A method for analyzing the chemical components and content determination of Chlorophytum comosum based on liquid chromatography-quadrupole-orbital trap tandem mass spectrometry

Abstract

The present invention discloses a method for analyzing the chemical composition and content determination of Chlorophytum comosum based on liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry, including preparing a reference solution, preparing a test solution, and chromatographic analysis, wherein the chemical composition analysis also includes mass spectrometry analysis and data processing. This application is the first to simultaneously quantitatively analyze three types of components in Chlorophytum comosum, namely flavonoids, phenylethanoid glycosides, and organic acids. The results show that the contents of these five components in medicinal materials from different origins vary greatly, which may be due to factors such as different origins, growth environments, climatic conditions, and harvesting times that affect the accumulation of compounds in plants. The specific reasons need further study.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product determination, and in particular relates to a method for analyzing the chemical components of Chlorophytum comosum and determining their contents based on liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry. Background Art

[0002] Stone spider plant (hereinafter referred to as SDL) is the dried aerial part of the Gesneriaceae plant stone spider plant, also known as black bone, stone cowpea, stone zelan, small zelan, rock cowpea, rock tea, rock zelan, rock orchid, Bayan grass, lung red grass, melon grass, stone flower, postpartum tea, mountain zelan, stone Panax notoginseng, stone tiger, rock ginseng, stone cowpea, stone bayberry, rock head Panax notoginseng, rock strips, bamboo thorns, Yuanlan, ground Kaihua. Medicinal material source: the whole herb of the Gesneriaceae plant stone spider plant. Latin plant animal mineral name: Lysionotuspauciflorus Maxim. Stone spider plant was first recorded in "Plant Names and Illustrations".

[0003] Non-patent literature [1]-[2] records that the spider plant has the effects of expectoration and cough relief, softening and dispersing nodules, and is often used to treat cough with sputum, scrofula and sputum nodules. Non-patent literature [3]-[8] found through modern chemical composition research that the spider plant mainly contains flavonoids, organic acids, phenylethanol glycosides and other components. Non-patent literature [9]-

[10] showed through pharmacological research that the spider plant has antibacterial, anti-inflammatory, anti-hepatotoxic, antihypertensive, anti-tuberculosis and lipid-lowering pharmacological effects. Non-patent literature

[11] reported that the ethyl acetate part of the spider plant has the effect of relieving cough and expectoration, and the active ingredients of the ethyl acetate part may be flavonoids and their glycosides; non-patent literature

[12] reported that the total polyphenols of the spider plant have antioxidant activity. It can be seen that different types of chemical components of the spider plant may have different pharmacological effects. Therefore, clarifying the main chemical composition of the spider plant is of great significance for subsequent pharmacodynamic research and promoting the development and utilization of medicinal resources. In the 2020 edition of the "Chinese Pharmacopoeia", the content of chelidonide is used as its quality control standard.

[0004] In addition, non-patent literature

[13] to

[15] determined the content of total flavonoids and flavonoids in spider plants. However, the chemical composition of spider plants is complex and contains many types of chemical components. The determination of a single component or a single type of component, such as total flavonoids, cannot reflect the overall quality characteristics of the medicinal material. In addition, the efficacy of traditional Chinese medicine is achieved through a complex mechanism of multiple components and multiple targets to exert a synergistic effect to achieve the purpose of treating diseases.

[0005] Note: Non-patent literature [1]: Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China: Volume 1 [S]. 2020 edition, Beijing: China Medical Science and Technology Press, 2020: 92.

[0006] Non-patent literature [2]: Guizhou Provincial Drug Administration. Quality Standards for Traditional Chinese and Ethnic Medicinal Materials in Guizhou Province (2003 Edition) [S]. Guiyang: Guizhou Science and Technology Press, 2003: 120.

[0007] Non-patent literature [3]: Liu Y, Wagner H, Bauer R. Nevadensin glycosides from Lysionotus pauciflorus [J]. Phytochemistry, 1996, 42(4): 1203.

[0008] Non-patent literature [4]: ​​Feng Weisheng, Li Qian, Zheng Xiaoke, et al. Chemical constituents from the plant Chimonanthus radiata[J]. Natural Product Research and Development, 2006(04):617-620.

[0009] Non-patent literature [5]: Li Jilong, Liu Jianhua, Gao Yuqiong, et al. Study on the volatile oil components of Chlorophytum comosum[J]. Chinese Pharmacy, 2011, 22(27): 2560-2562.

[0010] Non-patent literature [6]: Zhang JY, Yi P, Xiong Y, et al. A new acoranesesquiterpenes of Lysionotus pauciflorus maxim. Form Guizhou province, China [J]. Biochemical Systematics and Ecology, 2020, 93: 104165.

[0011] Non-patent literature [7]: Luo W, Wen YY, Tu YB, et al. A New Flavonoid Glycoside from Lysionotus pauciflorus [J]. Natural product communications, 2016, 11(5): 621-622.

[0012] Non-patent literature [8]: Hu Jun, Qi Mengdie, Zhang Quan, et al. Analysis of chemical components of the whole herb of Clerodendrum truncatum based on UPLC-Q-TOF-MS technology [J]. Modern Chinese Medicine, 2016, 18(05): 547-553+572.

[0013] Non-patent literature [9]: Zheng Xiaoke, Li Jun, Feng Weisheng, et al. Research progress of Gesneriaceae plants [J]. Chinese Journal of New Drugs, 2003(04):261-263.

[0014] Non-patent literature

[10] : Zhang YF, Liu QM, Liu B, et al. Dihydromyricetininhibited ovalbumin-induced mice allergic responses by suppressing the activation of mast cells [J]. Food & function, 2019, 10(11): 7131-7141.

[0015] Non-patent literature

[11] : Meng Xuefei, Li Yanfang. Study on the antitussive and expectorant effects of different polar extracts of Chlorophytum comosum[J]. West China Journal of Pharmaceutical Sciences, 2018, 33(05): 504-506.

[0016] Non-patent literature

[12] : Gou Tizhong, Tang Wenhua, Ren Yongquan, et al. Study on the antioxidant activity of total polyphenols from Chlorophytum comosum in vitro [J]. Food Industry Science and Technology, 2015, 36(05): 73-77.

[0017] Non-patent literature

[13] : Li Yan, Zhang Liyan, Yang Yuqin, et al. Comparative study on the content of flavonoids in Chlorophytum comosum seedlings from Guizhou Province [J]. Modern Chinese Medicine, 2011, 13(3): 11-13.

[0018] Non-patent literature

[14] : Zhou Hui, Luo Wei, Li Yanfang. Simultaneous determination of four flavonoid components in Clerodendrum truncatum from different origins by HPLC [J]. West China Journal of Pharmaceutical Sciences, 2021, 36(03): 361-364.

[0019] Non-patent literature

[15] : Sheng Weiguo, Xiong Yang, Xu Lianying. Determination of the content of chelidonin in chelidonium by HPLC [J]. Chinese Traditional Patent Medicine, 2009, 31(02): 321-322.

[0020] Therefore, the current methods for quality control of spider plants have certain limitations, cannot comprehensively control their intrinsic quality, and cannot provide a reasonable and controllable method for quality evaluation and control research of spider plants. Summary of the invention

[0021] In order to solve the above technical problems, the present invention provides a method for analyzing the chemical components of Chlorophytum comosum and determining the content thereof based on liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry.

[0022] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0023] A method for analyzing the chemical composition and content determination of Chlorophytum comosum based on liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry comprises preparing a reference solution, preparing a test solution, chromatographic analysis, and data processing, wherein the chemical composition analysis also includes mass spectrometry analysis.

[0024] Furthermore, the method for analyzing the chemical composition of Chlorophytum comosum specifically comprises the following steps:

[0025] ① Preparation of reference solution: accurately weigh forsythiaside B, physalisin, akebia phenylethanol glycoside B, plantain glycoside, verbascoside, chlorogenic acid, and neochlorogenic acid reference substances and place them in containers respectively, add methanol to stir and dissolve evenly and make up to volume to obtain reference substance stock solution, then add 50% methanol to dilute into reference substance solutions with concentrations of 422.20μg / mL, 32.63μg / mL, 201.12μg / mL, 21.09μg / mL, 31.63μg / mL, 100.00μg / mL, and 20.58μg / mL respectively;

[0026] ② Preparation of the test solution: Weigh the powder of the spider plant and place it in a container, add 50% methanol aqueous solution and weigh it, perform ultrasonic extraction for 40 minutes, cool to room temperature, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, centrifuge and take the supernatant, filter the filtrate through a microporous membrane to obtain the test solution;

[0027] ③ Determination: The reference solution and the test solution were subjected to chromatographic analysis, mass spectrometry analysis and data processing in turn, and the qualitative analysis of the chemical composition of the stone spider plant was completed through analysis.

[0028] Furthermore, the conditions for the chromatographic analysis are: the chromatographic column is Synergi TM Hydro-RP (100×2mm, 2.5μm); mobile phase A is acetonitrile, B is 0.1% formic acid water, elution gradient is 0~1min, 5~15%A; 1~6min, 15%A; 6~18min, 15~18%A; 18~20min, 18~25%A; 20~24min, 25~28%A; 24~27min, 28~32%A; 27~28min, 32~95%A; 28~34min, 95%A; 34~35min, 95~5%A; 35~40min, 5%A); column temperature: 40℃; flow rate: 0.2mL / min; injection volume: 2μL.

[0029] Furthermore, the conditions for the mass spectrometry analysis are as follows: the ion source is an electrospray ionization source; positive and negative ions are scanned separately; the scanning mode is: primary full scan-data-dependent secondary mass spectrometry scanning mode, with a scanning range of 100 to 1500 Da; capillary temperature: 320°C, spray voltage: 3.5 kV (positive mode), 2.5 kV (negative mode); sheath gas flow rate 35 arb; auxiliary gas flow rate 10 arb; auxiliary gas temperature: 350°C; in Ms / Ms mode, the collision energies in the positive, negative ion modes are all 20, 40, and 60 eV; the primary mass spectrometry resolution is 70,000 FWHM, and the secondary mass spectrometry resolution is 17,500 FWHM.

[0030] Furthermore, the data processing is as follows: the mass spectrometry data is processed using Thermo Xcalibur 4.4 and Compound Discoverer TM 3.2 Processing, import the raw data collected by mass spectrometry into Compoud Discoverer TM 3.2 The software performs preliminary calculations and compares the retention time of the main chromatographic peaks of the test solution, the quasi-molecular ions of the primary mass spectrometry, and the characteristic fragment ion information of the secondary mass spectrometry with the chromatographic retention time of the reference solution, the compound databases such as the mzCloud library, mzVault library, Chemspider library, and the mass spectrometry information of relevant literature to perform qualitative analysis on the main chromatographic peaks.

[0031] Furthermore, the determination of the chemical component content of the stone spider plant specifically includes the following steps:

[0032] (1) Preparation of reference substance solutions: accurately weigh neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and scutellaria baicalensis respectively and place them in containers, dissolve them with methanol and make up to volume to obtain reference substance stock solutions with concentrations of 1.0290 mg / mL, 1.0270 mg / mL, 1.0555 mg / mL, 2.0003 mg / mL, and 1.0876 mg / mL, respectively. Then, add 20% methanol solution to dilute them into reference substance solutions with concentrations of 25.725 μg / mL, 102.704 μg / mL, 253.003 μg / mL, 200.027 μg / mL, and 32.628 μg / mL, respectively;

[0033] (2) Preparation of the test solution: Accurately weigh the powder of the spider plant and place it in a container, add 50% methanol aqueous solution and weigh it, extract it by ultrasonication for 40 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake it well, filter it, take the supernatant after centrifugation, and filter the filtrate through a microporous membrane to obtain the test solution;

[0034] (3) Determination: The reference solution and the test solution are subjected to chromatographic analysis respectively, and then the experimental data are processed to complete the HPLC content determination.

[0035] Furthermore, in step ② and step (2), the mass ratio of the spider plant powder to methanol is 1:50.

[0036] Furthermore, in step ② and step (2), the centrifugation is to centrifuge the filtrate at 12000 r / min for 10 min.

[0037] Furthermore, in step ② and step (2), the filtration is through a 0.22 μm microporous filter membrane.

[0038] Furthermore, 84 compounds were identified in Chlorophytum comosum, including 27 flavonoids, 20 phenylethanol glycosides, 5 amino acids, 18 organic acids, 1 alkaloid, 6 nucleosides and 7 other compounds.

[0039] In this application, the purity of forsythiaside B is 98%, the purity of physalisin is 99%, the purity of oxalis phenylethanol glycoside B is 95%, the purity of plantain glycoside is 98%, the purity of verbascoside is 98%, the purity of chlorogenic acid is 98%, the purity of neochlorogenic acid is 98%, methanol, acetonitrile and formic acid are all chromatographically pure, the water used is ultrapure water, and the other reagents are analytically pure. The detailed information of 25 batches of physalis medicinal materials is shown in Table 1. All medicinal materials were identified as the dried aerial parts of physalis Lysionotuspauciflorus Maxim. by Associate Professor Liu Chunhua of the Key Laboratory of Drug Preparations of Guizhou Province.

[0040] Table 1 Information on each batch of Chlorophytum comosum medicinal materials

[0041] serial number Origin Collection time serial number Origin Collection time S1 Hezhang County, Guizhou 20200701 S14 Zhijin County, Guizhou 20210504 S2 Panzhou City, Guizhou 20200701 S15 Zhijin County, Guizhou 20210504 S3 Panzhou City, Guizhou 20200701 S16 Zhijin County, Guizhou 20210504 S4 Panzhou City, Guizhou 20200701 S17 Zhijin County, Guizhou 20210504 S5 Panzhou City, Guizhou 20200701 S18 Zhijin County, Guizhou 20210504 S6 Panzhou City, Guizhou 20200701 S19 Zhijin County, Guizhou 20210504 S7 Panzhou City, Guizhou 20200701 S20 Zhijin County, Guizhou 20210504 S8 Qianxi County, Guizhou 20210708 S21 Zhijin County, Guizhou 20210501 S9 Zhijin County, Guizhou 20210501 S22 Guanling County, Guizhou 20210722 S10 Zhijin County, Guizhou 20210502 S23 Changshun County, Guizhou 20210722 S11 Zhijin County, Guizhou 20210502 S24 Guiyang City, Guizhou 20210722 S12 Zhijin County, Guizhou 20210502 S25 Jinsha County, Guizhou 20210925 S13 Zhijin County, Guizhou 20210504

[0042] This application uses ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry (UHPLC-Q-Exactive Plus Orbitrap HRMS) technology for the first time to systematically analyze the chemical components of Chlorophytum comosum, and establishes an HPLC-UV content determination method for its main chemical components, which provides an efficient and rapid analysis method for the qualitative analysis of the chemical components of Chlorophytum comosum and its quality control. Since traditional Chinese medicine contains many chemical components and different compounds respond differently in different modes, positive and negative ion modes are used for scanning respectively, and the chemical components contained therein are analyzed more comprehensively through the complementation of structural information. Using SynergiTM Hydro-RP (100×2mm, 2.5μm) chromatographic column, acetonitrile-0.1% formic acid aqueous solution as mobile phase for gradient elution, flow rate 0.2mL·min -1, column temperature 40℃, mass spectrometry analysis using electrospray ionization source (ESI), data acquisition in positive and negative ion mode respectively; combined with the reference retention time, mass spectrometry ion information and related mass spectrometry data information in the database and literature for comparison, to identify the chemical components in the spider plant; further HPLC-UV method was used to simultaneously determine the contents of 5 components (neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, spider plant glycoside) in different batches of spider plants, and the detection wavelength was 330nm. A total of 84 compounds were identified from the spider plant test solution, including 27 flavonoids, 20 phenylethanol glycosides, 5 amino acids, 18 organic acids, 1 alkaloid, 6 nucleosides and 7 other compounds. The above five components have a good linear relationship with the peak area within a certain concentration range (r>0.999), and the linear ranges of each component are 3.22-102.90μg / mL, 12.84-410.82μg / mL, 31.63-1012.01μg / mL, 25.00-800.11μg / mL, and 4.08-130.51μg / mL, respectively. The instrument precision, method repeatability, and solution stability are all good. The average recovery rate ranges from 97.3% to 100.2%, and the RSD ranges from 0.95% to 2.36%. There are certain differences in the contents of the five components in different batches of Herba Chlorophytum from different origins in Guizhou, among which the average content of Forsythiaside B is the highest.

[0043] The main components are flavonoids, phenylethanol glycosides and organic acids. In addition, there are terpenoids, benzyl alcohol glycosides, anthraquinones, coumarins, nucleosides and amino acids. Among them, flavonoids have good responses in both positive and negative ion scanning modes, phenylethanol glycosides and organic acids have good responses in negative ion scanning mode, alkaloids and coumarin compounds have strong responses in positive ion mode and weak responses in negative ion mode. Flavonoid components represented by scutellaria sutchuenensis are used as indicators for quality control of medicinal materials. They have multiple effects such as antibacterial, anti-inflammatory, antitussive, antihypertensive, anti-tuberculosis and hypolipidemic effects (refer to non-patent literature

[11] ~

[39] ); phenylethanol glycoside components have anti-inflammatory and liver protection effects (refer to non-patent literature [9]). The qualitative analysis method established in this application can quickly and efficiently identify the chemical components of scutellaria sutchuenensis. The quantitative analysis method established is simple, fast and accurate, and can simultaneously determine the contents of 5 components in scutellaria sutchuenensis. This application helps to understand the material basis of its medicinal effects by identifying the chemical components in Chlorophytum comosum, and provides a scientific basis for further research on its material basis of medicinal effects, mechanism of action, clinical application development and medicinal material quality evaluation.

[0044] At present, the content determination of spider plant (refer to non-patent literature

[13] ~

[15] ) is mostly focused on the establishment of the content determination method of the main component spider plant, or the quality research is carried out with one type of component as the index component, which cannot evaluate its quality more comprehensively. Since spider plant mainly contains flavonoids, phenylethanol glycosides and organic acids, and these types of chemical components have significant biological activity, this application screened the main representative components of these components, neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside and spider plant, as index components to establish a content determination method, and carried out methodological verification experiments respectively. The results show that the method has strong specificity, good linear relationship, and the precision and repeatability meet the requirements. It can be used for the content determination of the above five chemical components in spider plant. This application is the first to simultaneously conduct quantitative analysis of three types of components in Stone Spider Plant, namely flavonoids, phenylethanol glycosides and organic acids. The results show that the contents of these five components in medicinal materials from different origins vary greatly. This may be due to factors such as different origins, growth environment, climatic conditions, and harvest time that affect the accumulation of compounds in plants. The specific reasons need further study.

[0045] Since the present invention adopts the above technical solution, it has the following beneficial effects:

[0046] (1) In view of the advantages of LC-MS technology such as high resolution, high mass accuracy, wide mass range and wide dynamic range, this application uses UHPLC-Q-Exactive Plus Orbitrap HRMS LC-MS technology to conduct a comprehensive analysis of the medicinal components of Clerodendrum truncatum, in order to provide a reliable basis for subsequent research on the material basis of related efficacy, mechanism of action and clinical application; and on this basis, a high performance liquid chromatography (HPLC-UV) method is established for the simultaneous determination of the contents of five components, namely, neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside and closaside, in different batches of Clerodendrum truncatum, for the main representative components of different types identified by mass spectrometry, so as to provide a reasonable and controllable method for the quality evaluation and control research of Clerodendrum truncatum.

[0047] (2) This application uses UHPLC-Q-Exactive Plus Orbitrap HRMS technology to identify 84 compounds in Clerodendrum truncatum, including 27 flavonoids, 20 phenylethanol glycosides, 5 amino acids, 18 organic acids, 1 alkaloid, 6 nucleosides and 7 other compounds; at the same time, an HPLC-UV content determination method for 5 different types of components in Clerodendrum truncatum (neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and Clerodendrum truncatum) was established, with good linearity, and the system applicability, stability, repeatability, precision and recovery rate can meet the requirements of quantitative analysis, and can be used for content determination and analysis of Clerodendrum truncatum medicinal materials. This application method provides a certain scientific basis for further research on Clerodendrum truncatum quality control methods, pharmacodynamic material basis, mechanism of action and new drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the examples of the present invention or the prior art, the drawings required in the implementation examples or the prior art description are briefly introduced below. Obviously, the drawings in the following description are only some examples of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without inventiveness:

[0049] Figure 1 The total ion chromatogram of SDL in UHPLC-Q-Exactive PlusOrbitrap HRMS in positive ion mode (A) and negative ion mode (B) for this application;

[0050] Figure 2 UHPLC-Q-ExactivePlus Orbitrap HRMS total ion chromatograms of the mixed reference substance in the positive ion mode (A) and negative ion mode (B) of this application;

[0051] Figure 3 MS / MS secondary mass spectrum of the chlorella extract in this application;

[0052] Figure 4 MS / MS mass spectrometry fragmentation pathway diagram of the chlorella in this application;

[0053] Figure 5 MS / MS secondary mass spectrum of psyllium glycosides in this application;

[0054] Figure 6 The MS / MS mass spectrometry fragmentation pathway diagram of psyllium glycosides in this application;

[0055] Figure 7 New chlorogenic acid MS / MS secondary mass spectrum of this application;

[0056] Figure 8 New chlorogenic acid MS / MS mass spectrometry fragmentation pathway diagram of this application;

[0057] Fig. 9 Adenosine MS / MS secondary mass spectrum of this application;

[0058] Fig.10 Adenosine MS / MS mass spectrometry fragmentation pathway diagram of this application;

[0059] Fig.11 MS / MS secondary mass spectrum of genipin-1-β-D-gentiobioside in this application;

[0060] Fig.12 MS / MS mass spectrometry fragmentation pathway diagram of genipin-1-β-D-gentiobioside in this application;

[0061] Fig.13 HPLC charts of the SDL sample solution (A) and mixed reference solution (B) of this application.

[0062] In the attached figure: 1-neochlorogenic acid; 2-chlorogenic acid; 3-forsythiaside B; 4-verbascoside; 5-chlorophyllin. DETAILED DESCRIPTION

[0063] The specific embodiments of the present invention are further described in detail below, but the present invention is not limited to these embodiments, and any improvement or substitution based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0064] Example

[0065] The method for analyzing the chemical components of Chlorophytum comosum and determining the content thereof based on liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry comprises preparing a reference solution, preparing a test solution, chromatographic analysis, and data processing, wherein the chemical component analysis also comprises mass spectrometry analysis; the method for analyzing the chemical components of Chlorophytum comosum comprises the following steps:

[0066] ① Preparation of reference solution: accurately weigh forsythiaside B, physalisin, akebia phenylethanol glycoside B, plantain glycoside, verbascoside, chlorogenic acid, and neochlorogenic acid reference substances and place them in containers respectively, add methanol to stir and dissolve evenly and make up to volume to obtain reference substance stock solution, then add 50% methanol to dilute into reference substance solutions with concentrations of 422.20μg / mL, 32.63μg / mL, 201.12μg / mL, 21.09μg / mL, 31.63μg / mL, 100.00μg / mL, and 20.58μg / mL respectively;

[0067] ② Preparation of the test solution: accurately weigh the powder of the spider plant and place it in a container, add 50% methanol aqueous solution and weigh it, ultrasonically extract for 40 minutes, cool to room temperature, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, centrifuge, take the supernatant, filter the filtrate through a microporous membrane, and obtain the test solution;

[0068] ③ Determination: The reference solution and the test solution were subjected to chromatographic analysis, mass spectrometry analysis and data processing in turn, and the qualitative analysis of the chemical composition of the stone spider plant was completed through analysis.

[0069] Furthermore, the conditions for the chromatographic analysis are: the chromatographic column is Synergi TM Hydro-RP (100×2mm, 2.5μm); mobile phase A is acetonitrile, B is 0.1% formic acid water, elution gradient is 0~1min, 5~15%A; 1~6min, 15%A; 6~18min, 15~18%A; 18~20min, 18~25%A; 20~24min, 25~28%A; 24~27min, 28~32%A; 27~28min, 32~95%A; 28~34min, 95%A; 34~35min, 95~5%A; 35~40min, 5%A); column temperature: 40℃; flow rate: 0.2mL / min; injection volume: 2μL. The conditions of mass spectrometry analysis are as follows: the ion source is an electrospray ionization source; positive and negative ions are scanned separately; the scanning mode is: primary full scan-data-dependent secondary mass spectrometry scanning mode, scanning range 100-1500Da; capillary temperature: 320°C, spray voltage: 3.5kV (positive mode), 2.5kV (negative mode); sheath gas flow rate 35arb; auxiliary gas flow rate 10arb; auxiliary gas temperature: 350°C; in Ms / Ms mode, the collision energy in positive and negative ion modes is 20, 40, and 60eV; the primary mass spectrometry resolution is 70000FWHM, and the secondary mass spectrometry resolution is 17500FWHM. The data processing is as follows: The mass spectrometry data is processed using Thermo Xcalibur 4.4 and Compound Discoverer TM 3.2 Processing, import the raw data collected by mass spectrometry into Compoud Discoverer TM 3.2 The software performs preliminary calculations and compares the retention time of the main chromatographic peaks of the test solution, the quasi-molecular ions of the primary mass spectrometry, and the characteristic fragment ion information of the secondary mass spectrometry with the chromatographic retention time of the reference solution, the compound databases such as the mzCloud library, mzVault library, Chemspider library, and the mass spectrometry information of relevant literature to perform qualitative analysis on the main chromatographic peaks.

[0070] Furthermore, the determination of the chemical component content of the stone spider plant specifically includes the following steps:

[0071] (1) Preparation of reference substance solutions: accurately weigh neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and scutellaria baicalensis respectively and place them in containers, dissolve them with methanol and make up to volume to obtain reference substance stock solutions with concentrations of 1.0290 mg / mL, 1.0270 mg / mL, 1.0555 mg / mL, 2.0003 mg / mL, and 1.0876 mg / mL, respectively. Then, add 20% methanol solution to dilute them into reference substance solutions with concentrations of 25.725 μg / mL, 102.704 μg / mL, 253.003 μg / mL, 200.027 μg / mL, and 32.628 μg / mL, respectively;

[0072] (2) Preparation of the test solution: Accurately weigh the powder of the spider plant and place it in a container, add 50% methanol aqueous solution and weigh it, extract it by ultrasonication for 40 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake it well, filter it, take the supernatant after centrifugation, and filter the filtrate through a microporous membrane to obtain the test solution;

[0073] (3) Determination: The reference solution and the test solution are subjected to chromatographic analysis respectively, and then the experimental data are processed to complete the HPLC content determination.

[0074] Furthermore, in step ② and step (2), the mass ratio of the spider plant powder to methanol is 1:50; the centrifugation is to centrifuge the filtrate at 12000r / min for 10min; and the filtration is through a 0.22μm microporous filter membrane.

[0075] 1. Mass spectrometry identification results

[0076] A total of 84 compounds were identified from the spider plant, including 27 flavonoids, 20 phenylethanol glycosides, 5 amino acids, 18 organic acids, 1 alkaloid, 6 nucleosides and 7 other compounds. Among them, 7 peaks were confirmed by comparison with the reference substance. Peaks 24, 28, 44, 49, 54, 55 and 83 were confirmed to be neochlorogenic acid, chlorogenic acid, plantagoside, forsythiaside B, verbascoside, akebia phenylethanol glycoside B and spider plant glycoside, respectively. The information of each chemical component is shown in Table 2. The total ion current of the test sample and the reference substance in the positive and negative ion modes are shown in Table 2. Figure 1 , Figure 2 shown.

[0077] Table 2 UHPLC-Q-Exactive Plus Orbitrap HRMS mass spectrometry information of chemical components in SDL

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Note: In Table 2: CA: caffeic acid; FA: ferulic acid; p-CoA: p-coumaric acid; Glc: glucose; GlcA: glucuronic acid; Gal: galactose; Ara: arabinose; Api: apiose; Xyl: xylose; a. flavonoids; b. phenylethanol glycosides; c. amino acids; d. organic acids; e. alkaloids; f. nucleosides; g. other categories; * confirmed by comparison with reference substances;

[0084]

[16] Zhang Yu, Dong Wenting, Huo Jinhai, et al. Analysis of chemical components of Pteris crenata L. based on UPLC-Q-TOF-MS technology[J]. Chinese Herbal Medicine, 2017, 48(02): 252-262.

[0085]

[17] Liu Xiangyi, Zeng Rui, Yang Changchuan, et al. Analysis of chemical components of Sancao Baogan Decoction based on UPLC-ESI-HRMS~n technology[J]. Chinese Journal of Experimental Traditional Chinese Medicine Formulae, 2020, 26(07): 141-147.

[0086]

[18] Wang Yi, Feng Liping, Huang Lilu, et al. Rapid identification of chemical components of Hibiscus mutabilis flowers by UPLC-Q-Orbitrap HRMS technology[J]. Natural Product Research and Development, 2021, 33(12): 2042-2052.

[0087]

[19] Sui Zhenyu, Hou Pengyi, Wang Bo. Rapid identification of chemical components in Polygonatum odoratum based on UPLC-Q-Orbitrap coupling technology[J]. Chinese Pharmacist, 2022, 25(04): 626-630.

[0088]

[20] Sun HY, Liu MX, Lin ZT, et al. Comprehensive identification of 125multifarious constituents in Shuang-huang-lian powder injection by HPLC-DAD-ESI-IT-TOF-MS. [J] JPharm Biomed Anal, 2015, 115: 86-106.

[0089]

[21] Han Xinlong, Cheng Xin, Sun Yingchao, et al. Analysis of chemical components of Qinghao Biejia Decoction based on UHPLC-Orbitrap-MS / MS[J]. Chinese Traditional Patent Medicine, 2021, 43(12): 3531-3535.

[0090]

[22] Yang Lumeng, Yang Lingjian, Jia Pu, et al. Analysis of chemical components of Ziziphus axillaris pulp based on HPLC-Q-TOF-MS / MS[J]. Journal of Second Military Medical University, 2016, 37(02): 159-166.

[0091]

[23] Xiang Qing, Wang Xiaohua, Lin Hui, et al. Qualitative and quantitative study of the main components of Yinhuang granules by HPLC-DAD-Q-TOF-MS / MS[J]. Chinese Traditional Patent Medicine, 2015, 37(01): 105-112.

[0092]

[24] Han De'en, Yue Zhongsheng, Li Hongwei, et al. Study on the antidepressant components and their mechanisms of action of Rehmannia glutinosa based on UPLC-Q-Orbitrap HRMS combined with network pharmacology[J]. China Journal of Traditional Chinese Medicine, 2022, 47(04): 1051-1063.

[0093]

[25] Xiao Guanlin, Jiang Jieyi, Xu Aili, et al. Differences in chemical components of Buzha leaves from different origins based on UPLC-Q-TOF-MS / MS combined with multivariate statistical analysis [J]. Hunan Journal of Traditional Chinese Medicine, 2021, 37(12): 171-177.

[0094]

[26] Ren Xiaolei, Huo Jinhai, Sun Guodong, et al. UPLC-Q-TOF-MS analysis of chemical components of Trollius brevis[J]. Chinese Herbal Medicine, 2015, 46(23): 3475-3481.

[0095]

[27] Hu Jun, Zhang Quan, Qi Mengdie, et al. Analysis of chemical components in Chimonanthus radiata L. based on UPLC-Q-TOF-MS technology[J]. China Journal of Traditional Chinese Medicine, 2016, 41(09): 1658-1669.

[0096]

[28] Xiao Qiming, Yu Qianqian, Yu Xinwei, et al. Analysis of metabolic characteristics of conycin in rats based on UPLC-Q-TOF / MS[J]. Journal of Wenzhou Medical University, 2022, 52(06): 478-485.

[0097]

[29] Zhang Yu, Yang Jian, Zhan Zhilai, et al. UPLC-MS / MS determination of phenylethanoid glycosides in Glechoma longituba from different origins[J]. Modern Chinese Medicine, 2017, 19(04): 504-508.

[0098]

[30] Sun Yujing, Huo Zhipeng, Wang Yu, et al. UPLC-Q-TOF / MS-E analysis of chemical components in Qixiao Granules for Colonitis[J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2021, 27(09): 157-167.

[0099]

[31] Zhang HJ, Yao W, Chen YY, et al. Simultaneous determination of fourmajor constituents of semen Vaccariae using HPLC-DAD[J]Nat Prod Commun,2012,7:1185-6.

[0100]

[32] Luo Yi, Zhang Le, Wang Weihua, et al. Identification of chemical components of Cassia seed by high performance liquid chromatography-ion trap-time of flight mass spectrometry[J]. Journal of Pharmaceutical Analysis, 2015, 35(08): 1408-1416.

[0101]

[33] Greenham J, Vassiliades DD, Harborne JB, et al. A distinctive flavonoid chemistry for the anomalous genus Biebersteinia[J] Phytochemistry, 2001,56:87-91.

[0102]

[34] Liu MC, Yang SJ, Jin LH, et al. Chemical constituents of the ethylacetate extract of Belamcanda chinensis(L.)DC roots and their antitumoractivities[J] Molecules,2012,17:6156-69.

[0103]

[35] Yang Fangqing, He Liying, Yang Dan, et al. UPLC-Q-Orbitrap HRMS analysis of flavonoid components in dried orange peel with different aging time[J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2021, 27(12): 125-132.

[0104]

[36] Yong L,Hildebert W,Rudolf B.Nevadensin glycosides from Lysionotuspauciflorus[J].Phytochemistry,1996,42(4):1203-1205.

[0105]

[37] Yong L,Hildebert W,Rudolf B.Phenylpropanoids and flavonoidglycosides from Lysionotus pauciflorus[J].Phytochemistry,1998,48(2):339-343.

[0106]

[38] Yang Li, Yuan Fengrui, Cao Lan, et al. Rapid identification of chemical components of Chai Shi Tuire Granules based on UPLC-ESI-Q-TOF-MS / MS technology[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2021, 27(17): 152-159.

[0107]

[39] Liang CJ, Zhang

[0108]

[40] Xi Lisha, Mu Taihua, Sun Hongnan. Research progress of chlorogenic acid at home and abroad[J]. Journal of Nuclear Agricultural Sciences, 2014, 28(02): 292-301.

[0109]

[41] Zeng Bin, Liu Hongmei, Liu Xiaomei, et al. Application of UPLC-Q-Exactive Orbitrap MS technology in traditional Chinese medicine analysis [J]. Chinese Medicinal Materials, 2020, 43(9): 2313-2319.

[0110] 1.1 Pyrolysis characteristics of flavonoids

[0111] Flavonoids are a class of compounds with a basic nucleus of 2-phenylchromone. Most flavonoid components exist in plants in the form of free or combined with sugars to form oxygen glycosides or carbon glycosides. A total of 27 flavonoids were identified from the spider plant, including compounds 31, 36-38, 43, 45, 51, 59, 64-68, 70-77 and 79-84. Compounds 31, 71, 74 and 83 were used as examples to analyze the fragmentation rules. The mass spectrometry fragmentation rules of flavonoid aglycones are mainly the loss of carbonyl CO, CO2, neutral water molecules H2O and side chain substituents in the C ring, and are prone to reverse Diels-Alder (RDA) fragmentation. For example, compound 71 obtained a quasi-molecular ion m / z 285.0405 [MH] in negative ion mode. - , its molecular formula is predicted to be C15 H 10 O6. In MS / MS, it loses CO and fragments to m / z 257.0444 [MH-CO] - , and RDA fragmentation occurred simultaneously to form the fragment ion m / z 151.0028 [MH-C8H6O2] - 、m / z 133.0282[MH-C7H4O4] - , m / z 151.0028 continued to lose CO2 and finally cracked into m / z 107.0125 [MH-C8H6O2-CO2] - . Based on the fragmentation characteristics of the compound's secondary mass spectrometry, after database retrieval and reference to non-patent literature

[23] , the compound was confirmed to be luteolin. The main fragmentation mode of flavonoid glycosides is to first lose sugar to obtain a strong aglycone ion peak, and then fragment according to the type of aglycone. The molecular ion peak of the glycoside does not appear. Taking compound 74 as an example, the retention time of compound 74 is 32.11min. In the positive ion mode, its quasi-molecular ion peak is m / z 653.2056 [M+H] + , its molecular formula is predicted to be C 30 H 36 O 16 The secondary mass spectrometry fragment ions are: 507.151 [M + H-Rha] + 、345.0961[M+H-Rha-Glc] + 、330.0723[M+H-Rha-Glc-CH3] + 、315.0492[M+H-Rha-Glc-2CH3] + 、300.0596[M+H-Rha-Glc-3CH3] + ,287.0544[M+H-Rha-Glc-2CH3-CO] + Combined with the reference non-patent literature

[37] and mass spectrometry database information, the compound was identified as Nevadensin 7-rutinoside. The retention time of flavonoid carbon glycoside compound 31 was 9.80min, and its quasi-molecular ion peak in negative ion mode was m / z 593.1509 [MH] - , its molecular formula is estimated to be C 27 H 30 O 15. The fragment ions m / z 503.1186 and m / z 473.1080 were observed in the secondary mass spectrum, indicating that they were generated by the loss of the 0-3 bond of hexose C3H6O3 (90) and the loss of the 0-2 bond of hexose C4H8O4 (120). At the same time, the fragment ion m / z 383.0771 [MH-C4H8O4-C3H6O3] was observed in the secondary mass spectrum. - and m / z 353.0663 [MH-C4H8O4-C4H8O4] - , it is speculated that the compound is a disaccharide carbonyl glycoside. m / z 353.0677 loses two molecules of carbonyl CO (28) in succession to generate a fragment peak m / z 325.0712 [MH-2C4H8O4-CO] - , m / z 297.0764[MH-2C4H8O4-2CO] - The mass spectrometry data is consistent with the report in the non-patent literature

[25] , and it is inferred that the compound is vicinin-2. The mass spectrometry fragmentation characteristics of polymethoxy-substituted flavonoids are obvious, and they can strongly lose -CH3 on each substituent. The RDA fragmentation of the C ring is not obvious. Taking compound 83 as an example, the retention time of compound 83 is 34.87min, and its quasi-molecular ion peak in negative ion mode is m / z 343.0818 [MH] - , its molecular formula is predicted to be C 18 H 16 O7. Secondary mass spectrometry fragment ions are: m / z 328.0585[MH-CH3]-, m / z 313.0351[MH-2CH3] - 、m / z298.0113[MH-3CH3] - 、m / z 270.0166[MH-3CH3-CO] - 、m / z 254.0216[MH-3CH3-CO-O] - , m / z254.0216[MH-2CH2O-CH2-CO] - Combined with the reference non-patent literature

[39] , the spectrum of the reference substance and the mass spectrum database information, the compound was identified as chelidonin. Its secondary mass spectrum and possible fragmentation rules are as follows: Figure 3 , Figure 4 The remaining flavonoids all had similar fragmentation patterns and were identified one by one based on the primary and secondary mass spectrometry fragment information. All the information is shown in Table 3.

[0112] 1.2 Pyrolysis characteristics of phenylethanol glycosides

[0113] Phenylethanol glycosides are a class of compounds with β-glucose as the parent nucleus, glycosylated with α-hydroxyphenylethyl or esterified with phenylacrylate at the same time; the central glucose group is often connected with acetyl, caffeoyl, feruloyl, coumaroyl or rhamnose, arabinose, apiose, xylose, glucose and other sugar groups. The differences in the types, positions and branching sequences of sugars attached to the sugar nucleus, as well as the differences in the substituent groups on the phenylacrylate and phenylethyl groups, result in the diversity of the structures of phenylethanol glycoside compounds. This application uses data information from positive and negative ion modes to structurally identify phenylethanol glycoside components, and finally identifies 20 phenylethanol glycoside components through the study of the cleavage rules of reference substances, combined with the isolated components of the genus Ceropegia and related mass spectrometry literature reports. The fragmentation patterns of the secondary mass spectra of these 20 phenylethanol glycosides are very similar, mostly showing that the 162Da caffeoyl group of the phenylpropanoid part is easily lost; the sugar part loses the neutral fragment ions of 132, 146 and 162Da, and the corresponding sugars are pentoses (C5H8O4) such as arabinose, celery sugar and xylose, deoxyhexose such as rhamnose (C6H 10 O4) and hexoses such as glucose (C6H 10 O5), etc. Taking verbascoside (peak 54), plantagoside (peak 44) and myricetin (peak 63) as examples, the quasi-molecular ion of verbascoside in negative ion mode is m / z 623.1971 [MH] - , the molecular formula is C 29 H 36 O 15 The main fragment ion in the secondary mass spectrum is m / z 461.1661[MH-caffeoyl] - 、m / z 179.0342[CA-H] - 、m / z 161.0233[CA-H2O] - , m / z 135.0439[MH-caffeoyl-Rha-Glc-H2O] - 、m / z 133.02821[CA-H2O-CO] - , which is consistent with the secondary fragment ion produced by the reference substance, so it is inferred that this compound is verbascoside. Similarly, the quasi-molecular ion of plantago glycoside in negative ion mode is m / z 639.1923[MH]-, and the molecular formula is C 29 H 36 O 16 The main fragment ion in the secondary mass spectrum is 477.1605 [MH-caffeoyl] - 、315.1085[MH-caffeoyl-Glc] - 、179.0342[CA-H] - 、161.0232[CA-H2O]- ,135.0438[MH-caffeoyl-2Glc-H2O] - Combined with the literature and the spectrum of the reference substance, it is inferred that the compound is psyllium glycoside. Its fragmentation pattern and secondary mass spectrum are shown in Figure 5 , Figure 6 . Quasi-molecular ion of myricetin m / z755.2395[MH] - , the molecular formula is C 34 H 44 O 19 The main difference between it and psyllium is that it contains an additional apiose. The main fragment ion produced in the secondary mass spectrometry is m / z 593.2086 [MH-caffeoyl] - , m / z 461.1656[MH-caffeoyl-Api] - , m / z 315.1075[MH-caffeoyl-Api-Rha] - , m / z135.0440[MH-caffeoyl-Api-Rha-Glc-H2O] - , which is basically consistent with the data in non-patent literature [8], and it is speculated that the compound is myricetin. The remaining phenylethanol glycosides were also identified one by one according to similar rules.

[0114] 1.3 Pyrolysis characteristics of amino acid compounds

[0115] Amino acids are a class of compounds containing amino and carboxyl groups and are the basic units of protein molecules. Therefore, the fragmentation characteristics of secondary mass spectrometry are usually the continuous loss of NH3, H2O, HCOOH and other fragments. Compound 1 produces a quasi-molecular ion peak m / z 175.1185 [M+H] in positive ion mode. + The secondary fragment is m / z 158.0921[M+H-NH3] + 、m / z 130.0974[M+H-NH3-CO] + 、m / z116.0706[M+H-NH3-CH2N2] + , m / z 70.0657[M+H-CH4N3-CO2-H2O] + , m / z 60.0564[M+H-NH3-COOH-C4H5] + , which was confirmed to be arginine by comparison with non-patent literature

[16] . The quasi-molecular ion peak of compound 18 in positive ion mode was m / z 166.0862 [M+H] + The fragment ions produced by the secondary mass spectrometry are m / z 149.0591 [M+H-H2O] +、m / z 131.0489[M+H-H2O-NH3] + 、m / z 120.0807[M+H-HCOOH] + , m / z 103.0544[M+H-NH3-HCOOH] + After comparison with databases and non-patent literature

[18] , the compound was identified as L-phenylalanine. Finally, a total of 5 amino acid components were identified from the spider plant.

[0116] 1.4 Pyrolysis characteristics of organic acid compounds

[0117] Organic acid compounds are acidic organic compounds widely found in plants in nature. They have strong antioxidant, antibacterial, anti-inflammatory and other pharmacological effects. They are prone to lose CO, CO2, -COOH, H2O, etc. in the mass spectrum to produce fragment ion peaks. A total of 18 organic acid compounds were identified from the extract of the stonewort, including salicylic acid, quinic acid, citric acid and chlorogenic acid. The retention time of compound 5 was 1.28min. In the negative ion mode, the quasi-molecular ion peak obtained by the first-level full scan was m / z 191.0550 [MH] - The molecular formula was fitted by Xcalibar4.1.31 software as C7H 11 O6, secondary fragment ion m / z 173.0442 [MH-H2O] - 、m / z 127.0388[MH-2H2O-CO] - 、m / z 109.0281[MH-3H2O-CO] - Combined with the relevant non-patent literature

[20] (Sun HY, Liu MX, Lin ZT, et al. Comprehensive identification of 125 multifarious constituents in Shuang-huang-lian powder injection by HPLC-DAD-ESI-IT-TOF-MS. [J] J Pharm Biomed Anal, 2015, 115: 86-106), it is speculated that it may be quinic acid (QA). The quasi-molecular ion peak of compound 29 is m / z 179.0340 [MH] -, the most likely molecular formula is inferred to be C9H7O4. The fragment ion m / z 135.0438 [MH-CO2]- appeared in its secondary fragment ions. After consulting the non-patent literature

[20] , it was confirmed to be caffeic acid (CA). Combined with the non-patent literature

[40] (Xi Lisha, Mu Taihua, Sun Hongnan. Research progress of chlorogenic acid substances at home and abroad [J]. Journal of Nuclear Agricultural Sciences, 2014, 28 (02): 292-301), chlorogenic acid compounds are esters formed by the condensation of one or more caffeic acid, ferulic acid, coumaric acid and quinic acid and their derivatives, and have multiple effects such as anti-inflammatory, antioxidant, antiviral, and anti-tumor. The HESI-MS2 of chlorogenic acid compounds has obvious characteristics, and the characteristic ions such as m / z 193, 191, 179, 173, 135, and 134 can be used as diagnostic ions of this type of compound. Among them, m / z 179 [CA-H] - or m / z 135 [CA-H-CO2] - Indicates the presence of caffeoyl groups, m / z 191 [QA-H] - or m / z 173 [QA-H-H2O] - Indicates the presence of quinic acid group, m / z 193 [FA-H] - ,134[FA-H-CH3-CO2] - The difference between the parent ion and the daughter ion mass number indicates the presence of a feruloyl group. At the same time, the type of organic acid that forms an ester with quinic acid can be inferred based on the difference between the parent ion and the daughter ion mass number. The difference between the parent ion and the daughter ion mass number is 162, 146, and 176 Da, indicating that there is a caffeoyl group, a coumaroyl group, and a feruloyl group in the structure, respectively. The quasi-molecular ion peaks of compounds 24 and 28 are both m / z 353 [MH] - , it is inferred that its molecular formula may be C 16 H 17 O9, MS2 fragmentation yielded m / z 191 [M-caffeoyl-H] - 、m / z 179[Caffeic acid-H] - 、m / z 173[M-caffeoyl-H2O-H] - The characteristic fragment ions showed that these two compounds were monocaffeoylquinic acid compounds and were isomers of each other. After comparison with the reference substance, compounds 24 and 28 were identified as neochlorogenic acid (5-CQA) and chlorogenic acid (3-CQA), respectively. The fragmentation rules were explained by taking neochlorogenic acid as an example. Figure 7 , Figure 8 shown.

[0118] In the negative ion mode, the retention times of compounds 26, 30, and 33 were 6.32, 9.75, and 11.97 min, respectively. The same quasi-molecular ion peak m / z 337.0928 [MH] was observed in the primary mass spectra of the three compounds. - , are isomers, and the calculated molecular formula is C 16 H 17 O8, and the secondary fragments are similar. The difference between the parent ion (m / z 337) and the daughter ion (m / z 191) in the secondary mass spectra of the three compounds is 146Da, indicating that a molecule of coumaroyl is lost, and the characteristic ions of quinic acid m / z 191 and m / z 173 appear, indicating that these compounds are coumaroylquinic acid compounds. The base peak ion in the secondary spectrum of compound 26 is m / z 163.0389, indicating that the compound is a 3-substituted quinic acid ester. Combined with non-patent literature

[20] , it is speculated that it is 3-pCoQA; the base peak ion in the secondary spectrum of compounds 30 and 33 is m / z 191.0551, indicating that the compound is a 5-substituted quinic acid ester. According to its retention time and reference to non-patent literature

[20] , the trans compound is eluted earlier than the cis compound, so compounds 30 and 33 are speculated to be trans-5-pCoQA and cis-5-pCoQA. The quasi-molecular ion peak of compound 32 is m / z 367.1031[MH] - , whose molecular formula may be C 17 H 19 O9, generating m / z 193.0495 [FA-H] in the secondary mass spectrum - , m / z 134.0360 [FA-CH3-CO2] - and m / z 191.0551 [MH-feruloyl] - , m / z 173.0445[MH-feruloyl-H2O] - The fragment ions of 5-FQA were detected, so it was inferred to be a feruloylquinic acid compound. According to non-patent literature

[20] , the base peak ion of 5-FQA in the secondary mass spectrum was m / z 191.0551, accompanied by a relatively weak m / z 173.0445. Therefore, compound 32 was inferred to be 5-FQA.

[0119] 1.5 Fragmentation rules of nucleoside compounds

[0120] Nucleoside compounds are a class of compounds that are prone to lose NH3, HCN or ribose. Combined with database search, a total of 6 nucleoside compounds were identified in Chlorophytum comosum. Compound 14 was used as an example to analyze its fragmentation rules. The quasi-molecular ion of compound 14 is m / z 268.1039[M+H] + , its molecular formula is estimated to be C 10 H 13N5O4, in the secondary mass spectrum, it is easy to see that one ribose is lost to form m / z 136.0616 [M+H-ribose] + The fragment ion continues to break up with high-energy collisions and loses NH3 to obtain the fragment ion m / z119.0354 [M+H-ribose-NH3] + According to non-patent literature

[20] , it is speculated to be adenosine. The secondary mass spectrum of adenosine and its possible fragmentation mode are shown in Fig. 9 , Fig.10 .

[0121] 1.6 Pyrolysis of other compounds

[0122] Other compounds were also identified in the spider plant, including iridoids, anthraquinones, benzyl alcohol glycosides and coumarins. Among them, iridoids are special monoterpenoids, which are usually linked to sugar groups to form iridoid glycosides. A total of one iridoid compound (peak 27) was analyzed in the spider plant. In the negative ion mode, the retention time of compound 27 was 6.38min, and the quasi-molecular ion peak was m / z 549.1825 [MH] - According to the elemental composition analysis, the molecular formula is C 23 H 33 O 15 The secondary mass spectrometry information showed that the quasi-molecular ion peak continuously removed two glucose groups to produce a fragment ion m / z 225.0761 [MH-2Glc] - , and then lose a water molecule to produce a fragment ion m / z 207.0660 [MH-2Glc-H2O] - , combined with the quasi-molecular ion peak, secondary fragment ion and relative retention time, which is basically consistent with the reference non-patent literature

[24] , it can be determined that compound 27 is genipin-1-β-D-gentiobioside. Fig.11 , Fig.12 shown.

[0123] 2. Content determination results

[0124] 2.1 Methodological investigation

[0125] 2.1.1 System suitability test

[0126] Weigh 0.5 g of sample (S8) accurately, prepare the test solution according to the method in 2.1.2, take the mixed reference solution prepared in 2.2.1, and analyze it according to the chromatographic conditions in 2.1.3. The separation between the adjacent peaks of neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and scutellariae is greater than 1.5, and the retention time is basically consistent with the reference spectrum (such as Fig.13 ), indicating that this method has good specificity.

[0127] 2.1.2 Standard curve drawing

[0128] Take the reference substance stock solution under 2.2.1 and dilute it with 50% methanol to form a series of mixed reference substance solutions. Inject and measure according to the chromatographic conditions under 2.1.3, and draw a standard curve with the concentration of the reference substance solution as the abscissa and the peak area as the ordinate. The regression equation, correlation coefficient and linear range of each component are shown in Table 3.

[0129] Table 3 Regression equations, correlation coefficients and linear ranges of the five components

[0130] Compound Linear equations Correlation coefficient r Linear range (μg / mL) Neochlorogenic Acid y=0.5419x+0.0652 1.0000 3.22～102.90 Chlorogenic acid y=0.4821x+1.1524 0.9999 12.84～410.82 Forsythiaside B y=0.2094x+0.8030 1.0000 31.63～1012.01 Verbascoside y=0.3010x+0.6535 1.0000 25.00～800.11 Stonewort y=0.7010x-0.4764 0.9997 4.08～130.51

[0131] 2.1.3 Intra-day and inter-day precision tests

[0132] Weigh 0.5g of sample (S8) accurately, prepare the test solution according to the method in 2.1.2, repeat the injection for 6 times under the chromatographic conditions in 2.1.3 for 3 consecutive days, and record the peak area. The RSD of the peak area of ​​neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and scutellaria sphaerocephala was less than 3%, indicating that the instrument had good precision.

[0133] 2.1.4 Repeatability test

[0134] Weigh 0.5 g of sample (S8) accurately, prepare 6 test solutions in parallel according to the method in 2.1.2, inject the sample under the chromatographic conditions in 2.1.3, and record the peak area. The RSDs of the peak areas of neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and scutellaria sphaerocephala were 0.61%, 1.80%, 0.66%, 0.27%, and 0.47%, respectively, indicating that the method has good repeatability.

[0135] 2.1.5 Stability test

[0136] Weigh 0.5 g of the test sample (S8) accurately, prepare the test solution according to the method in 2.1.2, inject the sample at 0h, 2h, 4h, 8h, 12h, and 24h according to the chromatographic conditions in 2.1.3, and record the peak area. The RSDs of the peak areas of neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and scutellaria sphaerocephala were 0.76%, 0.48%, 0.13%, 0.18%, and 0.35%, respectively, indicating that the test solution has good stability within 24h.

[0137] 2.1.6 Sample recovery test

[0138] Accurately weigh about 0.25g of the powder of the test sample of the known content of the stone spider plant, a total of 9 portions, add 50%, 100%, and 150% of the reference substance respectively, prepare the test solution according to the method under 2.1.2, inject and measure according to the chromatographic conditions under 2.1.3, record the peak area, and calculate the recovery rate. The average recovery rates of neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and stone spider plant were measured to be 100.0%, 99.7%, 100.2%, 97.5%, and 97.3%, respectively; the RSD of the peak area was 1.53%, 2.36%, 2.10%, 0.99%, and 0.95%, respectively, indicating that the method has good accuracy.

[0139] In order to better extract the various chemical components in the stone spider plant, the inventors of the present application investigated the effects of different extraction solvents (water, 50% methanol, 75% methanol and methanol), different extraction methods (Soxhlet extraction, heating reflux and ultrasonic extraction), and different ultrasonic extraction times (30min, 40min and 50min) on the extraction of medicinal materials. The results showed that the chromatographic peak of 50% methanol ultrasonic extraction for 40min had the richest information, high response and relatively stable baseline, so the standard curve drawing experimental method under 2.1.2 was selected. At the same time, the chromatographic conditions were also investigated, including different chromatographic columns (ACQUITYUPLC&BEH C18 (100×2.1mm, 1.7μm), SynergiTM Hydro-RP (100×2.0mm, 2.5μm)), mobile phase ratio and detection wavelength, and finally the chromatographic column SynergiTM Hydro-RP (100×2mm, 2.5μm) for gradient elution, and the detection wavelength was 330nm. UHPLC-Q-Exactive Plus Orbitrap HRMS technology has the characteristics of high resolution, good mass accuracy, strong qualitative and quantitative capabilities, etc., and can quickly separate, detect and identify the complex chemical components of traditional Chinese medicine without reference substances (refer to non-patent literature

[41] ). This application also optimizes the conditions of mass spectrometry, mainly including capillary temperature, spray voltage, sheath gas flow rate, auxiliary gas flow rate, auxiliary gas temperature, collision energy, etc., and finally selects the repeatability test method under the above 2.1.4.

[0140] 2.2 Sample content determination

[0141] Accurately weigh 0.5 g of the sample powder of each batch of Clerodendrum truncatum under Table 1, prepare the test solution according to the method under 2.1.2, inject according to the chromatographic conditions under 2.1.3, record the peak area, calculate the content according to the standard curve, and the results are shown in Table 4. In 25 batches of Clerodendrum truncatum medicinal materials, the contents of the above five components are 0.0007-0.4204 mg / g of neochlorogenic acid, 0.0841-13.1334 mg / g of chlorogenic acid, 0.1375-48.2631 mg / g of forsythiaside B, 0.1399-18.8918 mg / g of verbascoside, and 0.0161-4.7414 mg / g of Clerodendrum truncatum, among which the average content of forsythiaside B is the highest, and the average content of neochlorogenic acid is the lowest.

[0142] Table 4 Determination results of the contents of five components in 25 batches of SDL samples (mg / g)

[0143]

[0144]

[0145] In summary, this application uses UHPLC-Q-Exactive Plus Orbitrap HRMS technology to identify 84 compounds in spider plant, including 27 flavonoids, 20 phenylethanol glycosides, 5 amino acids, 18 organic acids, 1 alkaloid, 6 nucleosides and 7 other compounds; at the same time, an HPLC-UV content determination method for 5 components of different types in spider plant (neochlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, spider plant glycoside) was established, with good linear relationship, and the system applicability, stability, repeatability, precision and recovery rate can meet the requirements of quantitative analysis. It can be used for content determination and analysis of spider plant medicinal materials, and provides a certain scientific basis for further research on quality control methods, pharmacodynamic material basis, mechanism of action and new drug development of spider plant.

[0146] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from all points of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the equivalent meaning and scope of the claims be included within the scope of protection of the present invention.

Claims

1. A method for analyzing the chemical components and content determination of Chlorophytum comosum based on UHPLC-Q-Exactive Plus Orbitrap HRMS, characterized by: The method comprises preparing a reference solution, preparing a test solution, chromatographic analysis, and data processing, wherein the chemical component analysis also comprises mass spectrometry analysis; the method for analyzing the chemical components of the spider plant specifically comprises the following steps: ① Preparation of reference solution Accurately weigh forsythiaside B, physalisin, akebia phenylethanol glycoside B, plantain glycoside, verbascoside, chlorogenic acid, and neochlorogenic acid reference substances, respectively, and add methanol to each container, stir and dissolve evenly, and make up to volume to obtain reference substance stock solutions, and then add 50% methanol to dilute into reference substance solutions with concentrations of 422.20 μg / mL, 32.63 μg / mL, 201.12 μg / mL, 21.09 μg / mL, 31.63 μg / mL, 100.00 μg / mL, and 20.58 μg / mL, respectively; ② Preparation of test solution Weigh the powder of the spider plant accurately and place it in a container, add 50% methanol aqueous solution and weigh it, extract it by ultrasonic for 40 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake it well, filter it, take the supernatant after centrifugation, and take the filtrate through a microporous filter membrane to obtain the test solution; ③ Determination The reference solution and the test solution were subjected to chromatography analysis, mass spectrometry analysis, and data processing in turn, and the qualitative analysis of the chemical components of the spider plant was completed through analysis; The conditions of the chromatographic analysis are: the chromatographic column is Synergi TM Hydro-RP Its specifications are 100×2mm, 2.5μm; mobile phase A is acetonitrile, B is 0.1% formic acid water, and the elution gradient is 0-1min, 5-15%A; 1-6min, 15%A; 6-18min, 15-18%A; 18-20min, 18-25%A; 20-24min, 25-28%A; 24-27min, 28-32%A; 27-28min, 32-95%A; 28-34min, 95%A; 34-35min, 95-5%A; 35-40min, 5%A; column temperature: 40℃; flow rate: 0.2mL / min; injection volume: 2μL.

2. The method for analyzing the chemical components and content determination of Clerodendrum truncatum based on UHPLC-Q-Exactive Plus Orbitrap HRMS according to claim 1, characterized in that: The conditions for the mass spectrometry analysis are as follows: the ion source is an electrospray ionization source; positive and negative ions are scanned separately; the scanning mode is: primary full scan-data-dependent secondary mass spectrometry scanning mode, with a scanning range of 100 to 1500 Da; capillary temperature: 320°C, spray voltage: positive mode: 3.5 kV, negative mode: 2.5 kV; sheath gas flow rate 35 arb; auxiliary gas flow rate 10 arb; auxiliary gas temperature: 350°C; in Ms / Ms mode, the collision energies in the positive and negative ion modes are all 20, 40, and 60 eV; the primary mass spectrometry resolution is 70,000 FWHM, and the secondary mass spectrometry resolution is 17,500 FWHM.

3. The method for analyzing the chemical components and content determination of Clerodendrum truncatum based on UHPLC-Q-Exactive Plus Orbitrap HRMS according to claim 1, characterized in that: The data processing is as follows: mass spectrometry data were processed using Thermo Xcalibur 4.4 and Compound Discoverer TM 3.2 Processing, import the raw data collected by mass spectrometry into CompoudDiscoverer TM 3.2 The software performs preliminary calculations and compares the retention time of the main chromatographic peaks of the test solution, the quasi-molecular ions of the primary mass spectrometry, and the characteristic fragment ion information of the secondary mass spectrometry with the chromatographic retention time of the reference solution, the mzCloud library, mzVault library, Chemspider library compound database, and mass spectrometry information of relevant literature to perform qualitative analysis on the main chromatographic peaks.

4. The method for analyzing the chemical components and content determination of Clerodendrum truncatum based on UHPLC-Q-Exactive Plus Orbitrap HRMS according to claim 1, characterized in that: The determination of the chemical composition content of the stone spider plant specifically comprises the following steps: (1) Preparation of reference solution Accurately weigh new chlorogenic acid, chlorogenic acid, forsythiaside B, verbascoside, and scutellaria baicalensis respectively and place them in containers, dissolve them with methanol and make up to volume to obtain the reference substance stock solutions with concentrations of 1.0290 mg / mL, 1.0270 mg / mL, 1.0555 mg / mL, 2.0003 mg / mL, and 1.0876 mg / mL, respectively, and then add 20% methanol solution to dilute them into reference substance solutions with concentrations of 25.725 μg / mL, 102.704 μg / mL, 253.003 μg / mL, 200.027 μg / mL, and 32.628 μg / mL, respectively; (2) Preparation of test solution Weigh the powder of the spider plant accurately and place it in a container, add 50% methanol aqueous solution and weigh it, extract it by ultrasonic for 40 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake it well, filter it, take the supernatant after centrifugation, and take the filtrate through a microporous filter membrane to obtain the test solution; (3) Determination: The reference solution and the test solution are subjected to chromatographic analysis respectively, and then the experimental data are processed to complete the HPLC content determination.

5. The method for analyzing the chemical components and content determination of Clerodendrum truncatum based on UHPLC-Q-Exactive Plus Orbitrap HRMS according to claim 1 or 4, characterized in that: In step ② and step (2), the mass ratio of the spider plant powder to methanol is 1:

50.

6. The method for analyzing the chemical components and content determination of Clerodendrum truncatum based on UHPLC-Q-Exactive Plus Orbitrap HRMS according to claim 1 or 4, characterized in that: In step ② and step (2), the centrifugation is to centrifuge the filtrate at 12000r / min for 10min.

7. The method for analyzing the chemical components and content determination of Clerodendrum truncatum based on UHPLC-Q-Exactive Plus Orbitrap HRMS according to claim 1 or 4, characterized in that: In step ② and step (2), the filtration is through a 0.22 μm microporous filter membrane.

8. The method for analyzing the chemical components and content determination of Clerodendrum truncatum based on UHPLC-Q-Exactive Plus Orbitrap HRMS according to claim 1, characterized in that: A total of 84 compounds were identified in Chlorophytum comosum, including 27 flavonoids, 20 phenylethanol glycosides, 5 amino acids, 18 organic acids, 1 alkaloid, 6 nucleosides and 7 other compounds.

Images