Method for analyzing types and differences of volatile components of herba schizonepetae and processed products based on GC-IMS technology

Abstract

According to the present invention, the gas chromatography-ion mobility spectrometry (GC-IMS) technology is adopted to analyze the types and the differences of the volatile components of the herba schizonepetae and the processed product so as to provide reference for the characterization and the accurate identification of the volatile components of the herba schizonepetae and the processed product, and the GC-IMS is adopted to analyze and identify the volatile components in the herba schizonepetae, the fried herba schizonepetae and the herba schizonepetae charcoal, the difference of volatile components of the herba schizonepetae, the fried herba schizonepetae and the herba schizonepetae charcoal is analyzed in combination with chemometrics, the GC-IMS technology and the chemometrics analysis method are researched and utilized, the different volatile organic compounds of the herba schizonepetae, the fried herba schizonepetae and the herba schizonepetae charcoal are screened out, and an idea is

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and identification of Chinese herbal medicine pieces, and relates to a method for analyzing the types and differences of volatile components of Schizonepeta and processed products based on GC-IMS technology. Background Art

[0002] Nepeta is the dried aerial part of the Lamiaceae plant Schizonepeta tenuifolia Briq. It is a commonly used Chinese medicine in the past dynasties. It was first recorded in the Shennong Bencao Jing as "Jia Su" and is a common bulk medicinal material in my country. Various processing methods have been established according to different diseases in the past dynasties, including more than 10 processing methods such as cleaning, roasting and frying. Among the fried products, there are fried Nepeta and Nepeta charcoal. Raw Nepeta is often used to dispel wind and relieve heat. "Depei Materia Medica" records that "it is used raw to dispel wind"; fried Nepeta has a weakened pungent and dispersing effect, and focuses on dispelling wind and regulating blood; Nepeta charcoal has a weakened pungent and dispersing effect, and is good at stopping bleeding. "Yi Zong Shuo Yue" records that "it stops bleeding and is fried and used when it is black". Fried Nepeta and Nepeta charcoal are recorded in the processing specifications of many provinces. The 2020 edition of the Chinese Pharmacopoeia only includes Nepeta charcoal in the fried products, and fried Nepeta is mostly a special processing variety for clinical prescriptions.

[0003] Stir-frying can change the medicinal properties and increase the variety of clinical use of traditional Chinese medicine. Volatile oil is the main component of Schizonepeta, containing a variety of active ingredients. During the frying process of Schizonepeta, due to the influence of frying temperature, the type and content of volatile substances will change. At present, the research on the volatile components of Schizonepeta mainly focuses on the research of raw products and different medicinal parts of Schizonepeta, and the research on fried Schizonepeta is less. Therefore, the present invention uses GC-IMS technology to detect volatile organic compounds in Schizonepeta raw products and fried products, combines chemometric methods to find differential substances, compares the differences in volatile specific odor components in Schizonepeta, fried Schizonepeta, and Schizonepeta charcoal, to reveal the influence of processing on the volatile components of Schizonepeta, and provides a basis for the specific aroma substances and clinical application of Schizonepeta and processed products. Summary of the invention

[0004] The object of the present invention is to provide a method for analyzing the types and differences of volatile components of Nepeta and processed products based on gas chromatography-ion mobility spectrometry GC-IMS technology, characterized in that the steps are as follows:

[0005] (1) Preparation of materials and samples

[0006] Materials and Reagents 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone (all analytical grade, Aladdin); 2-methyl-3-heptanone (chromatographic grade, Aladdin);

[0007] Prepare raw Schizonepeta, fried Schizonepeta and Schizonepeta charcoal respectively.

[0008] (2) Headspace injection

[0009] Weigh 1 g of the sample separately and place it in a 20 mL headspace vial. Add 10 μL of 2-methyl-3-heptanone at 100 ppm as the internal standard. Incubate at 80 °C for 20 min and then inject the sample. Measure 3 parallel groups for each sample. Incubation temperature: 80 °C; Incubation time: 20 min; Injection volume: 500 μL; Splitless injection; Incubation rotation speed: 500 r·min -1 ; Injection needle temperature: 85 °C;

[0010] (3) Data processing

[0011] Detect a mixed standard of 6 ketones including 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone, establish a calibration curve for retention time and retention index. Subsequently, calculate the retention index of the substance through the retention time of the target compound. Use the built-in GC retention index (NIST2020) database and IMS migration time database in VOCal software to retrieve and compare, and conduct qualitative analysis on the target compound;

[0012] Use plugins such as Reporter, Gallery Plot and Nearest Neighbour in VOCal data processing software to generate three-dimensional spectra, two-dimensional spectra, difference spectra, fingerprint spectra and nearest neighbour analysis diagrams of volatile components respectively, for the comparison of volatile organic compounds between samples. Conduct PCA, OPLS-DA and cluster heat map analysis based on OriginPro2025 software, SIMCA14.1 and the microbiome informatics platform http: / / www.bioinformatics.com.cn / ;

[0013] (4) Gas-phase ion mobility spectrometry analysis

[0014] Based on GC-IMS, analyze the differences in volatile components in Schizonepeta tenuifolia Briq., stir-fried Schizonepeta tenuifolia Briq. and charred Schizonepeta tenuifolia Briq. The three-dimensional spectrum of GC-IMS, the three coordinate axes respectively represent the migration time (X-axis), retention time (Y-axis) and signal peak intensity (Z-axis). It can be directly seen from the figure that the volatile components of the processed products of Schizonepeta tenuifolia Briq. are similar, but the peak intensities are different; after normalizing the ion migration time and the position of the reaction ion peak (RIP), the gas-phase ion mobility spectrometry diagrams of Schizonepeta tenuifolia Briq. and its processed products are obtained. Each point on both sides of the RIP peak represents a volatile organic compound. The ordinate represents the retention time of gas chromatography, the abscissa represents the relative migration time, and the color represents the peak intensity of the substance. From blue to red, the darker the color, the greater the peak intensity. The results show that most signals appear within the retention time of 200 - 2000 s and the drift time of 1.0 - 2.0 ms. Select the spectrum of the sample Schizonepeta tenuifolia Briq. as the reference, and subtract the reference from the spectra of other samples to obtain the difference comparison diagrams of different samples. Compared with the raw product of Schizonepeta tenuifolia Briq., the contents of volatile organic compounds in stir-fried Schizonepeta tenuifolia Briq. and charred Schizonepeta tenuifolia Briq. are either increased or decreased;

[0015] (5) Qualitative identification and analysis

[0016] A total of 210 volatile components were obtained by gas-phase ion mobility spectrometry analysis, including 13 categories of compounds such as esters, alcohols, ketones, aldehydes, terpenes, pyrazines, sulfides, pyridines, furans, organic acids, benzenes, amines, and ethers. Some compounds have monomers M, dimers D, and trimers T. The volatile organic compounds detected in Nepeta samples with different processing degrees include: 33 aldehydes, accounting for 19.41%; 32 alcohols, accounting for 18.82%; 27 ketones, accounting for 15.88%; 21 esters, accounting for 12.35%; 18 terpenes, accounting for 10.59%; 12 pyrazines, accounting for 7.06%; 7 benzenes, accounting for 4.12%; 7 sulfides, accounting for 4.12%; 6 furans, accounting for 3.53%; and 7 others, accounting for 4.12%. Aldehydes, alcohols, and ketones are the main volatile components during the processing of Nepeta.

[0017] (6) GC-IMS fingerprint of Nepeta and its stir-fried products

[0018] Identify the characteristic peak regions of Nepeta and its stir-fried products through fingerprinting;

[0019] (7) Principal component analysis and nearest neighbor analysis

[0020] Perform principal component analysis and nearest neighbor analysis on the data of raw Nepeta and its stir-fried products;

[0021] (8) Orthogonal partial least squares-discriminant analysis

[0022] Taking the selected compounds as the dependent variables and different stir-fried products as the independent variables, through the OPLS-DA diagram, the effective differentiation of Nepeta and its stir-fried varieties was achieved. A total of 27 differential volatile components were screened out, namely (E)-2-hexenal D, 1-octen-3-ol D, 2,3,5-trimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-octanol D, 3-octanone D, acetone, β-phellandrene M, β-pinene D, β-pinene M, butyl hexanoate, camphor D, camphor M, carvone M, citronellal D, citronellal M, ethyl 3-hydroxyhexanoate D, isopulegone D, longifolene, limonene D, limonene M, myrcene T, neryl acetate, acetic acid D, acetic acid M, isopulegone M, and methyl acetate;

[0023] (9) Cluster heat map analysis of Nepeta and its stir-fried products

[0024] To more clearly distinguish Schizonepeta tenuifolia Briq. and its processed products, taking the 27 screened differential components as variables, a clustering heatmap analysis was performed on Schizonepeta tenuifolia Briq., stir-fried Schizonepeta tenuifolia Briq., and carbonized Schizonepeta tenuifolia Briq. samples using the microbial informatics platform http: / / www.bioinformatics.com.cn / . In the raw Schizonepeta tenuifolia Briq., the contents of (E)-2-hexenal D, 1-octen-3-ol D, 2,3,5-trimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-octanol D, 3-octanone D, acetone, β-phellandrene M, β-pinene D, β-pinene M, butyl hexanoate, camphor D, camphor M, carvone M, citronellal D, citronellal M, ethyl 3-hydroxyhexanoate D, isopulegone D, longifolene, limonene D, limonene M, myrcene T, and neryl acetate compounds were significantly higher than those in other samples; in stir-fried Schizonepeta tenuifolia Briq., the content of acetic acid D compound was significantly higher than that in other samples; in carbonized Schizonepeta tenuifolia Briq., the contents of acetic acid M, isopulegone M, and methyl acetate compounds were the highest among all samples.

[0025] The method for analyzing the types and differences of volatile components in Schizonepeta tenuifolia Briq. and its processed products based on GC-IMS technology. The preparation methods of the three samples of raw Schizonepeta tenuifolia Briq., stir-fried Schizonepeta tenuifolia Briq., and carbonized Schizonepeta tenuifolia Briq. are as follows:

[0026] (1) Preparation of raw Schizonepeta tenuifolia Briq.: The raw Schizonepeta tenuifolia Briq. decoction pieces JJ are the dried aerial parts of the plant Schizonepeta tenuifolia Briq. of the Lamiaceae family.

[0027] (2) Preparation of stir-fried Schizonepeta tenuifolia Briq.: Referring to the "Jiangsu Province Traditional Chinese Medicine Decoction Pieces Processing Specification" (the first volume, 2020 edition), take an appropriate amount of raw Schizonepeta tenuifolia Briq. and place it in a preheated frying pan at 100 °C. Stir-fry the raw Schizonepeta tenuifolia Briq. for 12 minutes until the surface is yellowish-brown, then take it out and let it cool to obtain stir-fried Schizonepeta tenuifolia Briq. CJJ.

[0028] (3) Preparation of carbonized Schizonepeta tenuifolia Briq.: Referring to the "Pharmacopoeia of the People's Republic of China" (2020 edition), take an appropriate amount of cleaned Schizonepeta tenuifolia Briq. and place it in a preheated frying pan at 210 °C. Stir-fry the raw Schizonepeta tenuifolia Briq. for 7 minutes until the surface is dark brown, then take it out and let it cool to obtain carbonized Schizonepeta tenuifolia Briq. JJC.

[0029] The method for analyzing the types and differences of volatile components in Schizonepeta tenuifolia and its processed products based on GC-IMS technology is characterized in that the contents of (E)-2-hexenal D, 1-octen-3-ol D, 2,3,5-trimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-octanol D, 3-octanone D, acetone, β-phellandrene M, β-pinene D, β-pinene M, butyl hexanoate, camphor D, camphor M, carvone M, citronellal D, citronellal M, ethyl 3-hydroxyhexanoate D, isopulegone D, longifolene, limonene D, limonene M, myrcene T and neryl acetate compounds in raw Schizonepeta tenuifolia are significantly higher than those in stir-fried Schizonepeta tenuifolia and carbonized Schizonepeta tenuifolia; the content of acetic acid D compound in stir-fried Schizonepeta tenuifolia is significantly higher than that in raw Schizonepeta tenuifolia and carbonized Schizonepeta tenuifolia; the contents of acetic acid M, isopulegone M and methyl acetate compounds in carbonized Schizonepeta tenuifolia are the highest among the three samples of raw Schizonepeta tenuifolia, stir-fried Schizonepeta tenuifolia and carbonized Schizonepeta tenuifolia.

[0030] After processing Schizonepeta tenuifolia, its color, smell and other external characters and medicinal properties change. Chemical components are the basis for exerting medicinal effects, and the change of chemical components will also affect the clinical efficacy before and after processing Schizonepeta tenuifolia. Through GC-IMS technology, the volatile components of Schizonepeta tenuifolia and its processed products can be quickly and conveniently detected in this experiment, which intuitively shows the differences in volatile components among the three. The differences in volatile components of Schizonepeta tenuifolia and its processed products are analyzed and compared by using multivariate statistical analysis methods, and the volatile organic compounds with significant differences are preliminarily determined, which not only provides data for the odor differences of Schizonepeta tenuifolia and its processed products, but also provides reference for the clinical application of Schizonepeta tenuifolia, stir-fried Schizonepeta tenuifolia and carbonized Schizonepeta tenuifolia. Description of the Drawings

[0031] Figure 1 Three-dimensional GC-IMS spectra of volatile components in Schizonepeta tenuifolia and its processed products

[0032] Figure 2 Gas-phase ion mobility diagrams of Schizonepeta tenuifolia and its processed products

[0033] Figure 3 Difference diagram of gas chromatography-ion mobility spectrometry of Schizonepeta tenuifolia and its processed products

[0034] Figure 4 Comparison of fingerprint spectra of volatile components in Schizonepeta tenuifolia and its stir-fried products

[0035] Figure 5 Principal component analysis diagram of Schizonepeta tenuifolia and its processed products

[0036] Figure 6 Nearest neighbor analysis diagram of Schizonepeta tenuifolia and its processed products

[0037] Figure 7 OPLS-DA (A) and model cross-validation results (B) of Schizonepeta tenuifolia and its processed products

[0038] Figure 8 Cluster heat map of volatile components of Schizonepeta tenuifolia and its processed products Detailed Implementation Modes

[0039] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, but these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0040] Embodiment 1:

[0041] 1 Materials and instruments

[0042] 1.1 Materials and reagents

[0043] 2-Butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone (all analytical grade, Aladdin); 2-methyl-3-heptanone (chromatographic grade, Aladdin).

[0044] 1.2 Equipment and Instruments

[0045] Gas phase ion mobility spectrometer (GAS); CTC-PAL3 static headspace automatic sampler (CTC Analytics AG); 20 mL headspace bottle (Shandong Haineng Scientific Instrument Co., Ltd.); BCE224-1CCN electronic balance (Sartorius Scientific Instrument Co., Ltd.), MXT-WAX (30m×0.53mm, 1.0μm) capillary chromatographic column (Restek, USA).

[0046] 2 Experimental methods

[0047] 2.1 Sample preparation

[0048] 2.1.1 Preparation of raw Nepeta

[0049] Schizonepeta tenuifolia Briq. was purchased from Anguo, Hebei Province, and was identified by Zhu Yufeng, chief Chinese medicine pharmacist of the Affiliated Hospital of Nanjing University of Chinese Medicine, as the dried aerial part of Schizonepeta tenuifolia Briq., a plant of the Lamiaceae family.

[0050] 2.1.2 Preparation of Stir-fried Nepeta

[0051] Referring to the first volume of "Jiangsu Province Traditional Chinese Medicine Preparation Specifications" (2020 edition), take an appropriate amount of raw Schizonepeta, place it in a preheated wok (100°C), and stir-fry the raw Schizonepeta until the surface is yellow-brown (about 12 minutes). Take it out and let it cool to obtain fried Schizonepeta (CJJ), which is homemade in the laboratory.

[0052] 2.1.3 Preparation of Nepeta charcoal

[0053] Refer to the Pharmacopoeia of the People's Republic of China (2020 Edition). Take an appropriate amount of crude Schizonepeta tenuifolia, place it in a preheated frying pan (210 °C), and fry the crude Schizonepeta tenuifolia until the surface is dark brown (about 7 minutes). Take it out and let it cool to obtain carbonized Schizonepeta tenuifolia (JJC), which is self-made in the laboratory.

[0054] 2.2 Headspace injection conditions

[0055] Weigh 1 g of the sample and place it in a 20 mL headspace vial. Add 10 μL of 100 ppm 2-methyl-3-heptanone as the internal standard. Incubate at 80 °C for 20 minutes and then inject the sample. Each sample is measured in 3 parallel groups. Incubation temperature: 80 °C; Incubation time: 20 minutes; Injection volume: 500 μL; Splitless injection; Incubation rotation speed: 500 r·min -1 ; Injection needle temperature: 85 °C.

[0056] 2.3 GC conditions:

[0057] Column temperature: 60 °C; Carrier gas: High-purity nitrogen (purity ≥ 99.999%); Programmed pressure rise: Initial flow rate 2.0 mL·min -1 Maintain for 2 minutes and linearly increase to 10.0 mL·min within 8 minutes -1 and linearly increase to 100.0 mL·min within 10 minutes -1 and maintain for 40 minutes. Chromatographic run time: 60 minutes; Injection port temperature: 80 °C.

[0058] 2.4 IMS conditions

[0059] Ionization source: Tritium source (3H); Drift tube length: 53 mm; Electric field strength: 500 V / cm; Drift tube temperature: 45 °C; Drift gas: High-purity nitrogen (purity ≥ 99.999%); Flow rate: 75.0 mL·min -1 ; Positive ion mode.

[0060] 2.5 Data processing

[0061] Detect a mixed standard of 6 ketones including 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone, establish a calibration curve for retention time and retention index, and then calculate the retention index of the substance through the retention time of the target. Use the GC retention index (NIST2020) database and IMS migration time database built into the VOCal software for retrieval and comparison to qualitatively analyze the target.

[0062] Using plugins such as Reporter, Gallery Plot, and Nearest Neighbour in VOCal data processing software, three-dimensional spectra, two-dimensional spectra, differential spectra, fingerprint spectra, and nearest neighbour analysis diagrams of volatile components were generated respectively for the comparison of volatile organic compounds between samples. PCA, OPLS-DA, and clustering heatmap analysis were carried out based on OriginPro2025 software, SIMCA14.1, and the Microbial Bioinformatics Platform (http: / / www.bioinformatics.com.cn / ).

[0063] 3 Results and Analysis

[0064] 3.1 Gas-phase Ion Mobility Spectrometry Analysis

[0065] Based on GC-IMS analysis of the differences in volatile components in Schizonepeta tenuifolia Briq., stir-fried Schizonepeta tenuifolia Briq., and carbonized Schizonepeta tenuifolia Briq., the results are shown in Figure 1 , that is, the three-dimensional spectrum of GC-IMS. The three coordinate axes represent migration time (X-axis), retention time (Y-axis), and signal peak intensity (Z-axis) respectively. It can be intuitively seen from the figure that the volatile components of the processed products of Schizonepeta tenuifolia Briq. are similar, but the peak intensities are different.

[0066] After normalizing the ion migration time and the position of the reaction ion peak (RIP), the gas-phase ion mobility spectra of Schizonepeta tenuifolia Briq. and its processed products were obtained, as shown in Figure 2 . Each point on both sides of the RIP peak represents a volatile organic compound. The ordinate represents the retention time of gas chromatography, and the abscissa represents the relative migration time. The color represents the peak intensity of the substance. From blue to red, the darker the color, the greater the peak intensity. It was found that most of the signals appeared within a retention time of 200 - 2000 s and a drift time of 1.0 - 2.0 ms. To further visually compare the differences in volatile components in the samples, the spectrum of sample JJ1 was selected as the reference, and the spectra of other samples were subtracted from the reference to obtain the differential comparison diagrams of different samples, as shown in Figure 3 . If the content of volatile organic compounds in the target sample and the reference is the same, the background after subtraction is white. Red represents that the concentration of this substance is higher in stir-fried Schizonepeta tenuifolia Briq. and carbonized Schizonepeta tenuifolia Briq. than in Schizonepeta tenuifolia Briq., and blue represents that the concentration of this substance is lower in stir-fried Schizonepeta tenuifolia Briq. and carbonized Schizonepeta tenuifolia Briq. than in Schizonepeta tenuifolia Briq. Compared with the raw product of Schizonepeta tenuifolia Briq., the content of volatile organic compounds in stir-fried Schizonepeta tenuifolia Briq. and carbonized Schizonepeta tenuifolia Briq. has increased or decreased.

[0067] 3.2 Qualitative Identification Analysis

[0068] A total of 210 volatile components were obtained by gas-phase ion mobility spectrometry analysis. However, only 170 components were identified due to limitations in the NIST and IMS databases, and 40 components remained to be confirmed. These included 13 types of compounds such as esters, alcohols, ketones, aldehydes, terpenes, pyrazines, sulfides, pyridines, furans, organic acids, benzenes, amines, and ethers. Some compounds existed as monomers (M), dimers (D), and trimers (T), as shown in Table 1. The volatile organic compounds detected in Schizonepeta tenuifolia samples with different processing degrees included: 33 aldehydes, accounting for 19.41%; 32 alcohols, accounting for 18.82%; 27 ketones, accounting for 15.88%; 21 esters, accounting for 12.35%; 18 terpenes, accounting for 10.59%; 12 pyrazines, accounting for 7.06%; 7 benzenes, accounting for 4.12%; 7 sulfides, accounting for 4.12%; 6 furans, accounting for 3.53%; and 7 others, accounting for 4.12%. This indicates that aldehydes, alcohols, and ketones are the main volatile components during the processing of Schizonepeta tenuifolia.

[0069] Table 1 Identification of volatile compounds in raw and processing Schizonepetae Herba

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] M - monomer; D - dimer; T - trimer; * - unclassified.

[0079] M - monomer; D - dimer; T - trimer; * - unclassified.

[0080] 3.3 GC-IMS Fingerprint of Schizonepeta tenuifolia and Its Stir-Fried Products

[0081] To further compare the differences in volatile flavor components between Schizonepeta tenuifolia Briq. and its stir-fried products, the characteristic peak regions of Schizonepeta tenuifolia Briq. and its stir-fried products were identified by fingerprint. Figure 4 Each column in Figure 4 represents the signal peaks of the same volatile organic compound in Schizonepeta tenuifolia Briq., stir-fried Schizonepeta tenuifolia Briq. and charred Schizonepeta tenuifolia Briq. Each row represents the sample name, and the digital number represents the unknown compound. Schizonepeta tenuifolia Briq. and its stir-fried products have their own characteristic peak regions as well as the same regions. The organic compounds in the red frame have a higher content in Schizonepeta tenuifolia Briq., and the content of the substances gradually decreases with the increase of the stir-frying degree; the organic compounds in the yellow frame have a higher content in stir-fried Schizonepeta tenuifolia Briq.; the organic compounds in the green frame have a higher content in charred Schizonepeta tenuifolia Briq. To sum up, the processing method can affect the content of volatile components in Schizonepeta tenuifolia Briq.

[0082] 3.4 Principal component analysis and nearest neighbor analysis

[0083] The data of raw Schizonepeta tenuifolia Briq. and its stir-fried products were subjected to principal component analysis and nearest neighbor analysis, as shown in Figure 5 Figure 6 . The contribution rates of PC1 and PC2 are 70.7% and 23.7% respectively. The cumulative contribution rate is 94.4%, indicating that PC1 and PC2 can better reflect the overall compound information of Schizonepeta tenuifolia Briq. and its stir-fried products and have good reliability. The aggregation degree among samples is relatively high. The Schizonepeta tenuifolia Briq. samples with different processing methods are clustered into one category respectively, indicating that there are significant differences in chemical components before and after the processing of Schizonepeta tenuifolia Briq., which can be effectively distinguished, and different processing methods have different effects on Schizonepeta tenuifolia Briq. The distance between stir-fried Schizonepeta tenuifolia Briq. and charred Schizonepeta tenuifolia Briq. is relatively close, indicating that the differences in volatile components of the Schizonepeta tenuifolia Briq. processed products obtained by the two processing methods are relatively small.

[0084] 3.5 Orthogonal partial least squares-discriminant analysis

[0085] Taking the selected compounds as the dependent variables and different stir-fried products as the independent variables, through the OPLS-DA diagram, the effective differentiation of Schizonepeta tenuifolia Briq. and its stir-fried varieties can be realized, as shown in Figure 7 . The fitting index (R 2 x ) of the independent variable in this analysis is 0.988, and the fitting index (R 2 y ) of the dependent variable is 1, and the model prediction index (Q 2 ) is 0.999. The samples of Schizonepeta tenuifolia Briq., stir-fried Schizonepeta tenuifolia Briq. and charred Schizonepeta tenuifolia Briq. are respectively aggregated in their respective regions and show obvious three categories, indicating that the volatile components have changed significantly before and after the processing of Schizonepeta tenuifolia Briq. After 200 times of permutation tests, as shown in the figure, the intercept of R 2 is 0.316, the intercept of Q 2 is -0.698, the R 2 and Q 2 on the leftThe values are all lower than those on the right side, indicating that the model does not have overfitting and has statistical significance. Taking VIP > 1 and P < 0.05 as indicators to screen the differential components of Schizonepeta tenuifolia Briq. and its stir-fried products, a total of 27 differential volatile components were screened out, namely (E)-2-hexenal D, 1-octen-3-ol D, 2,3,5-trimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-octanol D, 3-octanone D, acetone, β-phellandrene M, β-pinene D, β-pinene M, butyl hexanoate, camphor D, camphor M, carvone M, citronellal D, citronellal M, ethyl 3-hydroxyhexanoate D, isopulegone D, longifolene, limonene D, limonene M, myrcene T, neryl acetate, acetic acid D, acetic acid M, isopulegone M and methyl acetate.

[0086] 3.6 Cluster heat map analysis of Schizonepeta tenuifolia Briq. and its stir-fried products

[0087] To more clearly distinguish Schizonepeta tenuifolia Briq. and its processed products, taking the 27 differential components screened out as variables, the cluster heat map analysis of Schizonepeta tenuifolia Briq., stir-fried Schizonepeta tenuifolia Briq. and charred Schizonepeta tenuifolia Briq. samples was carried out using the microbioinformatics platform (http: / / www.bioinformatics.com.cn / ), and the results are shown in Figure 8 . 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 (E)-2-hexenal D, 1-octen-3-ol D, 2,3,5-trimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-octanol D, 3-octanone D, acetone, β-phellandrene M, β-pinene D, β-pinene M, butyl hexanoate, camphor D, camphor M, carvone M, citronellal D, citronellal M, ethyl 3-hydroxyhexanoate D, isopulegone D, longifolene, limonene D, limonene M, myrcene T and neryl acetate compounds in raw Schizonepeta tenuifolia Briq. are significantly higher than those in other samples; the content of acetic acid D compound in stir-fried Schizonepeta tenuifolia Briq. is significantly higher than that in other samples; the contents of acetic acid M, isopulegone M and methyl acetate compounds in charred Schizonepeta tenuifolia Briq. are the highest among all samples. In addition, the clustering relationship between stir-fried Schizonepeta tenuifolia Briq. and charred Schizonepeta tenuifolia Briq. is closer among the three groups of samples, clustering into one category, and raw Schizonepeta tenuifolia Briq. is clustered into a separate category, further indicating that stir-frying affects the volatile components of Schizonepeta tenuifolia Briq.

[0088] 4 Discussion

[0089] GC-IMS has high sensitivity and does not require enrichment and concentration. It has been applied to many fields such as food flavor, pharmaceuticals, and the environment, including specific aspects such as quality control during processing and food knowledge-based traceability analysis. Schizonepeta tenuifolia Briq. has a pungent taste, and its pungency should originate from volatile organic compounds. Volatile oil is the main component of Schizonepeta tenuifolia Briq., containing a variety of active ingredients. During the frying process of Schizonepeta tenuifolia Briq., the types and contents of volatile substances will change. Gong Min et al. analyzed and compared the volatile components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results showed that there were 12 common components between the two, and the content of esters decreased significantly after frying Schizonepeta tenuifolia Briq. After stir-frying Schizonepeta tenuifolia Briq. to charcoal, the volatile oil content decreased significantly, and the volatile components also changed. During the processing of Schizonepeta tenuifolia Briq., the original fragrant smell of the raw product gradually disappeared, and the color also changed accordingly, indicating that the frying temperature and frying time affected the component changes. Tian Tian et al. found that the fingerprint spectra of Schizonepeta tenuifolia Briq. and Schizonepetae Carbonis were quite different. After stir-frying Schizonepeta tenuifolia Briq. to charcoal, the types of components increased, and the contents of some substances decreased. In this study, GC-IMS technology was used to analyze and identify the flavor components in the raw and processed products of Schizonepeta tenuifolia Briq. A total of 210 flavor components were detected. According to qualitative analysis in the database, 170 flavor components were identified, including monomers and dimers and trimers of some substances. The GC-IMS flavor component fingerprint spectra showed that there were significant differences in the contents of volatile components among Schizonepeta tenuifolia Briq. and different processed varieties. The results of PCA and OPLS-DA showed that the raw, stir-fried, and carbonized products of Schizonepeta tenuifolia Briq. could be clustered into one category each, indicating that there were differences in their chemical compositions. The results of the nearest neighbor analysis showed that the chemical compositions of stir-fried Schizonepeta tenuifolia Briq. and Schizonepetae Carbonis were less different. The clustering heat map classified the three groups of samples into two categories, with the raw Schizonepeta tenuifolia Briq. clustering into one category alone, and stir-fried Schizonepeta tenuifolia Briq. and Schizonepetae Carbonis clustering into one category.

[0090] The frying temperature and frying time can affect the color and active ingredients of medicinal herb slices. If the frying temperature is too low, the frying time will be too long; if the frying temperature is too high, the frying degree will be difficult to control, and the content of active ingredients will be damaged and reduced. Aspects such as color change, component content change, and clinical efficacy will change with the change of frying methods. Color and components are regarded as being closely related to the quality of medicinal herbs, and the clinical efficacy of different fried products will also change. The raw product of Schizonepeta tenuifolia is light yellowish-green or light purplish-red, with a pungent taste, and is mainly used for antipyretic and analgesic effects clinically; fried Schizonepeta tenuifolia is yellowish-brown, and its pungent-dispersing effect is weakened, and it is mainly used for treating gynecological diseases clinically; charred Schizonepeta tenuifolia is dark brown, and its pungent-dispersing effect is also weakened, and it is mainly used for hemostasis clinically. In this study, the contents of volatile organic compounds in the raw product, fried product, and charred product of Schizonepeta tenuifolia changed, and different organic compound contents had obvious advantages in the raw product and its fried products of Schizonepeta tenuifolia. This study shows that the contents of ketones and alcohols in the raw product of Schizonepeta tenuifolia are relatively high. Alcohols have the aroma of flowers and fruits, and ketone substances have a strong smell. After frying Schizonepeta tenuifolia, the contents of alcohols and ketones decrease. It is speculated that the two may be the main material basis for the pungent taste of Schizonepeta tenuifolia. Components such as 1-octen-3-ol, limonene, and myrcene have pharmacological effects such as anti-inflammatory, antibacterial, and antiviral, and cooperate with other components to jointly play effects such as relieving exterior syndrome and dispelling wind. After frying Schizonepeta tenuifolia, the content of volatile oil decreases, its pungent-dispersing effect weakens, and the volatile organic compounds also change, which may be closely related to the clinical pharmacodynamic effects.

[0091] After processing Schizonepeta tenuifolia, its appearance traits such as color and smell and medicinal properties change. Chemical components are the basis for exerting pharmacological effects, and the change of chemical components will also affect the clinical efficacy before and after processing Schizonepeta tenuifolia. Through the GC-IMS technology in this experiment, the volatile components of Schizonepeta tenuifolia and its processed products can be detected quickly and conveniently, visually showing the differences in volatile components among the three. The multivariate statistical analysis method was used to analyze and compare the differences in volatile components of Schizonepeta tenuifolia and its processed products, and the volatile organic compounds with significant differences were initially determined, which not only provides data for the difference in smell of Schizonepeta tenuifolia and its processed products, but also provides a reference for the clinical application of Schizonepeta tenuifolia, fried Schizonepeta tenuifolia, and charred Schizonepeta tenuifolia.

[0092] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for analyzing the types and differences of volatile components in Schizonepeta tenuifolia and its processed products based on GC-IMS technology, characterized in that, Here are the steps: (1) Preparation of materials and samples Materials and reagents: 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone, all analytical grade, Aladdin; 2-methyl-3-heptanone, chromatographic grade, Aladdin; three samples of raw Nepeta, stir-fried Nepeta and Nepeta charcoal were prepared respectively; (2) Headspace injection Weigh 1 g of the sample separately and place it in a 20 mL headspace vial. Add 10 μL of 100 ppm 2-methyl-3-heptanone as the internal standard. After incubation at 80 °C for 20 min, inject the sample. Measure 3 parallel sets for each sample. Incubation temperature: 80 °C; Incubation time: 20 min; Injection volume: 500 μL; Splitless injection; Incubation rotation speed: 500 r·min -1 ; Injection needle temperature: 85 °C; (3) Data processing A mixed standard of six ketones, including 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone, was tested to establish a calibration curve of retention time and retention index. The retention index of the substance was then calculated based on the retention time of the target substance. The target substance was qualitatively analyzed by searching and comparing the built-in GC retention index NIST2020 database and IMS migration time database of the VOCal software. The Reporter, Gallery Plot and Nearest Neighbour plug-ins in the VOCal data processing software were used to generate three-dimensional spectra, two-dimensional spectra, difference spectra, fingerprints and nearest neighbor analysis diagrams of volatile components for comparison of volatile organic compounds between samples. PCA, OPLS-DA and cluster heat map analysis were performed based on OriginPro2025 software, SIMCA14.1 and the microbial information platform http: / / www.bioinformatics.com.cn / ; (4) Gas Phase Ion Mobility Spectrometry Based on GC-IMS analysis, the differences in volatile components of Nepeta, stir-fried Nepeta and Nepeta charcoal were analyzed. The three-dimensional spectrum of GC-IMS, with the three coordinate axes representing the migration time X-axis, retention time Y-axis and signal peak intensity Z-axis, shows that the volatile components of Nepeta processed products are similar, but the peak intensities are different. After normalizing the ion migration time and the RIP position of the reactive ion peak, the gas phase ion migration spectra of Nepeta and its processed products were obtained. Each point on both sides of the RIP peak represents a volatile organic compound. The ordinate represents the retention time of the gas chromatography, the abscissa represents the relative migration time, and the color represents the peak intensity of the substance. From blue to red, the darker the color, the greater the peak intensity. The results showed that most of the signals appeared in the retention time of 200-2000s and the drift time of 1.0-2.0ms. The spectrum of the Nepeta sample was selected as a reference, and the reference was deducted from the spectra of other samples to obtain the difference comparison diagram of different samples. Compared with the raw Nepeta, the content of volatile organic compounds in stir-fried Nepeta and Nepeta charcoal increased or decreased. (5) Qualitative identification analysis A total of 210 volatile components were obtained by gas-phase ion mobility spectrometry analysis, including 13 types of compounds such as esters, alcohols, ketones, aldehydes, terpenoids, pyrazines, sulfides, pyridines, furans, organic acids, benzenes, amines, and ethers. Some compounds have monomers M, dimers D, and trimers T. The volatile organic compounds detected in Nepeta cataria samples with different processing degrees include: 33 aldehydes, accounting for 19.41%; 32 alcohols, accounting for 18.82%; 27 ketones, accounting for 15.88%; 21 esters, accounting for 12.35%; 18 terpenoids, accounting for 10.59%; 12 pyrazines, accounting for 7.06%; 7 benzenes, accounting for 4.12%; 7 sulfides, accounting for 4.12%; 6 furans, accounting for 3.53%; and 7 other types, accounting for 4.12%. Aldehydes, alcohols, and ketone compounds are the main volatile components during the processing of Nepeta cataria. (6) GC-IMS fingerprint spectra of Nepeta cataria and its stir-fried products Identify the characteristic peak regions of Nepeta cataria and its stir-fried products through fingerprint spectra; (7) Principal component analysis and nearest neighbor analysis Perform principal component analysis and nearest neighbor analysis on the data of raw Nepeta cataria and its stir-fried products; (8) Orthogonal partial least squares-discriminant analysis Taking the selected compounds as the dependent variables and different stir-fried products as the independent variables, through the OPLS-DA diagram, the effective differentiation of Nepeta cataria and its stir-fried varieties is realized. A total of 27 differential volatile components are selected, namely (E)-2-hexenal D, 1-octen-3-ol D, 2,3,5-trimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-octanol D, 3-octanone D, acetone, β-phellandrene M, β-pinene D, β-pinene M, butyl hexanoate, camphor D, camphor M, carvone M, citronellal D, citronellal M, ethyl 3-hydroxyhexanoate D, isopulegone D, longifolene, limonene D, limonene M, myrcene T, neryl acetate, acetic acid D, acetic acid M, isopulegone M, and methyl acetate; (9) Cluster heat map analysis of Nepeta cataria and its stir-fried products To more clearly distinguish Nepeta cataria and its processed products, taking the 27 differential components selected as variables, the cluster heat map analysis of Nepeta cataria, stir-fried Nepeta cataria, and charred Nepeta cataria samples was carried out using the microsignal platform http: / / www.bioinformatics.com.cn / . The contents of (E)-2-hexenal D, 1-octen-3-ol D, 2,3,5-trimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-octanol D, 3-octanone D, acetone, β-phellandrene M, β-pinene D, β-pinene M, butyl hexanoate, camphor D, camphor M, carvone M, citronellal D, citronellal M, ethyl 3-hydroxyhexanoate D, isopulegone D, longifolene, limonene D, limonene M, myrcene T, and neryl acetate compounds in raw Nepeta cataria are significantly higher than those in other samples; the content of acetic acid D compound in stir-fried Nepeta cataria is significantly higher than that in other samples; the contents of acetic acid M, isopulegone M, and methyl acetate compounds in charred Nepeta cataria are the highest among all samples.

2. The method for analyzing the types and differences of volatile components of Schizonepeta tenuifolia and its processed products based on GC-IMS technology according to claim 1, characterized in that, The preparation methods of three samples of raw Nepeta cataria, stir-fried Nepeta cataria, and charred Nepeta cataria are as follows: (1) Preparation of crude Schizonepeta tenuifolia Briq.: The crude Schizonepeta tenuifolia Briq. slices JJ are the dried aerial parts of the plant Schizonepeta tenuifolia Briq. of the Lamiaceae family; (2) Preparation of stir-fried Schizonepeta tenuifolia Briq.: Referring to the "Jiangsu Province Traditional Chinese Medicine Decoction Pieces Processing Specification" 2020 Edition, Volume 1, take an appropriate amount of crude Schizonepeta tenuifolia Briq., place it in a preheated frying pan at 100 °C, stir-fry the crude Schizonepeta tenuifolia Briq. for 12 minutes, and take it out and let it cool when the surface is yellowish-brown to obtain stir-fried Schizonepeta tenuifolia Briq. CJJ; (3) Preparation of Schizonepeta tenuifolia Briq. charcoal: Referring to the "Pharmacopoeia of the People's Republic of China" 2020 Edition, take an appropriate amount of purified Schizonepeta tenuifolia Briq., place it in a preheated frying pan at 210 °C, stir-fry the crude Schizonepeta tenuifolia Briq. for 7 minutes, and take it out and let it cool when the surface is dark brown to obtain Schizonepeta tenuifolia Briq. charcoal JJC.

3. The method for analyzing the types and differences of volatile components of Schizonepeta tenuifolia and its processed products based on GC-IMS technology according to claim 1, wherein The contents of (E)-2-hexenal D, 1-octen-3-ol D, 2,3,5-trimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-octanol D, 3-octanone D, acetone, β-phellandrene M, β-pinene D, β-pinene M, butyl hexanoate, camphor D, camphor M, carvone M, citronellal D, citronellal M, ethyl 3-hydroxyhexanoate D, isopulegone D, longifolene, limonene D, limonene M, myrcene T, and neryl acetate compounds in crude Schizonepeta tenuifolia Briq. are significantly higher than those in stir-fried Schizonepeta tenuifolia Briq. and Schizonepeta tenuifolia Briq. charcoal; the content of acetic acid D compound in stir-fried Schizonepeta tenuifolia Briq. is significantly higher than that in crude Schizonepeta tenuifolia Briq. and Schizonepeta tenuifolia Briq. charcoal; the contents of acetic acid M, isopulegone M, and methyl acetate compounds in Schizonepeta tenuifolia Briq. charcoal are the highest among the three samples of crude Schizonepeta tenuifolia Briq., stir-fried Schizonepeta tenuifolia Briq., and Schizonepeta tenuifolia Briq. charcoal.

Images