Method for representing chemical components of schizonepeta and fried schizonepeta based on UPLC-Q-TOF-MS / MS

Abstract

The chemical components of the schizonepeta and the fried schizonepeta are characterized on the basis of an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) technology, the chemical component difference of the schizonepeta before and after frying is further compared in combination with a chemometrics analysis method, and the research characterizes the chemical components of the schizonepeta and the fried schizonepeta. And a chemometrics statistical analysis method is combined to analyze differential components between the two, so that a reference basis is provided for subsequent quality control and material basic research.

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 characterizing the chemical components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS). Background Art

[0002] Schizonepeta tenuifolia Briq. is the dried aerial part of the plant Schizonepeta tenuifolia Briq. of the Lamiaceae family. It has a pungent taste and a slightly warm nature, and has the effects of relieving exterior syndrome and dispelling wind, promoting eruption, dissipating swelling sores, etc. It is one of the traditional commonly used bulk medicinal materials. As the main medicine for relieving exterior syndrome and dispelling wind, it is clinically commonly used for expelling wind and relieving exterior syndrome, clearing the head and eyes, benefiting the throat, promoting eruption and relieving itching, etc. In the Ming Dynasty, the "Secretary of the Song's Gynecology" first recorded the frying method of "stir-frying" Schizonepeta tenuifolia Briq.; the "Compendium of Gynecology" recorded that "Coptis chinensis, Phellodendron amurense, lead oxide, and Schizonepeta tenuifolia Briq. are stir-fried slightly in equal parts and ground into fine powder, applied between the toes, and tied with cloth, and it will be naturally stable and painless", elaborating on the stir-frying method of "slightly stir-frying" Schizonepeta tenuifolia Briq. Fu Qing was good at using wind drugs in the treatment of menstrual diseases. For example, stir-fried Schizonepeta tenuifolia Briq. was used to soothe the liver and relieve depression, regulate the liver qi, and regulate qi movement, while rarely using overly pungent and dry products. The pungent-dispersing effect of stir-fried Schizonepeta tenuifolia Briq. is reduced, and it can strengthen the effect of entering the blood aspect of the liver meridian and enhance the effects of detoxification, dispelling wind and regulating blood, etc. Professor Xia Guicheng is also good at using stir-fried Schizonepeta tenuifolia Briq. in the treatment of gynecological diseases. For example, the Ziyin Shengan Decoction combined with Yueju Pills combined with Gouteng Decoction is often used for polycystic ovary syndrome type infertility - kidney yin deficiency.

[0003] Modern research shows that Schizonepeta tenuifolia Briq. contains chemical components such as volatile oils, flavonoids, and terpenoids, and has pharmacological effects such as antipyretic, anti-inflammatory and analgesic, and anti-tumor effects. During the frying process, the pungent-dispersing ability of Schizonepeta tenuifolia Briq. weakens, and its chemical components often also undergo complex changes. Researchers such as Gong Min used HS-SPME-GC-MS technology to analyze the volatile components before and after frying, and found that 14 new components were added and 15 components were reduced in stir-fried Schizonepeta tenuifolia Briq. Tian Tian et al. used UPLC technology to determine the contents of key components (including hesperidin, rosmarinic acid, and pulegone) in Schizonepeta tenuifolia Briq. decoction pieces with different processing times. The results showed that the longer the processing time of Schizonepeta tenuifolia Briq., the more obvious the decrease or even disappearance of the contents of hesperidin, rosmarinic acid, and pulegone. At present, the research on the chemical components of Schizonepeta tenuifolia Briq. and its processed products mainly focuses on the chemical components of Schizonepeta tenuifolia Briq. and Schizonepeta tenuifolia Briq. charred, and there is less research on stir-fried Schizonepeta tenuifolia Briq. and the differences in chemical components before and after frying of Schizonepeta tenuifolia Briq. Therefore, this patent uses UPLC-Q-TOF-MS / MS technology to analyze the chemical components contained in Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq., and screens the differential components of the two through chemometric analysis methods, in order to provide a scientific basis for the clinical application and processing research of Schizonepeta tenuifolia Briq. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for characterizing the chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS), and the steps are as follows:

[0005] (1) Preparation of stir-fried Schizonepeta tenuifolia Briq.

[0006] Referring to the first volume of the "Jiangsu Province Chinese Herbal Pieces Processing Specification" (2020 edition), take an appropriate amount of raw Schizonepeta tenuifolia Briq., place it in a preheated frying pan (100 °C), stir-fry the raw Schizonepeta tenuifolia Briq. for 12 minutes until the surface is yellowish-brown, take it out and let it cool to obtain stir-fried Schizonepeta tenuifolia Briq.

[0007] (2) Preparation of test solution

[0008] Weigh 0.5 g of Schizonepeta tenuifolia Briq. or stir-fried Schizonepeta tenuifolia Briq. powder (passing through No. 3 sieve) accurately, place it in a stoppered conical flask, add 10 mL of methanol solution, sonicate (250 W, 40 kHz) for 30 minutes, let it cool and then weigh, and make up the lost weight with methanol. Centrifuge at 12000 r·min -1 for 10 minutes, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the test solution;

[0009] (3) Preparation of reference solution

[0010] Weigh appropriate amounts of reference substances of apigenin, caffeic acid, oleanolic acid, chlorogenic acid, luteolin, madecassic acid, rutin, isoquercitrin, rosmarinic acid, hesperidin, and pulegone accurately, make up the volume with methanol, and prepare a mixed standard solution with mass concentrations of 5.245 μg·mL -1 , 7.079 μg·mL -1 , 4.723 μg·mL -1 , 5.114 μg·mL -1 , 6.086 μg·mL -1 , 6.115 μg·mL -1 , 5.276 μg·mL -1 , 7.120 μg·mL -1 , 4.640 μg·mL -1 , 4.830 μg·mL -1 , 4.861 μg·mL -1 ;

[0011] (4) Preparation of quality control samples

[0012] Take 1 mL of each of all the test solutions and mix them, centrifuge at 12000 r·min -1Centrifuge for 10 min, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate as the quality control (QC) sample. The QC sample, as the product of equal mixing of all samples, can represent the characteristics of all samples. By analyzing the QC sample, systematic errors can be corrected to ensure the stability and repeatability of the detection system.

[0013] (5) Perform ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) detection;

[0014] (6) Chemical component identification

[0015] The steps for component identification research include: First, collect information such as the chemical component names, structures, molecular weights, and chemical formulas of Schizonepeta tenuifolia and stir-fried Schizonepeta tenuifolia on websites such as TCMSP, Chemspider, ChemicalBook, and PubMed to construct a database containing Schizonepeta tenuifolia and its stir-fried products. Second, input the database information and the positive and negative ion mode data of the mass spectrometry into Peak View 2.1 software to perform peak matching. Finally, based on the matching results, combined with the comparison with reference standards, accurate molecular weights, and information such as specific fragment ions in the literature, identify the compounds;

[0016] (7) Screening of differential compounds

[0017] Export all the original data of UPLC-Q-TOF-MS / MS and perform preprocessing work such as peak extraction and peak alignment on these data using MS-DIAL software. Import the preprocessed positive and negative ion mode data into SIMCA 14.0 software respectively for multivariate statistical analysis; use principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) to perform comparative analysis on the samples of raw Schizonepeta tenuifolia and processed Schizonepeta tenuifolia; screen the components with significant differences through variable importance in projection (VIP) values, P values, and fold change (FC). Confirm and identify these differential components through database query and literature comparison;

[0018] (8) The differential components between Schizonepeta tenuifolia and stir-fried Schizonepeta tenuifolia are mainly organic acid components and flavonoid components; from the FC values, it can be seen that the contents of chemical components such as hesperidin, apigenin, linolenic acid, and apigenin 7-O-β-D-glucoside in Schizonepeta tenuifolia decrease after stir-frying, while isoferulic acid, luteolin, and diosmetin increase.

[0019] The method for characterizing the chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS): Liquid chromatography conditions: Waters CORTECS UPLC T3 chromatographic column (100 mm × 2.1 mm, 1.6 μm), column temperature 35 °C, injection volume 5 μL, flow rate 0.4 mL·min-1. The mobile phase is 0.1% formic acid in water (A)-acetonitrile (B); the gradient elution program is 0 - 4 min, 95% - 95% (A); 4 - 6 min, 95% - 85% (A); 6 - 18 min, 85% - 65% (A); 18 - 23 min, 65% - 50% (A); 23 - 28 min, 50% - 25% (A); 28 - 31 min, 25% - 25% (A); 31 - 36 min, 25% - 95% (A).

[0020] The method for characterizing the chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS): Mass spectrometry conditions: Detection is carried out using an AB SCIEX Triple TOF 5600 liquid chromatography-mass spectrometry instrument, with an electrospray ionization (ESI) source, and data is collected in positive and negative ion modes. Ionization temperature (TEM): 600 °C; nebulizing gas (GS1): 60 psi (1 psi ≈ 6.895 kPa); auxiliary heating gas (GS2): 60 psi; curtain gas (CUR): 35 psi; the acquisition range of the first-stage mass spectrometry is m / z 50 - 1000, with an accumulation time of 0.15 s; DP: 80 V; CE: 10 eV. The acquisition range of the second-stage mass spectrometry is m / z 25 - 1000, with an accumulation time of 0.035 s, DP: 80 V; CE: 40 eV; CES: 20; IRD: 67; IRW: 25. The relative molecular weight accuracy of the compound is automatically calibrated using the tuning liquid transfer system of AB SCIEX Company, USA.

[0021] The method for characterizing the chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on UPLC-Q-TOF-MS / MS: The differential components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. are mainly organic acid components and flavonoid components; after stir-frying, the contents of chemical constituents in Schizonepeta tenuifolia Briq. including hesperidin, apigenin, linolenic acid, and apigenin 7-O-β-D-glucoside decrease, while isoferulic acid, luteolin, and diosmetin increase.

[0022] This patent characterized the chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS), and further compared the differences in chemical constituents before and after stir-frying of Schizonepeta tenuifolia Briq. by combining chemometric analysis methods. UPLC-Q-TOF-MS / MS was used to analyze and identify the chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq., and principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) methods were used. With the variable importance in projection (VIP) value > 1, t-test (P < 0.05), and fold change (FC) > 1.2 or < 0.8 as the screening criteria, the differential components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. were screened through a self-built database and literature comparison. A total of 81 chemical constituents of Schizonepeta tenuifolia Briq. and 56 chemical constituents of stir-fried Schizonepeta tenuifolia Briq. were initially identified, including terpenoids, flavonoids, organic acids, etc. Fourteen differential components such as isoferulic acid, luteolin, and diosmetin were screened out, mainly organic acids and flavonoids. Among them, 10 components were down-regulated and 4 components were up-regulated in stir-fried Schizonepeta tenuifolia Briq. compared with Schizonepeta tenuifolia Briq. In summary, this study characterized the chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq., and analyzed the differential components between the two by combining chemometric statistical analysis methods, providing a reference basis for subsequent quality control and material basis research. Description of the Drawings

[0023] Figure 1 Total ion chromatogram (TIC) of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. Note: A. Total ion chromatogram (TIC) of Schizonepeta tenuifolia Briq. in positive ion mode; B. Total ion chromatogram (TIC) of Schizonepeta tenuifolia Briq. in negative ion mode; C. Total ion chromatogram (TIC) of stir-fried Schizonepeta tenuifolia Briq. in positive ion mode; D. Total ion chromatogram (TIC) of stir-fried Schizonepeta tenuifolia Briq. in negative ion mode Figure 2 Secondary mass spectrum of hesperidin and possible cleavage pathways

[0024] Figure 3 Secondary mass spectrum of rosmarinic acid and possible cleavage pathways

[0025] Figure 4 PCA score plot of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. Note: JJ. Schizonepeta tenuifolia Briq.; CJ. Stir-fried Schizonepeta tenuifolia Briq.; QC is the quality control sample. A. Positive ion mode; B. Negative ion mode.

[0026] Figure 5 OPLS-DA diagram and permutation test of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq.

[0027] Note: JJ - Schizonepeta tenuifolia Briq.; CJ - Stir-fried Schizonepeta tenuifolia Briq. Detailed Description of the Invention

[0028] 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 not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, but these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0029] Example 1:

[0030] 1 Materials

[0031] 1.1 Experimental Instruments

[0032] Exion LC UPLC instrument, Triple TOF 5600 mass spectrometer (AB SCIEX, USA), CPA225D electronic balance (Sartorius, Germany), Thermo Sorvall Legend Micro 17R centrifuge (Thermo, USA), WH2 micro vortex mixer (Shanghai Huxi Analytical Instrument Factory), Millipore Milli-Q Advantage A10 ultrapure water system (Milli-Q).

[0033] 1.2 Drugs and Reagents

[0034] Pulegone (batch number: 111706 - 201907, purity 99.8%), rosmarinic acid (batch number: 111871 - 202007, purity ≥98%), apigenin (batch number: 111901 - 202205, purity 98.4%), luteolin (batch number: 111520 - 202107, purity 96.3%), isoquercitrin (batch number: 111809 - 201804, purity 97.2%), oleanolic acid (batch number: 110709 - 202109, purity 95.8%), rutin (batch number: 100080 - 202012, purity 91.6%), hesperidin (batch number: 111538 - 202007, purity ≥98%), caffeic acid (batch number: 110885 - 201703, purity 99.7%), chlorogenic acid (batch number: 110753 - 202119, purity 96.3%) were all purchased from the National Institutes for Food and Drug Control; madecassic acid (batch number: 18449 - 41 - 7, purity 98%) was purchased from Chengdu Efar Biotechnology Co., Ltd. Methanol and acetonitrile (HPLC grade, Merck, Germany), formic acid (mass spectrometry grade, Fluka - Sigma - Aldrich, Switzerland), water was ultrapure water, and the rest of the reagents were all of analytical grade.

[0035] The Schizonepeta decoction pieces were purchased from different origins and identified by Chief Chinese Medicine Pharm. Dr. Zhu Yufeng of the Affiliated Hospital of Nanjing University of Chinese Medicine as the dried aerial parts of Schizonepeta tenuifolia Briq., a plant of the Lamiaceae family.

[0036] 2 Methods

[0037] 2.1 Sample preparation

[0038] 2.1.1 Preparation of processed products

[0039] According to the first volume of "Jiangsu Province Traditional Chinese Medicine Preparation Specifications" (2020 Edition), take an appropriate amount of raw Nepeta, put it in a preheated wok (100°C), and stir-fry the raw Nepeta for about 12 minutes until the surface turns yellow-brown. Take it out and let it cool to get the stir-fried Nepeta. Information on raw Nepeta and stir-fried Nepeta is shown in Table 1. In the following, J is the abbreviation for raw Nepeta, and CJ is the abbreviation for stir-fried Nepeta.

[0040] Table 1 Sample source information

[0041]

[0042]

[0043] 2.1.2 Preparation of test solution

[0044] Take 0.5 g of Nepeta or stir-fried Nepeta powder (pass through No. 3 sieve), weigh accurately, put into a stoppered conical flask, add 10 mL of methanol solution, ultrasonicate (250 W, 40 kHz) for 30 min, weigh after cooling, and make up the loss with methanol, and spin at 12000 r·min -1 Centrifuge for 10 min, filter through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0045] 2.1.3 Preparation of reference solution

[0046] Take appropriate amount of apigenin, caffeic acid, oleanolic acid, chlorogenic acid, luteolin, madecassoside, rutin, isoquercetin, rosmarinic acid, hesperidin, and pulegone reference substances, weigh them accurately, add methanol to make up to the volume, and prepare them to a mass concentration of 5.245 μg mL -1 , 7.079μg·mL -1 , 4.723 μg·mL -1 , 5.114 μg·mL -1 , 6.086μg·mL -1 、6.115μg·mL -1 , 5.276μg·mL -1 , 7.120μg·mL -1 , 4.640 μg·mL -1, 4.830 μg·mL -1 , 4.861 μg·mL -1 of the mixed standard solution.

[0047] 2.1.4 Preparation of quality control samples

[0048] Take 1 mL of each test solution and mix them. Centrifuge at 12000 r·min -1 for 10 min, filter through a 0.22 μm microporous membrane filter, and take the subsequent filtrate as the quality control (QC) sample. The QC sample, as the product of equal mixing of all samples, can represent the characteristics of all samples. By analyzing the QC samples, systematic errors can be corrected to ensure the stability and repeatability of the detection system.

[0049] 2.2 Liquid chromatography conditions

[0050] Waters CORTECS UPLC T3 chromatographic column (100 mm × 2.1 mm, 1.6 μm), column temperature 35 °C, injection volume 5 μL, flow rate 0.4 mL·min -1 . The mobile phase is 0.1% formic acid water (A) - acetonitrile (B); the gradient elution program is 0 - 4 min, 95% - 95% (A); 4 - 6 min, 95% - 85% (A); 6 - 18 min, 85% - 65% (A); 18 - 23 min, 65% - 50% (A); 23 - 28 min, 50% - 25% (A); 28 - 31 min, 25% - 25% (A); 31 - 36 min, 25% - 95% (A).

[0051] 2.3 Mass spectrometry conditions

[0052] Detection was performed using an AB SCIEX Triple TOF 5600 liquid chromatography - mass spectrometry instrument. The electrospray ionization (ESI) source was used to collect data in both positive and negative ion modes. Ionization temperature (TEM): 600 °C; nebulizing gas (GS1): 60 psi (1 psi ≈ 6.895 kPa); auxiliary heating gas (GS2): 60 psi; curtain gas (CUR): 35 psi. The acquisition range of the first - stage mass spectrometry was m / z 50 - 1000, with an accumulation time of 0.15 s; DP: 80 V; CE: 10 eV. The acquisition range of the second - stage mass spectrometry was m / z 25 - 1000, with an accumulation time of 0.035 s, DP: 80 V; CE: 40 eV; CES: 20; IRD: 67; IRW: 25. The relative molecular mass accuracy of the compounds was automatically calibrated using the tuning liquid transfer system of AB SCIEX Company, USA.

[0053] 2.4 Identification of chemical components

[0054] The steps for component identification research include: First, collect information such as the chemical component names, structures, molecular weights, chemical formulas, etc. of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. on websites such as TCMSP, Chemspider, ChemicalBook, and PubMed, and construct a database containing Schizonepeta tenuifolia Briq. and its stir-fried products. Second, input the database information and the positive and negative ion mode data of the mass spectrometry into Peak View 2.1 software to perform peak matching. Finally, based on the matching results, combined with information such as comparison with reference standards, accurate molecular weights, and specific fragment ions in the literature, identify the compounds.

[0055] 2.5 Screening of differential compounds

[0056] Export all the original data of UPLC-Q-TOF-MS / MS, and use MS-DIAL software to perform preprocessing work on these data such as peak extraction and peak alignment. Import the preprocessed positive and negative ion mode data into SIMCA 14.0 software respectively for multivariate statistical analysis. Use principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) to perform comparative analysis on raw Schizonepeta tenuifolia Briq. and processed Schizonepeta tenuifolia Briq. samples. Screen out the components with significant differences through variable importance in projection (VIP) values, P values, and fold change (FC). Confirm and identify these differential components through database query and literature comparison.

[0057] 3 Experimental results and analysis

[0058] 3.1 Results of component identification by UPLC-Q-TOF-MS / MS

[0059] In the positive and negative ion modes, the chromatograms of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. are basically similar. The high-resolution mass spectrometry data of the extracts of Schizonepeta tenuifolia Briq. and its stir-fried products collected were analyzed by means of comparison with reference standards, querying the self-built Schizonepeta tenuifolia Briq. compound database, and comparison with published literature. A total of 84 compounds were identified or deduced, including 28 flavonoid compounds, 48 organic acids (divided into 23 phenolic acids and 25 fatty acids), 5 terpenoid compounds, and 3 other types of compounds (including 2 sugars and 1 aldehyde). The total ion current chromatograms (TIC) of Schizonepeta tenuifolia Briq. (J1) and stir-fried Schizonepeta tenuifolia Briq. (CJ1) are shown in Figure 1 . The specific identified component information is shown in Table 2.

[0060] 3.1.1 Identification of Flavonoids

[0061] Flavonoid compounds in Schizonepeta tenuifolia Briq. are one of its key active ingredients, with multiple pharmacological activities such as analgesia, anti - inflammation, anti - aging, antioxidant, anti - cancer, and immune regulation. The structural parent nucleus of these compounds is 2 - phenylchromone, characterized by the connection of ring A to ring B through the first carbon atom on ring C. Ring C between rings A and B is prone to breakage due to the influence of the two adjacent rings. Compared with ring C, the structures of rings A and B are more stable, and neutral fragment ions such as glycosyl groups, CH3, CO, H2O, and CO2 are mainly removed through the retro - Diels - Alder reaction (RDA). Under the action of cleavage voltage, flavone monoglycoside compounds usually lose one sugar molecule, and the mass - to - charge ratios of the generated fragment ions are mainly 301 and 285.

[0062] Compound 32 (t R = 10.43 min) has a molecular ion peak m / z of 609.1821 [M - H] - , and its molecular formula is inferred to be C 28 H 34 O 15 . In its secondary mass spectrum, one molecule of glucose and rhamnose are lost, generating an ion fragment with m / z of 301.0696 [M - H - C6H 12 O6 - C6H 10 O4] - , and then losing a methyl group to generate an ion fragment with m / z of 286.0479 [M - H - C6H 12 O6 - C6H 10 O4 - CH3] – . At the same time, ring C cleaves to lose C 10 H8O3 to an ion fragment with m / z of 125.0239 [M - H - C6H 12 O6 - C6H 10 O4 - C 10 H8O3] – . By comparing with the literature reports and standards, this compound is identified as hesperidin, and its secondary mass spectrum and the speculated cleavage pathway are shown in Figure 2 .

[0063] Compound 43 (t R = 12.92 min) has a molecular ion peak m / z of 285.0407 [M - H] - , and its elemental composition is speculated to be C 15 H 10 O6. In the secondary mass spectrum, m / z of 151.0041 [M - H - C8H6O2] - and m / z of 133.0292 [M - H - C7H4O4] -Ion fragmentation peaks are typical features of the cleavage of ring C in flavonoid compounds. By comparing with the standard product, this compound was identified as luteolin.

[0064] Compound 23 (t R = 9.15 min) has a molecular ion peak m / z of 447.0932 [M-H] - , and its molecular formula is deduced to be C 21 H 20 O 11 . The molecular ion peak m / z is 447.0932 [M-H] - Under the action of collision energy, glucoside is removed to obtain the aglycone with m / z of 285.0402 [M-H-C6H 12 O6] - . In addition, compound 23 produces a major fragment ion at m / z of 151.0043 [M-H-C6H 10 O5-C7H2O3] - . After comparison with the literature, this compound is speculated to be luteoloside.

[0065] Compound 47 (t R = 19.18 min) has a molecular ion peak m / z of 269.0455 [M-H] - , and the mass-to-charge ratio is 16 less than that of luteolin. Its chemical formula is speculated to be C 15 H 10 O5. In the secondary mass spectrum, typical flavonoid RDA cleavage fragments m / z of 151.0025 [M-H-C8H6O] - appear, and the fragment ion with the strongest abundance m / z of 117.0344 [M-H-C7H4O4] - is also 16 less than the base peak fragment ion m / z of 133.0292 [M-H-C7H4O4] - of luteolin, indicating that this compound has one less hydroxyl group than luteolin. After comparison with the reference substance, this compound was identified as apigenin.

[0066] 3.1.2 Identification of organic acid compounds

[0067] Organic acid compounds usually contain an aromatic ring and a fatty acid structure substituted by phenolic hydroxyl groups. Such compounds tend to cleave at the carbonyl group to generate fragment ions during mass spectrometry cleavage, or neutral molecules such as H2O (18), COOH (45), and CO2 (44) are lost. A total of 48 organic acid components, including fatty acids and phenolic acids, were identified in this chapter.

[0068] 3.1.2.1 Identification of phenolic acid compounds

[0069] Compound 7 (t R= 5.75 min), and its molecular ion peak has an m / z value of 353.0876 [M-H] - , from which its molecular formula is deduced to be C 16 H 18 O9, and it is preliminarily judged to be a single caffeoylquinic acid. In its secondary mass spectrum, it loses quinic acid and caffeic acid to produce fragment ions with m / z of 191.0555 [M-H-C9H6O3] - and 179.0345 [M-H-C7H 10 O5] - , and then loses one molecule of CO2 to produce a fragment ion with m / z 135.0455 [M-H-C7H 10 O5-CO2] - . Based on the comparison with the standard sample, compound 9 (t R = 6.27 min) is identified as chlorogenic acid. According to the elution order of chlorogenic acid isomers on the reverse chromatographic column, compound 7 is identified as neochlorogenic acid.

[0070] Compound 36 (t R = 10.62 min) shows a molecular ion peak with m / z of 359.0772 [M-H] - , and its molecular formula is deduced to be C 18 H 16 O8. After the ester bond is broken, it produces a danshensu fragment with m / z of 197.0448 [C9H9O5] - and an ion fragment with m / z of 161.0235 [C9H5O3] - . Combining with the literature and comparison with the reference substance, it is identified as rosmarinic acid. Its secondary mass spectrum and the speculated cleavage pathway are shown in Figure 3 . The molecular ion peaks of compound 33 (t R = 10.49 min) and compound 35 (t R = 10.52 min) are both m / z 359 [M-H] - , and they have the same ion fragments as compound 36. Therefore, compound 33 and 35 are identified as rosmarinic acid or its isomers.

[0071] Compound 40 (t R = 11.95 min) shows a molecular ion peak with m / z of 717.1456 [M-H] - , and its molecular formula is deduced to be C 36 H 30 O 16 . The parent ion successively loses one molecule of danshensu and one molecule of caffeic acid to obtain fragment ions with m / z of 519.0872 and m / z of 339.0482. According to the fragment information and literature comparison, this compound is identified as salvianolic acid B.

[0072] 3.1.2.2 Identification of Fatty Acid Compounds

[0073] Fatty acids are compounds composed of a carboxyl group and an alkane group. Their cleavage usually first removes the carboxyl group by losing H2O (18) and CO2 (44), and then gradually sheds CH (13) or CH2 (14) molecules. The structural diversity of fatty acids, such as the different positions of hydroxyl groups or unsaturated bonds, leads to the formation of multiple isomers. These isomers are prone to breakage at the carboxyl position, generating characteristic fragment ions. The presence of unsaturated fatty acids increases the complexity of the mass spectrum because they are not only easily cleaved at the carboxyl group but also may undergo McLafferty rearrangement. Mass spectrometry data show that as the molecular mass increases, the abundance of the molecular ion peak of straight-chain alkanoic acids increases, and they are prone to losing H2O and COOH. The differences in mass spectra of unsaturated fatty acids with different double-bond positions are not obvious.

[0074] Compound 77 (t R = 31.83 min) shows a molecular ion peak at m / z 277.2181 [M-H] - , and its molecular formula is inferred to be C 18 H 30 O2. The parent ion successively loses one molecule of H2O and one molecule of CO2 to obtain fragment ion peaks at m / z 259.2073 [M-H-H2O] - and m / z 233.2297 [M-H-H2O-CO2] - . By comprehensive comparison with the Pubchem database and literature reports, this compound is identified as α-linolenic acid.

[0075] Compound 28 (t R = 9.95 min) has a molecular ion peak at m / z 187.0976 [M-H] - , and its molecular formula is inferred to be C9H 16 O4. This compound first loses one molecule of H2O to form a fragment ion at m / z 169.0890 [M-H-H2O] - , and then loses one molecule of CO2, H2O, and C2H4 successively to generate a fragment ion at m / z 97.0670 [M-H-H2O-CO2-C2H4] - . Consistent with the cleavage pattern reported in the literature, this compound is identified as azelaic acid.

[0076] 3.1.3 Identification of Terpenoid Compounds

[0077] Terpenoids are the most important chemical components in the volatile oil of Schizonepeta tenuifolia Briq. Based on the analysis of mass spectrometry data results, 5 pentacyclic triterpenoid compounds are speculated. Due to their similar molecular structures, especially the substituents (usually hydroxyl groups, carbonyl groups, or carboxyl groups) at the 3rd and 17th positions of the six-membered ring, they show similar fragment patterns.

[0078] Taking compound 78 (t R = 29.30 min) and compound 79 (t R = 29.49 min) as examples, their first-order mass spectrometry m / z in the negative ion mode is 455.3535 [M-H] - and 455.3524 [M-H] - . It is speculated that their elemental composition may be C 30 H 48 O3. In the second-order mass spectrometry, obvious fragment ion peaks m / z are 455 [M-H] - . Based on the mass spectrometry data and combined with the comparative analysis of literature data, compounds 78 and 79 were finally identified as oleanolic acid or ursolic acid. The structures of compounds 56 and 74 are similar to those of compounds 78 and 79, all of which are pentacyclic triterpenoids and have similar mass spectrometry fragmentation rules, which will not be elaborated here. It is speculated that they are hydroxy asiatic acid and maslinic acid. 3.1.4 Identification of other types of compounds

[0079] Three other types of components were identified from the extracts of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq., including 2 carbohydrate compounds (lactulose or isomers) and 1 aldehyde component (syringaldehyde).

[0080] Table 2 Identification information of chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq.

[0081] Tab.2 Identification of chemical constituents of Schizonepetae Herbaand Schizonepetae Tostum Herba

[0082]

[0083]

[0084]

[0085]

[0086] J. Schizonepeta tenuifolia Briq.; CJ. Stir-fried Schizonepeta tenuifolia Briq.

[0087] 3.2 Chemometric analysis

[0088] 3.2.1 Principal component analysis

[0089] Principal component analysis was performed on the cut pieces of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq., and the results are shown in Figure 4 . It can be seen from the figure that the aggregation of QC samples indicates that the method and instrument have good stability. In the positive ion mode, the cumulative interpretation rate R of the model in the X-axis direction of the score plot of 7 main components in principal component analysis (PCA2 X is 0.782, and the prediction ability Q 2 is 0.490. A score plot containing 7 principal components was also obtained under the negative ion mode, R 2 X is 0.707, Q 2 is 0.413. It can be seen from this that the raw and stir-fried products of Schizonepeta tenuifolia Briq. are clearly distinguishable, and there are differences in their components.

[0090] 3.2.2 Discriminant factor analysis

[0091] Discriminant factor analysis was performed on Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. slices, and 200 permutation tests were carried out. The results are shown in Figure 5 . It can be seen from the figure that the raw and stir-fried products of Schizonepeta tenuifolia Briq. are clearly distinguishable, the test is not overfitted, and the model has good prediction ability.

[0092] 3.3 Screening of differential components

[0093] Differential components were screened with VIP>1, P<0.05, FC>1.2 or <0.8 as the criteria. The msp file was imported into the MS-DIAL software, and the differential components were paired with databases and literatures including MassBank, PubChem, CASMI2016, etc. Finally, the differential components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. were identified, as shown in Table 3. According to Table 3, the differential components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. are mainly organic acid components and flavonoid components; from the FC values, it can be seen that the contents of chemical components such as hesperidin, apigenin, linolenic acid, apigenin 7-O-β-D-glucoside, etc. decreased after stir-frying Schizonepeta tenuifolia Briq., while isoferulic acid, luteolin, diosmetin, etc. increased.

[0094] Table 3 Differential components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq

[0095] Tab.3 Analysis of the differences of Schizonepetae Herba andSchizonepetae Tostum Herba

[0096]

[0097] 4 Analysis and discussion

[0098] At present, there are many studies on Schizonepeta tenuifolia Briq. and carbonized Schizonepeta tenuifolia Briq., and the reports on chemical components mainly focus on the research of volatile oils. The research on other components and the components of stir-fried Schizonepeta tenuifolia Briq. is not comprehensive. As a traditional diaphoretic medicinal material, after stir-frying, Schizonepeta tenuifolia Briq. will inevitably cause the cleavage or transformation of different components, thus bringing about the change of its medicinal properties, which also fully proves why traditional Chinese medicine uses raw or stir-fried Schizonepeta tenuifolia Briq. according to different diseases and syndromes in clinical practice.

[0099] UPLC-Q-TOF-MS / MS combines the separation ability of ultra-high performance liquid chromatography and the discrimination ability of time-of-flight mass spectrometry for compounds, enabling this technology to rapidly separate and identify the structures of complex components, with characteristics such as high sensitivity, fast analysis speed, and powerful qualitative and quantitative capabilities. In this experiment, UPLC-Q-TOF-MS / MS technology was used to perform screening analysis using precise relative molecular masses, compare the primary mass-to-charge ratios and secondary fragment ions, and refer to the fragmentation patterns of secondary mass spectra, the mass spectrometry data of chemical standards, and relevant literature reports, as well as databases such as PubChem, MassBank, and ChemicalBook. A total of 84 compounds were identified in Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq., mainly divided into flavonoids, organic acids, and terpenoids. The unique components of Schizonepeta tenuifolia Briq. mainly include phenolic acids (succinic acid, pimelic acid, p-coumaric acid, monobutyl phthalate, cinnamic acid), flavonoids (apigenin 7-O-(6”-O-malonyl) β-D-glucoside, buddleoside), and fatty acids (DGMG 18:3, Cis-parinaric acid, (10E,12Z)-9-hydroxy-10,12-octadecadienoic acid, arachidic acid / eicosanoic acid). Research shows that the chemical component p-coumaric acid has a wide range of pharmacological activities and good anti-inflammatory and analgesic effects; buddleoside may exert an anti-inflammatory effect by inhibiting the activation of NF-κB; eicosanoids are important inflammatory factors and are also closely related to cancer, and can affect the occurrence and development of tumors through mechanisms such as regulating the proliferation of tumor cells, influencing the tumor inflammatory microenvironment, and tumor immunosuppression. The differences in these components may be the reasons for the different medicinal properties of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq., and the specific mechanism of action still needs further exploration. The unique component of stir-fried Schizonepeta tenuifolia Briq. is syringaldehyde. Aldehydes generally have a fresh, fruity, and sweet fragrance and can affect the overall characteristic flavor. Stir-fried Schizonepeta tenuifolia Briq. weakens the pungency of Schizonepeta tenuifolia Briq., while syringaldehyde has a pleasant fragrance, which may be one of the reasons for the difference in the odors of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq.

[0100] Combined with chemometric analysis, it was found that Schizonepeta tenuifolia and stir-fried Schizonepeta tenuifolia were clearly separated on the PCA axis, which was consistent with the results of OPLS-DA, with obvious classification effect and obvious component differences. The differential components between the raw and stir-fried products of Schizonepeta tenuifolia were mainly flavonoids and organic acid compounds. The flavonoid components, such as hesperidin, apigenin, apigenin 7-O-β-D-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, and the organic acid components, such as rosmarinic acid and linolenic acid, were reduced to varying degrees; while the contents of isoferulic acid, luteolin, and diosmetin increased to varying degrees. The relationship between the changes in component contents and the pharmacodynamic effects needs further study. Schizonepeta tenuifolia is pungent in taste and has a dispersing effect; it belongs to the lung meridian and exerts a therapeutic effect on exterior syndromes through the function of "the lung governing qi". Volatile oil is its main component, and it is speculated that its volatile oil may be the main material basis for inducing sweating and relieving exterior syndrome. Schizonepeta tenuifolia belongs to the pungent-warm exterior-relieving herbs. During the frying process, the volatile oil will be reduced to varying degrees. The longer the frying time, the higher the loss of volatile oil and the weaker the pungent-dispersing effect. "Explanation of Materia Medica Classics" records: "Schizonepeta tenuifolia is pungent to reach the qi of wind-wood, warm to disperse the stagnation of ministerial fire. When the stagnant fire is dispersed and the wind is calmed, all symptoms will be alleviated." "Summary of Materia Medica" further proposed the mechanism of Schizonepeta tenuifolia in treating wind hidden in the liver to regulate blood. Clinically, due to the excessive pungency and dryness of Schizonepeta tenuifolia, stir-fried Schizonepeta tenuifolia is used in the treatment of gynecological diseases such as leucorrhea syndrome, polycystic ovary syndrome, and irregular menstruation. At present, a large number of studies have shown that natural flavonoid components have obvious antiviral effects. Liu et al. found that apigenin and luteolin have strong inhibitory effects on influenza virus. In addition, differential components such as hesperidin, luteolin, diosmetin, rosmarinic acid, and isoferulic acid have various pharmacological activities such as antibacterial, anti-inflammatory, antioxidant, antitumor, and hypoglycemic effects. Salvianolic acid B has the effects of promoting blood circulation and removing blood stasis and reducing liver injury. The content of this component increased significantly after Schizonepeta tenuifolia was stir-fried. Salvianolic acid B can regulate the intestinal flora to play a role in reducing liver injury by increasing the relative abundance of Lactobacillus, decreasing the relative abundance of Faecalibacterium and Bifidobacterium, and restoring the content of some short-chain fatty acids. Han Xiujuan et al. simulated the in vivo blood-brain barrier model and screened out active components such as isoferulic acid by examining the transmembrane resistance value and permeability of cells in the model. The content of isoferulic acid increased significantly after Schizonepeta tenuifolia was stir-fried, and it is speculated that the change in the content of this component is the reason for the enhanced blood-regulating effect of stir-fried Schizonepeta tenuifolia.

[0101] In summary, the UPLC-Q-TOF-MS / MS method established in this paper can better separate and identify the chemical components in Schizonepeta tenuifolia and stir-fried Schizonepeta tenuifolia. The main differential components before and after stir-frying of Schizonepeta tenuifolia were screened out by chemometric analysis methods, providing a basis for the study of the processing theory of stir-fried Schizonepeta tenuifolia. Although the UPLC-Q-TOF-MS / MS technology has advantages such as fast analysis speed and high resolution, due to the self-built database, the similar secondary fragment ions and fragmentation rules of isomers in Schizonepeta tenuifolia herbs, and the lack of verification of reference substances, there are still some components that have not been identified. In addition, further studies on the differences in medicinal properties are needed to provide a more complete reference basis for clinical applications.

[0102] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for characterizing the chemical constituents of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on UPLC-Q-TOF-MS / MS, characterized in that, The steps are as follows: (1) Preparation of stir-fried Schizonepeta tenuifolia Briq. Refer to the first volume of the "Jiangsu Province Traditional Chinese Medicine Decoction Pieces Processing Specification" (2020 edition). Take an appropriate amount of raw Schizonepeta tenuifolia Briq., 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.; (2) Preparation of test solution Respectively take 0.5 g of Schizonepeta tenuifolia Briq. or stir-fried Schizonepeta tenuifolia Briq. powder, sieve through No. 3 sieve, weigh accurately, place in a stoppered conical flask, add 10 mL of methanol solution, ultrasonicate for 30 min, the ultrasonic conditions are 250 W and 40 kHz, let it cool and then weigh, and make up the lost weight with methanol, 12000 r·min -1 Centrifuge for 10 min, filter through a 0.22 μm microporous membrane, take the subsequent filtrate to obtain the test solution; (3) Preparation of reference solution Weigh appropriate amounts of reference substances of apigenin, caffeic acid, oleanolic acid, chlorogenic acid, luteolin, madecassic acid, rutin, isoquercitrin, rosmarinic acid, hesperidin, and pulegone accurately, dissolve them in methanol and make up the volume to obtain mixed standard solutions with mass concentrations of 5.245 μg·mL -1 , 7.079 μg·mL -1 , 4.723 μg·mL -1 , 5.114 μg·mL -1 , 6.086 μg·mL -1 , 6.115 μg·mL -1 , 5.276 μg·mL -1 , 7.120 μg·mL -1 , 4.640 μg·mL -1 , 4.830 μg·mL -1 , 4.861 μg·mL -1 respectively; (4) Preparation of quality control samples Mix 1 mL of each of the test article solutions, centrifuge at 12,000 r·min -1 for 10 min, filter through a 0.22 μm microporous membrane filter, and take the subsequent filtrate as the quality control sample, i.e., the QC sample. As the product of equal mixing of all samples, it can represent the characteristics of all samples. By analyzing the QC sample, systematic errors can be corrected to ensure the stability and repeatability of the detection system; (5) Perform ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) detection; (6) Chemical component identification The steps for component identification research include: First, collect information such as the chemical component names, structures, molecular weights, and chemical formulas of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. on websites such as TCMSP, Chemspider, ChemicalBook, and PubMed, and construct a database containing Schizonepeta tenuifolia Briq. and its stir-fried products. Secondly, input the database information and the positive and negative ion mode data of the mass spectrometry into PeakView 2.1 software and perform peak matching. Finally, based on the matching results, combined with the comparison of reference standards, accurate molecular weights, and information such as specific fragment ions in the literature, identify the compounds; (7) Screening of differential compounds Export all the original data of UPLC-Q-TOF-MS / MS, and use MS-DIAL software to perform preprocessing work on these data, such as peak extraction and peak alignment. Import the preprocessed positive and negative ion mode data into SIMCA 14.0 software respectively for multivariate statistical analysis; use principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) to compare and analyze the samples of raw Schizonepeta tenuifolia Briq. and processed Schizonepeta tenuifolia Briq.; screen the components with significant differences through variable importance in projection (VIP value), P value, and fold change (FC), and confirm and identify these differential components through database query and literature comparison; (8) The differential components between Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. are mainly organic acid components and flavonoid components; from the FC value, it can be seen that the contents of chemical components such as hesperidin, apigenin, linolenic acid, and apigenin 7-O-β-D-glucoside in Schizonepeta tenuifolia Briq. decrease after stir-frying, while isoferulic acid, luteolin, and diosmetin increase.

2. The method for characterizing the chemical components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on UPLC-Q-TOF-MS / MS according to claim 1, wherein, Liquid chromatography conditions: Waters CORTECS UPLC T3 chromatographic column, with conditions of 100 mm × 2.1 mm, 1.6 μm, column temperature 35 °C, injection volume 5 μL, and flow rate 0.4 mL·min -1 . The mobile phase was 0.1% formic acid in water (A) - acetonitrile (B); the gradient elution program was 0 - 4 min, 95% - 95% (A); 4 - 6 min, 95% - 85% (A); 6 - 18 min, 85% - 65% (A); 18 - 23 min, 65% - 50% (A); 23 - 28 min, 50% - 25% (A); 28 - 31 min, 25% - 25% (A); 31 - 36 min, 25% - 95% (A).

3. The method for characterizing the chemical components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on UPLC-Q-TOF-MS / MS according to claim 1, wherein Mass spectrometry conditions: Detection was carried out using an AB SCIEX Triple TOF 5600 liquid chromatography-mass spectrometry instrument. The electrospray ionization (ESI) source was used, and data were collected in positive and negative ion modes. Ionization temperature TEM: 600 °C; nebulizing gas GS1: 60 psi, where 1 psi ≈ 6.895 kPa; auxiliary heating gas GS2: 60 psi; curtain gas CUR: 35 psi; the acquisition range of the first-stage mass spectrometry was m / z 50 - 1000, and the accumulation time was 0.15 s; DP: 80 V; CE: 10 eV. The acquisition range of the second-stage mass spectrometry was m / z 25 - 1000, the accumulation time was 0.035 s, DP: 80 V; CE: 40 eV; CES: 20; IRD: 67; IRW: 25; the relative molecular weight accuracy of the compound was automatically calibrated using the tuning liquid delivery system of AB SCIEX Company, USA.

4. The method for characterizing the chemical components of Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. based on UPLC-Q-TOF-MS / MS according to claim 1, wherein, The differential components between Schizonepeta tenuifolia Briq. and stir-fried Schizonepeta tenuifolia Briq. are mainly organic acid components and flavonoid components; after stir-frying, the contents of chemical components in Schizonepeta tenuifolia Briq. including hesperidin, apigenin, linolenic acid, and apigenin 7-O-β-D-glucoside decreased, while isoferulic acid, luteolin, and diosmetin increased.

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