.bioinformatics.com.cn / .

9. The method for identifying different processed products of Astragalus membranaceus based on analyzing characteristic odor substances by GC-IMS according to claim 1, wherein The different processed astragalus products are raw astragalus or processed astragalus. Among astragalus and its processed products, astragalus is raw astragalus, and the processed astragalus is rice-fried astragalus. The preparation method of rice-fried astragalus is: take raw astragalus, that is, clean astragalus slices, put rice in a preheated frying pan, set the temperature to 240°C, put in rice and fry for 7 minutes, fry the rice until it is slightly yellow, then put in clean astragalus, fry the rice and medicine together, fry for 10 minutes until the rice turns yellow, the astragalus slices turn dark in color and have an aroma, take out, sieve out the rice, spread to cool, and the product is ready.

10. The method for identifying different processed products of Astragalus membranaceus based on GC-IMS analysis of characteristic odor substances according to claim 1, wherein The contents of methylpropionaldehyde, isovaleraldehyde and 5-methyl-2-acetylfuran compounds in raw Astragalus were significantly higher than those in stir-fried Astragalus with rice. The contents of (E,E)-2,4-hexadienal compounds in stir-fried Astragalus with rice were significantly higher than those in raw Astragalus with rice.

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