Method for rapidly identifying non-volatile components in ledum palustre and preparation thereof and application

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for rapidly identifying non-volatile components in ledum palustre and a preparation thereof and application, the method comprises the following steps: taking a ledum palustre sample, adding an extraction solvent A, soaking, boiling, distilling, filtering, concentrating an extracting solution, and freeze-drying to obtain ledum palustre A extract freeze-dried powder; drying and crushing filter residues, adding an extraction solvent B, performing reflux extraction, recovering the solvent B, and performing freeze drying to obtain ledum palustre B extract freeze-dried powder; respectively taking the ledum palustre A extract freeze-dried powder and the ledum palustre B extract freeze-dried powder, adding an extraction solvent C, carrying out ultrasonic and centrifugal treatment, and taking supernate to obtain a test solution; injecting the test solution and the reference solution into an ultra-high performance liquid chromatograph and a high-resolution mass spectrometer for determination; and analyzing the collected data. 118 non-volatile chemical components are identified, 102 non-volatile chemical components are unreported components in ledum palustre, and reference is provided for ledum palustre pharmacodynamic substance research, quality control and product development.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method and application for rapidly identifying non-volatile components in Ledum palustre and its preparations based on UHPLC-Q-TOF-MS technology. Background Art

[0002] Ledum palustre Harmaja is a perennial evergreen shrub of the genus Rhododendron L. in the family Ericaceae, and its aliases include narrow-leaved Ledum palustre, broad-leaved Ledum palustre, and trip-wire Ledum palustre. Ledum palustre is distributed in the temperate or cold temperate regions of the Northern Hemisphere. In China, it is mainly concentrated in the Greater and Lesser Hinggan Mountains and the Changbai Mountains. The resources of Ledum palustre in the Greater Hinggan Mountains are rich, and Ledum palustre accounts for more than 70% of the total plant density in this area. Ledum palustre is pungent, bitter, and cold in nature, and belongs to the lung meridian; it has the effects of resolving phlegm, relieving cough, and calming asthma, and is often used to treat whooping cough, bronchitis-induced asthma, podagra, rheumatism, moist eczema, acne, etc. It is also used for quitting smoking and drug addiction, and is also used as a substitute tea for anti-cancer. In the Changbai Mountains region of China, there is also a custom of using Ledum palustre as the main material to treat irregular menstruation and infertility. Ledum palustre mainly contains chemical components such as volatile oils, triterpenoids, coumarins, flavonoids, and phytosterols. Modern pharmacological studies have found that Ledum palustre has anti-inflammatory, antitussive, expectorant, transdermal absorption, anti-radiation, antiviral, anti-diabetic, anti-drug addiction, antibacterial, antioxidant, and anti-tumor effects.

[0003] At present, the research on Ledum palustre mainly focuses on the volatile components and pharmacological effects of its leaves. For example, Chinese patent applications CN109897730A (a method for refining volatile oil of Ledum palustre), CN107751252A (application of total volatile oil of Ledum palustre branches and leaves in controlling stored grain pests), CN117487625A (a high-efficiency extraction method for Ledum palustre essential oil), CN115322837A (a refining process for Ledum palustre essential oil and its antibacterial application), CN104173417A (a preparation method for microcapsules of narrow-leaved Ledum palustre volatile oil and its anti-inflammatory and analgesic application), CN103045371A (a preparation method for purifying essential oil and ursolic acid from wild Ledum palustre in the Greater Hinggan Mountains), CN1969934A (a Ledum palustre oil dropping pill for treating chronic bronchitis and its preparation method), etc. The research focuses of these literature materials mainly lie in the extraction and application of volatile components in Ledum palustre. CN103558324A discloses a method for extracting and identifying aesculin, fraxin, aesculetin, and resveratrol from narrow-leaved Ledum palustre. This invention uses an On-line HPLC-ABTS+ antioxidant component screening system, and can only extract and identify 4 antioxidant active components, namely aesculin, fraxin, aesculetin, and resveratrol.

[0004] In the article "Optimization of the Extraction Process and Component Analysis of Ledum palustre Essential Oil" published by Li Zhengyuan et al., the essential oil components of Ledum palustre branches and leaves were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) technology. However, this method could only analyze 25 key compounds in Ledum palustre essential oil. In the article "Analysis of Chemical Constituents and Antibacterial Activity of Volatile Oils from Ledum palustre Leaves" published by Wang Tingjie et al., the chemical constituents of the volatile oils from Ledum palustre leaves were separated and identified by gas chromatography-mass spectrometry. However, this method could only identify 46 chemical constituents in the volatile oils from Ledum palustre broad-leaved leaves. In the article "Enzyme-Assisted Extraction Process and Antioxidant Activity of Ledum palustre Essential Oil" published by Kang Hedi et al., the chemical constituents in the essential oil of Ledum palustre leaves were analyzed by gas chromatography-mass spectrometry. However, this method could only analyze 44 chemical constituents in the Ledum palustre essential oil obtained by enzyme-assisted extraction. In the article "Research on the Extraction Process of Ledum palustre Volatile Oil and GC-MS Analysis of Its Components" published by You Liyan et al., the chemical constituents in the extracted volatile oils from Ledum palustre stems and leaves were analyzed and identified by gas chromatography-mass spectrometry, and a total of 27 chemical constituents were separated and identified. In the paper "Preparation of Ledum palustre Essential Oil and Pyrolysis Oil by Microwave Method and Their Inhibitory Activity against Fusarium" published by Wang Xun, the Ledum palustre essential oil was prepared by solvent-free microwave-assisted rotary distillation method, the Ledum palustre pyrolysis oil was prepared by microwave fixed-bed anaerobic thermochemical conversion method, and the Ledum palustre essential oil and pyrolysis oil were analyzed by gas chromatography-mass spectrometry. 21 compounds were analyzed in the Ledum palustre pyrolysis oil, and a total of 27 compounds were detected in the Ledum palustre essential oil. In the article "Preparation and Sustained Release Performance of Porous Starch Immobilized Ledum palustre Essential Oil" published by Zhang Qian et al., the main chemical constituents in the crude extract of Ledum palustre angustum essential oil were determined and analyzed by GC-MS. The chemical composition of the crude extract of Ledum palustre angustum essential oil was relatively complex, containing at least more than 30 chemical constituents. In the article "Response Surface Optimization of the Extraction Process of Ledum palustre and Its Antioxidant Activity Research" published by Xie Yang et al., the extraction process of Ledum palustre antioxidants was optimized by response surface, the total flavonoid content in Ledum palustre was determined by ultraviolet spectrophotometry, etc., and the polyphenol components in the Ledum palustre antioxidants were determined by Folin-Ciocalteu reagent method, but the specific chemical constituents were not identified. In the article "Optimization of the Experimental Conditions for Extracting Volatile Oils from Ledum palustre angustum by CO2 Supercritical Extraction" published by Li Peng et al., the optimal extraction conditions for extracting volatile oils from Ledum palustre angustum by CO2 supercritical (SFE-CO2) were disclosed. However, the chemical constituents in the Ledum palustre volatile oils were not analyzed. In the article "Analysis of Chemical Constituents of Volatile Oils from Ledum palustre angustum Leaves by GC-MS" published by Gao Yan et al., the chemical constituents in the volatile oils from Ledum palustre angustum leaves were analyzed by gas chromatography-mass spectrometry, but only 31 compounds were identified.In the paper "Pharmacological Effects and Chemical Constituents of the Effective Parts of Ledum palustre L. in the Treatment of Acute Bronchitis" published by Yang Deqiang, the systematic solvent extraction method was used to prepare petroleum ether, ethyl acetate, n-butanol and water components in Ledum palustre L. samples, and column chromatography such as silica gel and ODS, as well as preparative HPLC were used to systematically separate the chemical constituents of the n-butanol component and ethyl acetate component of Ledum palustre L., according to. 1 H and 13 C-NMR spectra, combined with HRESI-MS mass spectra, a total of 35 monomeric compounds were isolated and identified, including 20 flavonoid compounds. In the article "Process Study and Component Analysis of Ultrasonic-Assisted Extraction of Ledum palustre L. Essential Oil" published by Ma Chenghui et al., a gas chromatography-mass spectrometer was used to analyze the components of the crude extract of Ledum palustre L. essential oil, and 19 compounds were obtained. In the article "Study on the Chemical Constituents of Ledum palustre L. (Ⅱ)" published by Huang Ying et al., silica gel column chromatography was used to separate and purify the compounds in the branches and leaves of Ledum palustre L., and the compounds were identified by combining ultraviolet spectroscopy, infrared spectroscopy and liquid chromatography-mass spectrometry. Only 5 compounds were separated and identified. In the article "Separation and Identification of Flavonoid Chemical Constituents in Ledum palustre L." published by Yang Deqiang et al., column chromatography methods such as silica gel and ODS and preparative HPLC were used to separate the components in the branches of Ledum palustre L., and 1 H and 13 C-NMR spectra combined with high-resolution electrospray ionization mass spectrometry data were used to identify the structures of the compounds, and 5 flavonoid compounds were separated. In the article "Study on the Chemical Constituents of Ledum palustre L." published by Zhang Hongcai et al., silica gel, ODS column, HPLC column and recrystallization were used to separate and purify the above-ground part of Ledum palustre L., and the structures of the obtained compounds were identified by nuclear magnetic resonance spectroscopy. Only 8 compounds were separated from it.

[0005] To sum up, the existing technology for the research of traditional Chinese medicine Ledum palustre L. mainly focuses on the extraction methods, analysis and identification, and pharmacological effect research of its leaf volatile components. There are research reports that its volatile oil and monomer components in the volatile oil have certain toxic effects on p-cymene. There is less research on the non-volatile components of Ledum palustre L., and there are no literature reports on the systematic characterization and structure identification of its non-volatile components. Therefore, it is urgent to develop a method that can rapidly, accurately and comprehensively characterize and identify the non-volatile components in traditional Chinese medicine Ledum palustre L. and its preparations. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method and application for rapidly identifying the non-volatile components in Ledum palustre L. and its preparations. The present invention can rapidly, accurately and comprehensively characterize and identify the non-volatile components in traditional Chinese medicine Ledum palustre L., and the obtained non-volatile components are very diverse, providing a reference for its pharmacodynamic substance research, quality control and product development.

[0007] The technical solution of the present invention is:

[0008] A method for rapidly identifying non-volatile components in Ledum palustre L., comprising the following steps:

[0009] S1 Preparation of test solution: Take Ledum palustre L. samples, add extraction solvent A, soak, boil, distill by steam distillation method, filter to obtain an extract, concentrate the obtained extract, freeze-dry after concentration to obtain a freeze-dried powder of Ledum palustre L. extract A; dry the filter residue after filtration, crush the dried filter residue, add extraction solvent B to the crushed filter residue, reflux extract, freeze-dry after recovering extraction solvent B to obtain a freeze-dried powder of Ledum palustre L. extract B; respectively take the freeze-dried powder of Ledum palustre L. extract A and the freeze-dried powder of Ledum palustre L. extract B, add extraction solvent C, perform ultrasonic treatment and centrifugation treatment, and take the supernatant to obtain the test solution;

[0010] S2 Preparation of reference solution: Take a reference substance, add a solvent to make a reference solution;

[0011] S3 Inject the test solution and the reference solution into an ultra-high performance liquid chromatograph and a high-resolution mass spectrometer for determination respectively;

[0012] S4 Analyze the collected data by UHPLC-Q-TOF-MS.

[0013] Further, the Ledum palustre L. samples in step S1 are Ledum palustre L. leaves and tender branches.

[0014] Further, the extraction solvent A in step S1 is water, the soaking time is 0.5 - 2 h, the distillation time by steam distillation method is 4 - 6 h, decompression concentration is used when concentrating the obtained extract, the vacuum pressure is -0.09 to -0.1 MPa, the concentration temperature is 75 - 85 °C, and the concentration is up to a relative density of 1.06 - 1.10. The extraction solvent B is anhydrous ethanol, the reflux extraction temperature is 80 - 90 °C, and the reflux extraction time is 1 - 3 h.

[0015] Further, the mass-to-volume ratio of the Ledum palustre L. samples to the extraction solvent A in step S1 is 1:15 - 25 g / mL; the mass-to-volume ratio of the crushed filter residue to the extraction solvent B is 1:15 - 25 g / mL; the mass-to-volume ratio of the freeze-dried powder of Ledum palustre L. extract A to the extraction solvent C is 1:15 - 25 g / mL; the mass-to-volume ratio of the freeze-dried powder of Ledum palustre L. extract B to the extraction solvent C is 1:15 - 25 g / mL.

[0016] Further, when taking the freeze-dried powder of Ledum palustre A extract, adding extraction solvent C, and performing ultrasonic treatment and centrifugation treatment in step S1, extraction solvent C is an aqueous methanol solution with a volume fraction of 15-25%, the ultrasonic power is 250-350 W, the ultrasonic time is 25-35 min, the centrifugation speed is 10,000-13,000 r / min, and the centrifugation time is 3-7 min; when taking the freeze-dried powder of Ledum palustre B extract, adding extraction solvent C, and performing ultrasonic treatment and centrifugation treatment, extraction solvent C is methanol, the ultrasonic power is 250-350 W, the ultrasonic time is 25-35 min, the centrifugation speed is 10,000-13,000 r / min, and the centrifugation time is 3-7 min.

[0017] The components of traditional Chinese medicine are very complex, and it is very difficult to comprehensively characterize and identify the components in traditional Chinese medicine. The preparation method of the test solution has a great influence on the qualitative and quantitative analysis of the components in the subsequent Ledum palustre samples. The extraction of non-volatile components in Ledum palustre is a very complex process, and how to retain as many original non-volatile components in the Ledum palustre samples as possible is a difficult point. The preparation process of the test solution in the present invention includes: taking Ledum palustre samples, adding extraction solvent A, soaking, boiling, distilling by steam distillation method, filtering to obtain an extract, concentrating the obtained extract, freeze-drying after concentration to obtain the freeze-dried powder of Ledum palustre A extract; drying the filter residue after filtration, crushing the dried filter residue, adding extraction solvent B to the crushed filter residue, refluxing and extracting, freeze-drying after recovering extraction solvent B to obtain the freeze-dried powder of Ledum palustre B extract; respectively taking the freeze-dried powder of Ledum palustre A extract and the freeze-dried powder of Ledum palustre B extract, adding extraction solvent C, performing ultrasonic treatment and centrifugation treatment, and taking the supernatant to obtain the test solution; through the above series of operation steps, combined with specific operation parameters, conditions such as extraction solvent, etc., it is possible to retain the original non-volatile components in the Ledum palustre samples to the greatest extent in the test solution, laying a foundation for characterizing and structurally identifying more compounds in the subsequent analysis.

[0018] Further, the reference substances in step S2 include hyperoside, aesculetin, aesculin, quercitrin, chlorogenic acid, protocatechuic acid, arbutin, epicatechin, catechin, neochlorogenic acid, cryptochlorogenic acid, citric acid, adenosine, gallic acid, gallocatechin, protocatechualdehyde, salidroside, procyanidin B3, epigallocatechin, procyanidin B4, aesculoside, procyanidin B2, kalankoside, myricetin 3-O-β-galactoside, procyanidin A1, resveratrol-3-O-β-D-glucoside, isoquercitrin, guaijaverin, taxillusflavone, asiatic acid, eupatilin, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone, oleanolic acid and ursolic acid.

[0019] Traditional Chinese medicine chemical reference substances are physical references for the research, quality inspection, and quality control of the quality standards of traditional Chinese medicine, and play an extremely crucial role in the development of new drugs and the evaluation of old drugs. Since the specific chemical components contained in the non-volatile components of Ledum palustre are unknown, the prior art has no technical inspiration for the selection of reference substances when identifying the non-volatile components of traditional Chinese medicine Ledum palustre. Through a large number of creative experiments, the present invention finally obtains 36 reference substances suitable for the present invention, which is conducive to the rapid, comprehensive, and accurate identification of the non-volatile components of Ledum palustre.

[0020] Further, the chromatographic conditions in step S3 are as follows: Waters ACQUITY UPLC HSS T3 chromatographic column: 2.1 mm × 100 mm, 1.8 μm; the mobile phase is composed of formic acid aqueous solution (A) with a volume fraction of 0.05 - 0.2% and acetonitrile (B); gradient elution, 0 - 3 min, 3% B; 3 - 7 min, 3% - 8% B; 7 - 20 min, 8% - 15% B; 20 - 40 min, 15% - 26% B; 40 - 55 min, 26% - 65% B; 55 - 61 min, 65% - 95% B; 61 - 64 min, 95% B; column temperature 25 - 35°C; flow rate 0.2 - 0.4 mL / min; injection volume 1 - 3 μL; detection wavelength 280, 190 - 400 nm.

[0021] The chromatographic separation conditions are important factors affecting the stability and reproducibility of the chromatogram, and the setting of the mobile phase and the gradient elution program among them is the key to determining parameters such as the number of peaks eluted by complex components and the analysis time. For traditional Chinese medicine, its structure is complex, and the separation of active ingredients is more difficult. There are many influencing factors for the gradient elution method. The present invention has found through a large number of creative studies that when the mobile phase is composed of formic acid aqueous solution (A) with a volume fraction of 0.05 - 0.2% and acetonitrile (B), under the gradient elution conditions of 0 - 3 min, 3% B; 3 - 7 min, 3% - 8% B; 7 - 20 min, 8% - 15% B; 20 - 40 min, 15% - 26% B; 40 - 55 min, 26% - 65% B; 55 - 61 min, 65% - 95% B; 61 - 64 min, 95% B, it is not only conducive to the separation of non-volatile substances in the test solution of Ledum palustre, but also conducive to increasing the number of peaks eluted, and the non-volatile substances in the test solution of Ledum palustre are separated to the greatest extent, greatly improving the comprehensiveness and accuracy of the method of the present invention for identifying non-volatile components in Ledum palustre.

[0022] Further, the mass spectrometry conditions in step S3 are as follows: mass spectrometry detection mode: ESI, positive and negative ion modes, mass scanning range of m / z 50 - 1700, nebulizer gas pressure of 50 psi, auxiliary heating gas pressure of 50 psi, curtain gas pressure of 35 psi, ionization voltage of -4500 / 5000 V, ion source temperature of 500 °C, declustering voltage of 100 V, collision voltage of 10 eV; secondary mass scanning range of m / z 50 - 1250, declustering voltage of 100 V, collision voltage of ±40 eV, collision voltage swing of 20 eV, ion release delay of 30 ms, ion beam width of 15 ms.

[0023] Further, in step S4, UHPLC-Q-TOF-MS is used to analyze the collected data, including using Analyst TF 1.7.1 software to collect data, PeakView 1.2 software to process data. When identifying, the mass spectrometry data is preferentially matched with the Natural Products HR-MS / MS Spectral Library 1.0 database, and the reference substance is compared with the identification result according to information such as retention time and fragment ions. The compounds are preliminarily screened according to the score information of each chromatographic peak, and further confirmed according to the primary and secondary information of each chromatographic peak, etc. For the compounds not included in the database, they are identified according to literature reports, mass spectrometry fragmentation rules, etc.

[0024] Due to the complex components and diverse structures in Chinese herbal medicines, the analysis and identification of their active ingredients have become difficult problems. At present, there are few studies on the non-volatile components in Ledum palustre, and there are no literature reports on the systematic characterization and structural identification of its non-volatile components. Therefore, it is very difficult to accurately, comprehensively and quickly identify the non-volatile components in the Chinese medicine Ledum palustre. Through a large number of creative studies, the present invention obtains a method for preparing a test solution, which can retain the original non-volatile components in the Ledum palustre sample to the greatest extent; through a large number of creative studies, the composition of the mobile phase system and chromatographic conditions such as gradient elution conditions are obtained, so that the non-volatile substances in the Ledum palustre test solution are separated to the greatest extent, greatly improving the comprehensiveness and accuracy of the method of the present invention for identifying non-volatile components in Ledum palustre. The present invention has established a complete method system for identifying non-volatile components in Ledum palustre (Ledum palustre leaves, tender branches of Ledum palustre), and each step and condition synergistically enhance the efficiency, speed, comprehensiveness and accuracy of the identification. The present invention uses UHPLC-Q-TOF-MS technology to systematically characterize and structurally identify the non-volatile chemical components in the tender branches and leaves of Ledum palustre. According to high-resolution mass spectrometry data, database, reference substance comparison, and derivation and identification methods such as characteristic ions and neutral small molecule losses, a total of 118 non-volatile chemical components have been identified, including 35 flavonoid glycosides, 15 phenolic glycosides, 12 flavonoids, 7 phenolic acids, 7 phenyl ethanol glycosides, 6 tannins, 6 phospholipids, 5 coumarins, 5 monoterpene glycosides, 6 triterpenes, 3 fatty acids, 11 other compounds. Among them, 102 are unreported components in Ledum palustre, 36 are identified by comparison with reference substances. The chemical components contained in the B extract and A extract of Ledum palustre leaves and tender branches are slightly different, and 84 chemical components are common components, accounting for about 71.2% of the 118 identified chemical components. No obvious toxic components were found in the identification results, and most of the detected components showed strong pharmacological activities in the literature reports. For example, 1-oleoyl-sn-glycero-3-phosphocholine in phospholipid components has the activity of regulating glucagon-stimulated adenylate cyclase in the liver plasma membrane of rats.

[0025] Another object of the present invention is to provide the application of the method for rapidly identifying non-volatile components in the Chinese medicine Ledum palustre in detecting non-volatile components in Ledum palustre or preparations containing Ledum palustre.

[0026] Furthermore, the preparations containing Ledum palustre include granules, capsules, pills, tablets, oral liquids, injections, ointments, suppositories, extracts, buccal tablets, powders, pills, suspensions, powders, creams, sprays, drops, sustained-release agents, patches, liniments, film-forming agents, tinctures.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) The present invention can rapidly, accurately and comprehensively characterize and identify the non-volatile components in Ledum palustre L., and a large number of non-volatile components are obtained. A total of 118 non-volatile chemical components are identified.

[0029] (2) The present invention establishes a complete method system for identifying the non-volatile components in Ledum palustre L. (Ledum palustre L. leaves and tender branches). Each step and condition synergistically enhance the effect. The method is simple, rapid, and improves the identification efficiency, speed, comprehensiveness and accuracy, and has broad application prospects and market value.

[0030] (3) The present invention systematically characterizes and identifies the non-volatile chemical components in Extract A and Extract B of Ledum palustre L. for the first time, providing a reference for the study of its pharmacodynamic substances, quality control and product development. Description of the Drawings

[0031] Figure 1 It is a Venn diagram of the identification information of the non-volatile chemical components of Ledum palustre L.;

[0032] Figure 2 It is the base peak chromatogram (BPC) - negative ion mode of the UPLC-HRMS of the sample of Extract B of Ledum palustre L. leaves;

[0033] Figure 3 It is the base peak chromatogram (BPC) - negative ion mode of the UPLC-HRMS of the sample of Extract A of Ledum palustre L. leaves;

[0034] Figure 4 It is the base peak chromatogram (BPC) - negative ion mode of the UPLC-HRMS of the sample of Extract B of Ledum palustre L. tender branches;

[0035] Figure 5 It is the base peak chromatogram (BPC) - negative ion mode of the UPLC-HRMS of the sample of Extract A of Ledum palustre L. tender branches;

[0036] Figure 6 It is the chromatogram of the reference substance of Mixture 1;

[0037] Figure 7 It is the chromatogram of the reference substance of Mixture 2;

[0038] Figure 8 It is the chromatogram of the reference substance of Mixture 3;

[0039] Figure 9 It is the chromatogram of the reference substance of resveratrol 3-O-β-D-glucoside;

[0040] Figure 10 It is the chromatogram of the reference substance of luteolin;

[0041] Figure 11 It is the chromatogram of the reference substance of protocatechuic acid;

[0042] Figure 12 Chromatogram of the reference substance of the mixture of epigallocatechin and epicatechin gallate 4

[0043] Figure 13 Chromatogram of the reference substance of citric acid

[0044] Figure 14 Chromatogram of the reference substance of adenosine

[0045] Figure 15 Chromatogram of the reference substance of viscumoside

[0046] Figure 16 Chromatogram of the reference substance of 5-hydroxy-3,7,3′,4′-tetramethoxyflavone

[0047] Figure 17 Chromatogram of the reference substance of the mixture of quercitrin and oleanolic acid / ursolic acid 5

[0048] Figure 18 Chromatogram of the reference substance of fraxoside

[0049] Figure 19 Possible fragmentation pattern diagram of flavonoid compound 104

[0050] Figure 20 Possible fragmentation pattern diagram of flavonoid glycoside compound 50

[0051] Figure 21 Possible fragmentation pattern diagram of tannin compound 59

[0052] Figure 22 Secondary mass spectrum diagram of tannin compound 59 Detailed implementation manners

[0053] The present invention will be further described below through the description of the detailed implementation manners, but this is not a limitation to the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, but as long as they do not depart from the basic idea of the present invention, they are all within the scope of the present invention.

[0054] 1. Experimental materials

[0055] 1.1 Medicinal materials

[0056] The Ledum palustre L. samples were collected from Jiagedaqi, Daxing'anling, Heilongjiang Province. The medicinal materials were stored in the Science and Technology R & D Department of China National Traditional Chinese Medicine Co., Ltd. and identified as Ledum palustre L. by Professor Tu Pengfei.

[0057] 1.2 Reference substances and reagents

[0058] Acetonitrile (mass spectrometry grade, batch number I1232729230, Merck KGaA, Germany); methanol (mass spectrometry grade, batch number I1243235239, Merck KGaA, Germany); water (purified water, batch number 20240127C, Guangzhou Watson's Food & Beverage Co., Ltd.); absolute ethanol (analytical grade, batch number 20221207, Sinopharm Chemical Reagent Co., Ltd.); formic acid (mass spectrometry grade, batch number B2307771, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0059] There are 36 reference substances in total, including hyperoside (batch number 111521-202310, purity 94.7%), esculin (batch number 110741-201708, purity 99.9%), fraxin (batch number 110740-201806, purity 92.4%), quercitrin (batch number 111538-202308, purity 95.3%), chlorogenic acid (batch number 110753-202119, purity 96.3%), protocatechuic acid (batch number 110809-201906, purity 97.7%), arbutin (batch number 111951-201301, purity 98.7%), epicatechin (batch number 110878-201703, purity 99.7%) were all purchased from the National Institutes for Food and Drug Control, China; Catechin (batch number P02A9F57645, purity 98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; Neochlorogenic acid (batch number 17221), Cryptochlorogenic acid (batch number 14332), Citric acid (batch number 7792), Adenosine (batch number 17630G), Gallic acid (batch number 15673), Gallocatechin (batch number 2501), Protocatechualdehyde (batch number 15318G), Salidroside (batch number 18310G), Procyanidin B3 (batch number 16503G), Epigallocatechin (batch number 17140G), Procyanidin B4 (batch number 16504G), Fraxetin (batch number 14688G), Procyanidin B2 (batch number 17527G), Gypsophila paniculata glycoside (batch number 14878), Myricetin 3-O-β-galactoside (batch number 17945G), Procyanidin A1 (batch number 8925), Resveratrol-3-O-β-D-glucoside (batch number 14806G), Isoquercitrin (batch number 12870), Punicalagin (batch number 12619G), Avicularin (batch number 17211G), Astragalin (batch number 19167-P240701), Pteryxin (batch number 17828G), Asiatic acid (batch number 18703G), Eupalitin (batch number 16553G), 5-Hydroxy-3,7,3′,4′-tetramethoxyflavone (batch number 18642G), Oleanolic acid (batch number 17811G), Ursolic acid (batch number 16710G) were all sourced from Shanghai Standard Technology Service Co., Ltd., and their purities were all ≥98%.

[0060] 1.3 Experimental Instruments

[0061] Waters H-Class ultra-high performance liquid chromatograph (Waters Corporation, USA); AB Sciex Triple 4600 high-resolution mass spectrometer (SCIEX Corporation, USA); ME104 electronic balance [Mettler-Toledo International Trade (Shanghai) Co., Ltd.]; KQ-300BD ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); H1650-W high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); RE-5205A rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); VFD freeze dryer (Beijing Boyikang Experimental Instrument Co., Ltd.); BD / BC-423DKEM(E) freezer (Hefei Midea Refrigerator Co., Ltd.).

[0062] Example 1. A Method for Rapid Identification of Non-volatile Components in Ledum palustre L.

[0063] The method for rapid identification of non-volatile components in Ledum palustre L. includes the following steps:

[0064] 1. Preparation of test solution

[0065] Weigh 150 g of dried Ledum palustre L. leaves, place them in a 10000 mL round-bottom flask, add 3000 mL of distilled water, soak for 0.5 h, boil, and distill for 4 h by steam distillation method. After filtration, obtain the extract. Concentrate the obtained extract under reduced pressure, with the vacuum pressure ranging from -0.09 to -0.1 MPa and the concentration temperature at 75 °C, until the relative density reaches 1.06. After concentration, perform freeze-drying to obtain the freeze-dried powder of Ledum palustre L. leaf A extract; dry the filter residue after filtration at a drying temperature of 60 °C for 6 h, crush the dried filter residue, pass it through a 20-mesh sieve, add absolute ethanol to the crushed filter residue, with the mass-volume ratio of the crushed filter residue to absolute ethanol being 1:20 g / mL, reflux extract at 80 °C for 3 h, recover absolute ethanol and then perform freeze-drying to obtain the freeze-dried powder of Ledum palustre L. leaf B extract.

[0066] The freeze-drying procedure is shown in Table 1:

[0067] Table 1 Freeze-drying procedure

[0068]

[0069]

[0070] The preparation methods of the freeze-dried powder of Ledum palustre L. twig A extract and the freeze-dried powder of Ledum palustre L. twig B extract are similar to those of the freeze-dried powder of Ledum palustre L. leaf A extract and the freeze-dried powder of Ledum palustre L. leaf B extract respectively, with the only difference being that Ledum palustre L. leaves are replaced by Ledum palustre L. twigs.

[0071] Weigh accurately 0.5 g each of the freeze-dried powder of Ledum palustre L. leaf extract A and the freeze-dried powder of Ledum palustre L. twig extract A, place them in a 50 mL stoppered conical flask, add 7.5 mL of a methanol-water solution with a volume fraction of 15%, and ultrasonically treat (power 250 W, frequency 40 KHz) for 25 min. Take out, let it cool, shake well, take 2 mL and put it into a centrifuge tube, and centrifuge at high speed (10 000 r·min -1 ) for 7 min. Take the supernatant to obtain the test solution for liquid chromatography-mass spectrometry analysis.

[0072] Weigh accurately 0.5 g each of the freeze-dried powder of Ledum palustre L. leaf extract B and the freeze-dried powder of Ledum palustre L. twig extract B, place them in a 50 mL stoppered conical flask, add 7.5 mL of methanol, and ultrasonically treat (power 250 W, frequency 40 KHz) for 25 min. Take out, let it cool, shake well, take 2 mL and put it into a centrifuge tube, and centrifuge at high speed (10 000 r·min -1 ) for 7 min. Take the supernatant to obtain the test solution for liquid chromatography-mass spectrometry analysis.

[0073] 2. Preparation of reference solution

[0074] Weigh accurately appropriate amounts of 36 reference substances respectively, add methanol with different concentrations to prepare reference solutions with different mass concentrations for liquid chromatography-mass spectrometry analysis. The concentrations of each reference solution and the solvent information used are shown in Table 2.

[0075] Table 2 Concentrations of each reference solution and solvent information used

[0076]

[0077]

[0078]

[0079] 3. Inject the test solution and the reference solution into a Waters H-Class ultra-high performance liquid chromatograph and an ABSciex Triple 4600 high-resolution mass spectrometer for determination, where:

[0080] The chromatographic conditions were as follows: Waters ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm); the mobile phase consisted of 0.05% formic acid aqueous solution (A) and acetonitrile (B) by volume; gradient elution (0 - 3 min, 3% B; 3 - 7 min, 3% - 8% B; 7 - 20 min, 8% - 15% B; 20 - 40 min, 15% - 26% B; 40 - 55 min, 26% - 65% B; 55 - 61 min, 65% - 95% B; 61 - 64 min, 95% B); column temperature 25°C; flow rate 0.2 mL·min -1 ; injection volume 1 μL; detection wavelengths 280, 190 - 400 nm;

[0081] The mass spectrometry conditions were as follows: mass spectrometry detection mode: ESI, positive and negative ion modes, mass scanning range m / z 50 - 1700, Gas1 (nitrogen) nebulizing gas pressure 50 psi, Gas 2 (argon) auxiliary heating gas pressure 50 psi, curtain gas pressure 35 psi, ionization voltage -4500 / 5000 V, ion source temperature 500°C, declustering voltage 100 V, collision voltage 10 eV; secondary mass scanning range m / z 50 - 1250, declustering voltage 100 V, collision voltage ±40 eV, collision voltage swing 20 eV, ion release delay 30 ms, ion beam width 15 ms.

[0082] 4. UHPLC-Q-TOF-MS was used to analyze the collected data, including using Analyst TF 1.7.1 software to collect data, PeakView 1.2 software for data processing. When identifying, the mass spectrometry data was preferentially matched with the Natural Products HR-MS / MS Spectral Library 1.0 database. The reference substance was compared with the identification results based on information such as retention time and fragment ions. The compounds were preliminarily screened according to the score information of each chromatographic peak, and further confirmed according to the primary and secondary information of each chromatographic peak, etc. For compounds not included in the database, they were identified according to literature reports, mass spectrometry fragmentation rules, etc.

[0083] Example 2. A method for rapid identification of non-volatile components in Ledum palustre L.

[0084] The method for rapid identification of non-volatile components in Ledum palustre L. includes the following steps:

[0085] 1. Preparation of test solution

[0086] Weigh 150 g of dried Ledum palustre leaves, place them in a 10,000 mL round-bottom flask, add 3,000 mL of distilled water, soak for 3 h, boil, and distill for 6 h by steam distillation method. After filtration, obtain the extract. Concentrate the obtained extract under reduced pressure, with a vacuum pressure of -0.09 to -0.1 MPa and a concentration temperature of 85 °C, concentrate to a relative density of 1.10, and then freeze-dry to obtain the freeze-dried powder of Ledum palustre leaf A extract; dry the filter residue after filtration at a drying temperature of 60 °C for 7 h, crush the dried filter residue, pass through a 20-mesh sieve, add absolute ethanol to the crushed filter residue, and the mass-volume ratio of the crushed filter residue to absolute ethanol is 1:25 g / mL. Reflux and extract at 90 °C for 1 h, recover absolute ethanol and then freeze-dry to obtain the freeze-dried powder of Ledum palustre leaf B extract.

[0087] The freeze-drying procedure is the same as that in Table 1 of Example 1.

[0088] The preparation methods of the freeze-dried powder of Ledum palustre twig A extract and the freeze-dried powder of Ledum palustre twig B extract are similar to those of the freeze-dried powder of Ledum palustre leaf A extract and the freeze-dried powder of Ledum palustre leaf B extract respectively. The only difference is that Ledum palustre leaves are replaced by Ledum palustre twigs.

[0089] Weigh 0.5 g each of the freeze-dried powder samples of Ledum palustre leaf A extract and Ledum palustre twig A extract accurately, place them in a 50 mL stoppered conical flask, add 12.5 mL of a methanol aqueous solution with a volume fraction of 20%, ultrasonically treat (power 350 W, frequency 40 KHz) for 35 min, take out, cool to room temperature, shake well, take 2 mL and put it into a centrifuge tube, centrifuge at high speed (13,000 r·min -1 ) for 3 min, take the supernatant, and obtain the test solution for liquid chromatography-mass spectrometry analysis.

[0090] Weigh 0.5 g each of the freeze-dried powder samples of Ledum palustre leaf B extract and Ledum palustre twig B extract accurately, place them in a 50 mL stoppered conical flask, add 12.5 mL of methanol, ultrasonically treat (power 350 W, frequency 40 KHz) for 35 min, take out, cool to room temperature, shake well, take 2 mL and put it into a centrifuge tube, centrifuge at high speed (13,000 r·min -1 ) for 3 min, take the supernatant, and obtain the test solution for liquid chromatography-mass spectrometry analysis.

[0091] 2. Preparation of reference solution

[0092] The preparation method of the reference solution is the same as that in Example 1.

[0093] 3. Inject the test solution and the reference solution into a Waters H-Class ultra-high performance liquid chromatograph and an ABSciex Triple 4600 high-resolution mass spectrometer for determination, where:

[0094] The chromatographic conditions were as follows: Waters ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm); the mobile phase was composed of 0.2% formic acid aqueous solution (A) and acetonitrile (B) by volume; gradient elution (0 - 3 min, 3% B; 3 - 7 min, 3% - 8% B; 7 - 20 min, 8% - 15% B; 20 - 40 min, 15% - 26% B; 40 - 55 min, 26% - 65% B; 55 - 61 min, 65% - 95% B; 61 - 64 min, 95% B); column temperature 35 °C; flow rate 0.4 mL·min -1 ; injection volume 3 μL; detection wavelengths 280, 190 - 400 nm;

[0095] The mass spectrometry conditions were as follows: mass spectrometry detection mode: ESI, positive and negative ion modes, mass scanning range m / z 50 - 1700, Gas1 (nitrogen) spray gas pressure 50 psi, Gas 2 (argon) auxiliary heating gas pressure 50 psi, curtain gas pressure 35 psi, ionization voltage -4500 / 5000 V, ion source temperature 500 °C, declustering voltage 100 V, collision voltage 10 eV; secondary mass scanning range m / z 50 - 1250, declustering voltage 100 V, collision voltage ±40 eV, collision voltage swing 20 eV, ion release delay 30 ms, ion beam width 15 ms.

[0096] 4. UHPLC-Q-TOF-MS was used to analyze the collected data, including using Analyst TF 1.7.1 software to collect data, PeakView 1.2 software to process data. During identification, the mass spectrometry data was preferentially matched with the Natural Products HR-MS / MS Spectral Library 1.0 database. The reference substance was used to compare the identification results based on information such as retention time and fragment ions. The chromatographic peak score information was used to preliminarily screen the compounds, and further confirmation of the compounds was carried out according to the primary and secondary information of each chromatographic peak, etc. For compounds not included in the database, identification was carried out according to literature reports, mass spectrometry fragmentation rules, etc.

[0097] Example 3. A method for rapid identification of non-volatile components in Ledum palustre

[0098] The method for rapid identification of non-volatile components in Ledum palustre includes the following steps:

[0099] 1. Preparation of test solution

[0100] Weigh 150 g of dried Ledum palustre leaves, place them in a 10000 mL round-bottom flask, add 3000 mL of distilled water, soak for 1 h, boil, and distill for 5 h by steam distillation method. After filtration, obtain the extract. Concentrate the obtained extract under reduced pressure, with a vacuum pressure of -0.09 to -0.1 MPa and a concentration temperature of 80 °C, concentrate to a relative density of 1.08, and then freeze-dry after concentration to obtain the freeze-dried powder of Ledum palustre leaf A extract; dry the filter residue after filtration, with a drying temperature of 60 °C and a drying time of 8 h. Crush the dried filter residue, pass through a 20-mesh sieve, add absolute ethanol to the crushed filter residue, and the mass-volume ratio of the crushed filter residue to absolute ethanol is 1:20 g / mL. Reflux and extract at 85 °C for 2 h, recover absolute ethanol and then freeze-dry to obtain the freeze-dried powder of Ledum palustre leaf B extract.

[0101] The freeze-drying procedure is the same as that in Table 1 of Example 1.

[0102] The preparation methods of the freeze-dried powder of Ledum palustre twig A extract and the freeze-dried powder of Ledum palustre twig B extract are similar to those of the freeze-dried powder of Ledum palustre leaf A extract and the freeze-dried powder of Ledum palustre leaf B extract respectively, with the only difference being that Ledum palustre leaves are replaced by Ledum palustre twigs.

[0103] Weigh 0.5 g each of the freeze-dried powder samples of Ledum palustre leaf A extract and Ledum palustre twig A extract accurately, place them in a 50 mL stoppered conical flask, add 10 mL of a methanol aqueous solution with a volume fraction of 20%, and ultrasonically treat (power 300 W, frequency 40 KHz) for 30 min. Take out, cool to room temperature, shake well, take 2 mL and transfer it to a centrifuge tube, and centrifuge at high speed (12 000 r·min -1 ) for 5 min. Take the supernatant to obtain the test solution for liquid chromatography-mass spectrometry analysis.

[0104] Weigh 0.5 g each of the freeze-dried powder samples of Ledum palustre leaf B extract and Ledum palustre twig B extract accurately, place them in a 50 mL stoppered conical flask, add 10 mL of methanol, and ultrasonically treat (power 300 W, frequency 40 KHz) for 30 min. Take out, cool to room temperature, shake well, take 2 mL and transfer it to a centrifuge tube, and centrifuge at high speed (12000 r·min -1 ) for 5 min. Take the supernatant to obtain the test solution for liquid chromatography-mass spectrometry analysis.

[0105] 2. Preparation of reference solution

[0106] The preparation method of the reference solution is the same as that in Example 1.

[0107] 3. Inject the test solution and the reference solution into a Waters H-Class ultra-high performance liquid chromatograph and an ABSciex Triple 4600 high-resolution mass spectrometer for determination, where:

[0108] The chromatographic conditions were as follows: Waters ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm); the mobile phase was composed of 0.1% formic acid aqueous solution (A) and acetonitrile (B) by volume; gradient elution (0 - 3 min, 3% B; 3 - 7 min, 3% - 8% B; 7 - 20 min, 8% - 15% B; 20 - 40 min, 15% - 26% B; 40 - 55 min, 26% - 65% B; 55 - 61 min, 65% - 95% B; 61 - 64 min, 95% B); column temperature 30 °C; flow rate 0.3 mL·min -1 ; injection volume 2 μL; detection wavelengths 280, 190 - 400 nm;

[0109] The mass spectrometry conditions were as follows: mass spectrometry detection mode: ESI, positive and negative ion modes, mass scanning range m / z 50 - 1700, Gas1 spray gas (nitrogen) pressure 50 psi, Gas 2 auxiliary heating gas (argon) pressure 50 psi, curtain gas pressure 35 psi, ionization voltage -4500 / 5000 V, ion source temperature 500 °C, declustering voltage 100 V, collision voltage 10 eV; secondary mass scanning range m / z 50 - 1250, declustering voltage 100 V, collision voltage ±40 eV, collision voltage swing 20 eV, ion release delay 30 ms, ion beam width 15 ms.

[0110] 4. The collected data were analyzed by UHPLC-Q-TOF-MS, including data collection by Analyst TF 1.7.1 software and data processing by PeakView 1.2 software. During identification, the mass spectrometry data was matched with the Natural Products HR-MS / MS Spectral Library 1.0 database first. The identification results were compared with the reference materials according to the retention time, fragment ion and other information. The compounds were preliminarily screened based on the score information of each chromatographic peak, and the compounds were further confirmed based on the primary and secondary information of each chromatographic peak. The Natural Products HR-MS / MS Spectral Library 1.0 database contains multi-level mass spectra of reference materials from Shanghai Shidande Standard Technology Service Co., Ltd. or other sources, including different acquisition modes, different adduct ions, different collision energies, etc. The compound information is comprehensive and the matching results are highly accurate. Compounds not included in the database were identified by searching literature reports on platforms such as CNKI (https: / / www.cnki.net / ), PubMed (https: / / pubmed.ncbi.nlm.nih.gov / ), and PubChem (https: / / pubchem.ncbi.nlmnih.gov / ), as well as mass spectrometry fragmentation patterns.

[0111] 5. Results and Analysis

[0112] The high-resolution data of Ledum A and B extracts collected in positive and negative ion modes were analyzed by comparison with reference substances, database retrieval and literature comparison. A total of 118 compounds were identified or deduced from the Ledum samples, including 110 chemical components from the alcohol-extracted freeze-dried powder of Ledum leaves, 107 chemical components from the water-extracted freeze-dried powder of Ledum leaves, 101 chemical components from the alcohol-extracted freeze-dried powder of Ledum twigs, and 103 chemical components from the water-extracted freeze-dried powder of Ledum twigs. The 118 compounds include 35 flavonoid glycosides, 14 phenolic glycosides, 12 flavonoids, 8 phenolic acids, 7 phenylethanol glycosides, 6 tannins, 6 phospholipids, 5 coumarins, 5 monoterpene glycosides, 6 triterpenes, 3 fatty acids, and 11 other compounds. Among them, 36 compounds were identified by comparison with reference substances. The identification results are shown in Table 3. Figure 1 , the negative ion current diagram of Ledum leaf and twig A extract and B extract is shown in Figures 2 to 5 , 36 reference substance chromatograms are shown in Figures 6 to 18 ,in, Figure 6 It is obtained by injecting proanthocyanidin B3, proanthocyanidin B4 and proanthocyanidin B2 as mixed reference substances (mixed solution 1 reference substance). Figure 7It is obtained by injecting neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid as mixed reference substances (mixed solution 2 reference substances). Figure 8 The sample was obtained by injecting arbutin, gallic acid, protocatechuic aldehyde, salidroside, aesculin, catechin, aesculetin, kalanchoe glycoside, epicatechin, myricetin 3-O-β-galactoside, proanthocyanidin A1, hyperoside, isoquercetin, guajava glycoside, astragaloside, asiatic acid and euphorbia lutea as mixed reference substances (mixed solution 3 reference substances). Figure 12 It is obtained by injecting gallocatechin and epigallocatechin as mixed reference substances (mixed solution 4 reference substances). Figure 17 It is obtained by injecting quercetin, oleanolic acid and ursolic acid as mixed reference substances (mixed solution 5 reference substances).

[0113] Table 3 Identification results of main non-volatile components of Ledum palustris samples

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Note: 1) Compounds identified from Ledum palustris B extract; 2) Compounds identified from Ledum palustris A extract; 3) Compounds identified from Ledum palustris B extract; 4) Compounds identified from Ledum palustris shoots A extract; 5) Compounds compared with reference substances; 6) Compounds that have been reported in the literature.

[0125] From Table 3 and Figure 1It can be seen that a total of 118 chemical components were identified in the A extract and B extract of Ledum palustre leaves, which were consistent with the 118 chemical components identified in the A extract and B extract of the tender branches of Ledum palustre. There were 84 chemical components in common in the A extract and B extract of Ledum palustre leaves and tender branches, accounting for about 71.2% of the 118 identified chemical components. In addition to the 84 chemical components, there were 11 chemical components in common in the A extract and B extract of Ledum palustre leaves and the A extract of the tender branches of Ledum palustre, 4 chemical components in common in the A extract and B extract of Ledum palustre leaves and the B extract of the tender branches of Ledum palustre, 2 chemical components in common in the A extract and B extract of the tender branches of Ledum palustre and the A extract of Ledum palustre leaves, 6 chemical components in common in the A extract of Ledum palustre leaves and the A extract of the tender branches of Ledum palustre, and 11 chemical components in common in the B extract of Ledum palustre leaves and the B extract of the tender branches of Ledum palustre.

[0126] Figures 6 to 18Chinese: 3. Citric acid; 4. Adenosine; 5. Arbutin; 6. Gallic acid; 11. Protocatechuic acid; 14. Gallocatechin; 19. Neochlorogenic acid; 20. Protocatechualdehyde; 22. Salidroside; 23. Fraxin; 26. Procyanidin B3; 27. Epigallocatechin; 30. Chlorogenic acid; 31. Catechin; 32. Esculin; 34. Cryptochlorogenic acid; 37. Procyanidin B4; 38. Fraxetin; 40. Procyanidin B2; 41. Gypsophila paniculata glycoside; 44. Epicatechin; 56. Myricetin 3-O-β-galactoside; 59. Procyanidin A1; 62. Resveratrol 3-O-β-D-glucoside; 64. Hyperoside; 65. Isoquercitrin; 67. Punicalagin; 71. Avicularin; 73. Astragalin; 74. Quercitrin; 99. Pteryxin; 100. Asiatic acid; 103. Eupalitin; 108. 5-Hydroxy-3,7,3′,4′-tetramethoxyflavone; 118. Oleanolic acid / ursolic acid.

[0127] 5.1 Identification of flavonoid glycosides and flavonoid components

[0128] Flavonoid glycosides have stronger activities than flavonoid components and possess strong antioxidant effects. Most of them are formed by adding glycosides to flavonoid aglycones. Small molecule glycosylation can enhance the ability of organisms to resist external environmental stresses. The most abundant compounds identified in Ledum palustre plants in the present invention are flavonoid glycosides. Some, such as catechin-3-O-glucoside and catechin-7-O-glucoside, are derived from the glycosylation of catechin. This may contribute to improving their tolerance to abiotic stresses such as low temperature, and it is also possible that their solubility and bioavailability increase. Based on the structures of Ledum palustre flavonoid aglycones, glycosyl groups, and flavonoid components identified in the present invention, the structural characteristics, characteristic fragment ions, and the rules of neutral fragment ion loss are summarized as follows.

[0129] ① The flavonoids mainly include flavan-3-ols, flavones, isoflavones, and flavonols. The cleavage rules of flavan-3-ols (catechin and gallocatechin) are consistent with the literature reports, and they are prone to undergo retro-Diels-Alder (RDA) cleavage of ring C and the loss of ring B. Other flavonoids have methoxylation substitutions at different hydroxyl sites; the main cleavage fragment ions are [M-H] - , [M-CH3] - , [M-CO] - , [M-CH3-CO] - , [M-H2CO2] - , [M-H2O] - ; flavonols with a C-2′ methoxy group are prone to lose this methoxy group [M-OCH3] - to form a new stable five-membered heterocycle, such as compound 102 and compound 104. The possible cleavage rules are shown in Figure 19 .

[0130] ② The flavonoid glycosides mainly include flavonol glycosides, flavanol glycosides, and dihydroflavonol glycosides (taxifolin-like) components. Most of the flavonoid glycoside components in Ledum palustre are glycosylated at the C-3 or C-7 hydroxyl sites of their aglycones, and C-3′ hydroxyl substitution is occasionally seen. Flavonoid glycosides lose glycosyl fragment ions such as glucosyl (C6H 10 O5) (162), galactosyl (C6H 10 O5) (162), rhamnosyl (C6H 10 O4) (146), arabinosyl (C5H8O4) (132), etc.; at the same time, the hydroxyl groups on the glycosyl groups of flavonoid glycosides have acetylation (C2H3O) (43), coumaroylation (C9H7O2) (147), caffeoylation (C9H7O3) (163), and feruloylation (C 10Substituted by H9O3)(177), flavonoid glycosides have fragment ions with the loss of these substituents or glycosyl substituents, resulting in flavonoid glycoside fragment ions or flavonoid aglycone fragment ions. Typical flavonol aglycone fragment ions include C 15 H9O7(301), C 15 H8O7(300), C 14 H7O6(271), C 14 H7O5(255), etc.

[0131] A total of 35 flavonoid glycosides and 12 flavonoid compounds were identified or deduced from Ledum palustre in this invention. Compounds 56, 64 - 65, 67, 71, 73 - 74 were identified as flavonoid glycoside components by comparison with reference substances, and compounds 14, 27, 31, 44, 99, 103 were confirmed as flavonoid components by comparison with reference substances.

[0132] Compound 64 (t R = 23.61 min) showed a molecular ion peak at m / z 463.090 1 [M-H] - , and its molecular formula was deduced to be C 21 H 20 O 12 (with an error of 4.1×10 -6 ). Its characteristic fragment ion m / z 301.034 3 [M-H-C6H 10 O5] - was produced by the loss of one molecule of galactose from the parent ion. Then, by neutral loss of 2H, 1 molecule of CO or loss of 1 molecule of H2O, 1 molecule of CO, fragment ions m / z 300.026 2 [M-H-C6H 10 O5-H] - , 271.023 8 [M-H-C6H 10 O5-2H-CO] - , 255.0280 [M-H-C6H 10 O5-H2O-CO] - were obtained respectively. By comparison with the reference substance, it was found that the retention time, parent ion and secondary fragment characteristics of this compound were consistent with those of hyperoside and the literature reports. Therefore, compound 64 was identified as hyperoside.

[0133] Compound 73 (t R = 27.67 min) showed a molecular ion peak at m / z 447.095 1 [M-H] - , and its molecular formula was deduced to be C 21 H 20 O 11 (with an error of 4.0×10 -6)。 Its characteristic fragment ion m / z 285.038 9 [M-H-C6H 10 O5] - is generated by the loss of one molecule of pyranose glucose from the parent ion. After further neutral loss of 2H, 1 molecule of CO, and 1 molecule of CO, fragment ions m / z 284.031 4 [M-H-C6H 10 O5-H] - , 255.028 6 [M-H-C6H 10 O5-2H-CO] - , 227.033 9 [M-H-C6H 10 O5-2H-2CO] - are obtained respectively. By comparing with the reference substance, it is found that the retention time, parent ion, and secondary fragment characteristics of this compound are consistent with those of astragalin and the literature reports. Therefore, compound 73 is identified as astragalin.

[0134] Compound 50 (t R = 17.03 min) shows a molecular ion peak at m / z 465.105 5 [M-H] - , and its molecular formula is deduced to be C 21 H 22 O 12 (with an error of 3.7×10 -6 ). Its characteristic fragment ion m / z 285.040 5 [M-H-C6H 10 O5-H2O] - is generated by the loss of one molecule of glucose and then one molecule of H2O from the parent ion. After further RDA cleavage, the vinyl group contained in the B-ring ion unique to dihydroflavonols is obtained, and then after neutral loss of 2H and 1 molecule of CO2, characteristic fragment ions are obtained. Based on the parent ion and secondary fragment characteristics, compound 50 is identified as taxifolin 3-O-glucoside, and its possible cleavage pattern is shown in Figure 20 .

[0135] 5.2 Identification of phenolic glycoside components

[0136] Phenolic glycoside compounds are glycoside compounds formed by the dehydration condensation of phenolic hydroxyl groups in the aglycone molecule with the hemiacetal or hemiketal hydroxyl groups of sugars. In the negative ion mode, the quasi-molecular ions of phenolic glycoside compounds are basically all [M-H] - , and there are also [M-H+COOH] with relatively high responses -。Under the cleavage voltage, glycosyl groups are often lost, and the characteristic fragment ions are glycosyl groups and aglycones after the loss of glycosyl groups; or small molecules C4H8O4 (120) on the glycosyl group are lost. A total of 15 phenolic glycoside compounds were identified from Ledum palustre L. Among them, compounds 5 and 62 were confirmed by comparison with reference substances and were arbutin and resveratrol-3-O-β-D-glucoside, respectively.

[0137] Compound 5 (t R = 2.06 min) showed a molecular ion peak at m / z 317.089 0 [M-H+COOH] - , m / z 271.084 3 [M-H] - , and its molecular formula was inferred to be C 12 H 16 O7 (the error was 3.8×10 -6 ). Its characteristic fragment ions m / z 161.045 9 and 108.021 9 were generated by the 1,4-benzoquinone obtained by removing one molecule of pyranose glucose group from the parent ion and removing 2 H from hydroquinone, respectively. By comparing with the reference substance, it was found that the retention time, parent ion, and secondary fragment characteristics of this compound were consistent with those of arbutin, so compound 5 was identified as arbutin.

[0138] Compound 62 (t R = 23.93 min) showed a molecular ion peak at m / z 435.132 2 [M-H+COOH] - , and its molecular formula was inferred to be C 20 H 22 O8 (the error was 5.7×10 -6 ). Its characteristic fragment ion m / z 227.071 8 [M-H-C6H 10 O5] - was generated by removing one molecule of glucose group from the parent ion. After neutral loss of one molecule of C2H2O (42), the fragment ion m / z 185.059 4 [M-H-C2H2O] - was obtained. By comparing with the reference substance, it was found that the retention time, parent ion, and secondary fragment characteristics of this compound were consistent with those of resveratrol-3-O-β-D-glucoside and the literature reports, so compound 62 was identified as resveratrol-3-O-β-D-glucoside.

[0139] 5.3 Identification of phenolic acid components

[0140] For phenolic acid compounds in the negative ion mode, their quasi-molecular ions are basically all [M-H]- , at the cleavage voltage, it often breaks at the carboxyl group to form characteristic fragment ions, and then further loses neutral small molecules such as H2O, CO2, and CO. Seven phenolic acid compounds were identified from Ledum palustre L. in this invention, and compounds 6, 11, 19 - 20, 30, and 34 were confirmed by comparison with reference substances.

[0141] Compound 19 (t R = 8.81 min) has a molecular ion peak at m / z 353.087 8 [M - H] - , and its molecular formula is deduced to be C 16 H 18 O9 (with an error of 0×10 -6 ). Its characteristic fragment ions m / z 191.055 3 [M - H - C9H6O3] - , 179.034 8 [M - H - C7H 10 O5] - are obtained by the parent ion losing the quinic acid group and caffeic acid group respectively, and further losing H2O and CO2 respectively to get m / z 173.044 7 [M - H - C9H6O3 - H2O] - , 135.045 1 [M - H - C7H 10 O5 - CO2] - . After comparison with the reference substance, compound 19 was identified as neochlorogenic acid. Compound 30 (t R = 11.81 min) and compound 34 (t R = 12.79 min) show molecular ion peaks both at m / z 353 [M - H] - , and according to their characteristic fragment ions and comparison with the reference substance, compound 30 was identified as chlorogenic acid, and compound 34 was identified as cryptochlorogenic acid.

[0142] Compound 6 (t R = 2.96 min) has a molecular ion peak at m / z 169.014 7 [M - H] - , and its molecular formula is deduced to be C7H6O5 (with an error of 3.0×10 -6 ). After losing CO2, CO, and H2O, it obtains fragment ions m / z 125.024 5 [M - H - CO2] - , 97.029 6 [M - H - CO2 - CO] - , 79.018 7 [M - H - CO2 - CO - H2O] -By comparing with the reference substance, it was found that the retention time, parent ion, and secondary fragment characteristics of this compound were all consistent with those of gallic acid. Therefore, compound 6 was identified as gallic acid.

[0143] 5.4 Identification of phenylethanoid glycoside components

[0144] Phenylethanoid glycoside compounds are glycoside compounds formed by the combination of phenylethanoid glycoside aglycone and glycosyl groups. In the negative ion mode, their quasi-molecular ions are basically all [M-H] - , often accompanied by [M-H+COOH] with a relatively high response - . They often lose glycosyl groups, and the characteristic fragment ions are glycosyl groups and phenylethanoid glycoside aglycone after losing glycosyl groups; further loss of neutral small molecules such as H2O (18) and H2CO (30). A total of 7 phenylethanoid glycoside compounds were identified from Ledum palustre in this invention, and compound 22 was confirmed by comparison with the reference substance.

[0145] The molecular ion peak of compound 22 (t R = 9.50 min) is m / z 345.119 2 [M-H+COOH] - , m / z 299.1199 [M-H] - , and its molecular formula was inferred to be C 14 H 20 O7 (the error is 0.3×10 -6 ). Its characteristic fragment ions m / z 161.045 5 [M-H-C8H 10 O2] - , 137.060 5 [M-H-C6H 10 O5] - were obtained by the parent ion losing glucose group and 4-hydroxyphenylethyl group respectively, and further losing H2O (18) to get m / z 119.049 3 [M-H-C6H 10 O5-H2O] - . After comparison with the reference substance, compound 22 was identified as salidroside.

[0146] 5.5 Identification of tannin components

[0147] Tannins are complex polyphenolic compounds. The main tannin precursors identified in Ledum palustre in this invention are catechin-like substances, and their tannins are polymers condensed by carbon-carbon bonds, all of which are dimers. Based on their molecular ion peaks and characteristic fragment ions, they were speculated to be proanthocyanidin type A and type B, that is, dimers formed by C-O-C bonds between C2 and C7 or C2 and C5 respectively and dimers connected by C4-C8 or C4-C6 bonds. A total of 6 tannin compounds were identified from Ledum palustre in this invention, and compounds 26, 37, 40, and 59 were confirmed by comparison with the reference substance.

[0148] Compound 40 (t R = 14.30 min) has a molecular ion peak at m / z 577.136 8 [M-H] - , and its molecular formula is deduced to be C 30 H 26 O 12 (with an error of 2.9×10 -6 ). Its characteristic fragment ion m / z 451.101 9 [M-H2O-C6H6O3] - is obtained by losing one molecule of H2O from the parent ion and then undergoing an HRF (heterocyclic ring fission) reaction to lose one molecule of phloroglucinol. m / z 425.087 1 [M-C8H8O3(152)] - is generated by the RDA reaction of the parent ion. After losing one molecule of H2O, the fragment ion m / z 407.0771 [M-C8H8O3-H2O] - is produced by the cleavage of the carbon-carbon bond of the parent ion to generate the fragment ion m / z289.070 7 [M-C 15 H 12 O6(288)] - . By comparing with the reference substance, it is found that the retention time, parent ion and secondary fragment characteristics of this compound are consistent with those of procyanidin B2 and the literature reports. Therefore, compound 40 is identified as procyanidin B2.

[0149] Compound 59 (t R = 20.91 min) has a molecular ion peak at m / z 575.122 5 [M-H] - , and its molecular formula is deduced to be C 30 H 24 O 12 (with an error of 5.2×10 -6 ). Its characteristic fragment ion m / z 539.098 7 [M-H2O-H2O] - is obtained by losing two molecules of H2O from the parent ion. m / z 449.088 0 [M-C6H6O3] - is obtained by the HRF reaction of the parent ion to lose one molecule of phloroglucinol [C6H6O3(126)]. The fragment ion m / z 289.072 5 [M-C 15 H 10 O6(286)] - , and its possible cleavage pattern is shown in Figure 21 , and its secondary mass spectrum is shown in Figure 22。By comparing with the reference substance, it was found that the retention time, parent ion, and secondary fragment characteristics of this compound were consistent with those of procyanidin A1 and the literature reports. Therefore, compound 59 was identified as procyanidin A1.

[0150] 5.6 Identification of Coumarin Components

[0151] Coumarin is a general term for a class of natural compounds with a benzopyranone-α pyrone nucleus. Structurally, it can be regarded as the lactone formed by the dehydration of cis-o-hydroxycinnamic acid. Many coumarins and their glycosides have various biological activities. In the negative ion mode, their quasi-molecular ions are basically all [M-H] - , and often after losing the sugar group, they further lose neutral small molecules such as CO2, CH3, and CO in the lactone structure of coumarin. Five coumarin compounds were identified from Ledum palustre in this invention, and compounds 23, 32, and 38 were confirmed by comparing with the reference substance.

[0152] The molecular ion peak of compound 23 (t R = 9.89 min) is m / z 339.071 5 [M-H] - , and its molecular formula was deduced to be C 15 H 16 O9 (the error is -2.1×10 -6 ). Its characteristic fragment ion m / z 177.018 8 [M-H-C6H 10 O5] - is obtained by the parent ion losing the glucose group, and further losing CO2 and CO respectively to obtain m / z 133.0298 [M-H-C6H 10 O5-CO2] - , 105.034 5 [M-H-C6H 10 O5-CO2-CO] - . After comparing with the reference substance, compound 23 was identified as aesculin.

[0153] The molecular ion peak of compound 38 (t R = 13.59 min) is m / z 369.083 4 [M-H] - , and its molecular formula was deduced to be C 16 H 18 O 10 (the error is 1.9×10 -6 ). Its characteristic fragment ion m / z 207.028 9 [M-H-C6H 10 O5] -It is obtained by the loss of a glucosyl group from the parent ion, and further loses CO2 and CH3 respectively to obtain m / z 163.003 2 [M-H-C6H 10 O5-CO2] - 、192.005 3 [M-H-C6H 10 O5-CH3] - , and m / z163.003 2 further loses CO to obtain m / z 135.008 5 [M-H-C6H 10 O5-CO2-CO] - . After comparison with the reference substance, compound 38 was identified as fraxetin.

[0154] 5.7 Identification of Triterpenoid Components

[0155] Triterpenoids are widely distributed in Chinese herbal medicines. They are polymerized from isoprene and mainly include tetracyclic triterpenoids and pentacyclic triterpenoids. Based on their molecular ion peaks and characteristic fragment ions, it is speculated that the triterpenoid compounds in the Ledum palustre extract of the present invention are all pentacyclic triterpenoid components. Their molecular ions are prone to losing neutral small molecules such as H2O and CO2 or undergoing RDA cleavage to obtain characteristic fragment ions. A total of 6 triterpenoid compounds were identified from Ledum palustre in the present invention, and compounds 100 and 118 were confirmed by comparison with the reference substance.

[0156] The molecular ion peak of compound 100 (t R =50.33 min) is m / z 533.350 0 [M-H+COOH] - , m / z487.343 1 [M-H] - , and its molecular formula is inferred to be C 30 H 48 O5 (the error is 3.0×10 -6 ). Its characteristic fragment ion m / z425.338 6 [M-H-H2O-CO2] - is obtained by the loss of 1 molecule of H2O and 1 molecule of CO2 from the parent ion. After comparison with the reference substance, compound 100 was identified as asiatic acid.

[0157] 5.8 Others

[0158] Through database automatic matching results and comparison with reference substances, a total of 24 other compounds were identified: 2 organic acids (among which citric acid was identified by comparison with the reference substance), 1 ester, 3 fatty acids, 1 carbohydrate, 2 lignans, 5 phospholipids, 1 nucleoside (by comparison with the reference substance), 1 phenolic acid glycoside, 5 monoterpene glycosides, 1 sesquiterpene glycoside (by comparison with the reference substance), and 2 unknown components, as shown in Table 3 above.

[0159] The present invention uses UHPLC-Q-TOF-MS technology, combined with reference substance comparison and databases, to characterize and identify the chemical components of Extract A and Extract B from the tender branches and leaves of Ledum palustre. From the identification results, the chemical components of Extract A and Extract B in the leaves are more than those in the tender branches. There are basically no triterpenoid and phospholipid components in Extract A from the leaves and tender branches, and there are no organic acid components with strong polarity in Extract B from the leaves and tender branches, suggesting that the influence of the extraction solvent on the qualitative and quantitative determination of characteristic components should be considered when establishing the quality standard; the current research mainly focuses on the medicinal part of Ledum palustre leaves. According to the research results of the present invention, the tender branches of Ledum palustre also contain many active ingredients, and no toxic components are found in the leaves and tender branches after extracting the volatile oil. These suggest that in the future comprehensive utilization of Ledum palustre, the tender branches can also be used as its medicinal part, and at the same time, the influence of the extraction process on the extraction effect of chemical components should be considered.

[0160] The compounds identified in the present invention are mainly flavonoid glycosides, phenolic glycosides, flavonoids, phenolic acids, phenyl ethanol glycosides, tannins, phospholipids, coumarins, monoterpene glycosides and triterpenoids. Both flavonoid glycosides and flavonoid compounds have pharmacological effects such as anti-inflammatory, antioxidant, immunomodulatory and anti-tumor. Phenolic glycoside and phenolic acid compounds both have phenolic hydroxyl groups, and phenolic hydroxyl groups can capture and stabilize the oxidative free radicals in the body, thus playing an antioxidant role. Oxidative stress is an important cause of aging and many diseases. Phenyl ethanol glycoside compounds have effects such as antibacterial, anti-inflammatory, antiviral, anti-tumor, antioxidant, immunomodulatory, memory enhancement, liver protection and cardiotonic, especially the antibacterial activity is the most significant. Monoterpene glycoside compounds have pharmacological effects such as antibacterial, anti-inflammatory, anti-tumor, liver protection and hypoglycemic. These substances are related to the anti-inflammatory, antibacterial, antioxidant and anti-tumor effects of Ledum palustre, and may be the material basis for its pharmacological effects.

[0161] There are many identifications of structural similar isomers in the present invention. For example, Compound 13 and Compound 22, Compound 47, Compound 51 and Compound 56, Compound 66 and Compound 71, Compound 68 and Compound 73, Compound 116 and Compound 117, etc. are all isomers, and some compounds are determined by comparing with reference substances. In summary, the present invention analyzes the non-volatile chemical components of Ledum palustre based on UHPLC-Q-TOF-MS / MS technology, and a total of 118 components are identified. These components provide a reference for the research on the medicinal substances, quality control and product development of Ledum palustre.

Claims

1. A method for rapidly identifying non-volatile components in the Chinese medicine Ledum palustris, characterized in that: The following steps are involved: S1 Preparation of test solution: Take a sample of Ledum palustris, add extraction solvent A, soak, boil, distill by steam distillation, filter to obtain an extract, concentrate the obtained extract, freeze-dry after concentration to obtain freeze-dried powder of Ledum palustris A extract; dry the filter residue after filtration, crush the dried filter residue, add extraction solvent B to the crushed filter residue, reflux extraction, recover the extraction solvent B and freeze-dry to obtain freeze-dried powder of Ledum palustris B extract; take freeze-dried powder of Ledum palustris A extract and freeze-dried powder of Ledum palustris B extract respectively, add extraction solvent C, perform ultrasonic treatment and centrifugation, take the supernatant to obtain the test solution; Preparation of S2 reference solution: Take the reference substance and add solvent to make a reference solution; S3 injects the test solution and the reference solution into an ultra-high performance liquid chromatograph and a high-resolution mass spectrometer for determination respectively; S4 used UHPLC-Q-TOF-MS to analyze the collected data.

2. The method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to claim 1, characterized in that: The Ledum samples in step S1 are Ledum leaves and Ledum twigs.

3. The method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to claim 1, characterized in that: In step S1, the extraction solvent A is water, the soaking time is 0.5-2h, the steam distillation time is 4-6h, and the obtained extract is concentrated by reduced pressure concentration, the vacuum pressure is -0.09 to -0.1MPa, the concentration temperature is 75-85°C, and the relative density is concentrated to 1.06-1.

10. The extraction solvent B is anhydrous ethanol, the reflux extraction temperature is 80-90°C, and the reflux extraction time is 1-3h.

4. The method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to claim 1, characterized in that: In step S1, the mass volume ratio of the Ledum sample to the extraction solvent A is 1:15-25 g / mL; the mass volume ratio of the crushed filter residue to the extraction solvent B is 1:15-25 g / mL; the mass volume ratio of the Ledum A extract freeze-dried powder to the extraction solvent C is 1:15-25 g / mL; the mass volume ratio of the Ledum B extract freeze-dried powder to the extraction solvent C is 1:15-25 g / mL.

5. The method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to claim 1, characterized in that: In the step S1, when taking the freeze-dried powder of the Ledum palustris A extract, adding the extraction solvent C, and performing ultrasonic treatment and centrifugal treatment, the extraction solvent C is a methanol aqueous solution with a volume fraction of 15-25%, the ultrasonic power is 250-350W, the ultrasonic time is 25-35min, the centrifugal speed is 10000-13000r / min, and the centrifugal time is 3-7min; when taking the freeze-dried powder of the Ledum palustris B extract, adding the extraction solvent C, and performing ultrasonic treatment and centrifugal treatment, the extraction solvent C is methanol, the ultrasonic power is 250-350W, the ultrasonic time is 25-35min, the centrifugal speed is 10000-13000r / min, and the centrifugal time is 3-7min.

6. The method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to claim 1, characterized in that: The reference substances in step S2 include hyperoside, aesculetin, aesculetin, quercetin, chlorogenic acid, protocatechuic acid, arbutin, epicatechin, catechin, neochlorogenic acid, cryptochlorogenic acid, citric acid, adenosine, gallic acid, gallocatechin, protocatechualdehyde, salidroside, proanthocyanidin B3, epigallocatechin, proanthocyanidin B4, aesculetin, proanthocyanidin B2, kalanchoe glycoside, myricetin 3-O-β-galactoside, proanthocyanidin A1, resveratrol-3-O-β-D-glucoside, isoquercitrin, guava glycoside, broad parasitoid glycoside, astragalin, jatropha flavescens, asiatic acid, zedoaria flavescens, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone, oleanolic acid and ursolic acid.

7. The method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to claim 1, characterized in that: The chromatographic conditions in step S3 are as follows: Waters ACQUITY UPLC HSS T3 chromatographic column: 2.1 mm×100 mm, 1.8 μm; the mobile phase is composed of a formic acid aqueous solution (A) and acetonitrile (B) with a volume fraction of 0.05-0.2%; gradient elution, 0-3 min, 3% B; 3-7 min, 3%-8% B; 7-20 min, 8%-15% B; 20-40 min, 15%-26% B; 40-55 min, 26%-65% B; 55-61 min, 65%-95% B; 61-64 min, 95% B; column temperature 25-35° C.; flow rate 0.2-0.4 mL / min; injection volume 1-3 μL; detection wavelengths 280, 190-400 nm.

8. The method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to claim 1, characterized in that: The mass spectrometry conditions in step S3 are as follows: mass spectrometry detection mode: ESI, positive and negative ion modes, mass scanning range of m / z 50-1700, spray gas pressure of 50 psi, auxiliary heating gas pressure of 50 psi, curtain gas pressure of 35 psi, ionization voltage of -4500 / 5000 V, ion source temperature of 500° C., declustering voltage of 100 V, collision voltage of 10 eV; secondary mass scanning range of m / z 50-1250, declustering voltage of 100 V, collision voltage of ±40 eV, collision voltage swing of 20 eV, ion release delay of 30 ms, and ion beam width of 15 ms.

9. Use of the method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to any one of claims 1 to 8 in detecting non-volatile components in Ledum palustris or a preparation containing Ledum palustris.

10. The use of the method for rapid identification of non-volatile components in the Chinese medicine Ledum palustris according to 9 in detecting non-volatile components in Ledum palustris or a preparation containing Ledum palustris, characterized in that: The preparations containing Ledum palustris include granules, capsules, pills, tablets, oral liquids, injections, ointments, suppositories, granules, lozenges, powders, pills, suspensions, powders, creams, sprays, drops, sustained-release preparations, patches, liniments, coatings, and tinctures.

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