Method for extracting and enriching phenylethanoid glycoside and lignan effective components from fructus forsythiae fresh leaves as well as enriched product and application of enriched product

Abstract

The invention provides a method for extracting and enriching phenylethanoid glycoside and lignan effective components from fructus forsythiae fresh leaves. The method comprises the following steps: extracting phenylethanoid glycoside and lignan compounds, enriching MCI resin components, analyzing by liquid chromatography-mass spectrometry, and identifying. The enrichment extracted by the method provided by the invention is a mixture containing phenylethanoid glycoside and lignan compounds respectively, and can be used as a medicine for resisting the human coronavirus HCoV-229E.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, in particular, to a method for extracting and enriching phenylethanoid glycosides and lignans from forsythia suspensa fresh leaves, and the enriched product and use thereof. BACKGROUND

[0002] Forsythia suspensa is a deciduous shrub of Oleaceae in Dilleniopsida, and the medicinal part of F. suspensa used in the 2020 edition of Chinese Pharmacopoeia is dry fruit, which has the effects of clearing heat and resolving toxicity, and eliminating carbuncle and resolving mass, and is often used for treating acute wind-heat syndrome, carbuncle and sore, lymphatic tuberculosis, and urinary tract infection. Phenylethanoid glycosides and their glycosides are rich in F. suspensa, which are representative chemical components of F. suspensa, and have significant anti-inflammatory activity, immunomodulatory effect and hepatoprotective activity. The structure of phenylethanoid glycosides is usually centered on β-glucose, and the basic skeleton is composed of phenylethanoid and glycosyl.

[0003] Characteristic ion fragments and neutral loss refer to characteristic fragment ions or neutral loss molecules that can represent the type of chemical components generated in the mass spectrometry cracking process. According to the principle that compounds of the same type often have similar secondary fragments or neutral molecule loss, the structure type of the target compound can be quickly and accurately locked in the identification process, and the compound can be further identified. In the diagnosis process of chemical components of traditional Chinese medicine, the accurate identification of the structure has good specificity by combining the mother nucleus structure type, the type of substituent group, the common neutral loss and the characteristic ion fragments generated in the mother nucleus cracking process.

[0004] Mass Defect Filter (MDF) is another commonly used means and technique in data mining. Mass defect refers to the difference between the accurate molecular weight of a compound and the integer part, that is, the mass defect related to the element composition of the chemical component. The mass defect of components with the same type of mother nucleus structure differs in the change of substituent group. MDF relies on the characteristics of compounds of the same structure to classify complex chemical components, and improves the filtering and screening of target compounds by setting a specific range of MDF screening window, and automatically filters interfering ions to rapidly mine target component groups and target compounds from complex matrix background.

[0005] Exploring the complex chemical components of traditional Chinese medicine by mass spectrometry can more comprehensively characterize the material basis of traditional Chinese medicine, improve the analysis efficiency, expand the analysis range, and improve the accuracy of the analysis work, which has shown immeasurable potential in the qualitative analysis of traditional Chinese medicine, and has made great contribution to the development of modernization of traditional Chinese medicine. SUMMARY

[0006] In order to overcome the problems in the prior art, the present application provides a method for extracting and enriching phenylethanoid glycosides and lignans from forsythia fresh leaves, and the enriched product and use thereof, which can be used as a medicine for resisting human coronavirus HCoV-229E.

[0007] The present application provides the following technical solutions:

[0008] A method for extracting and enriching phenylethanoid glycosides and lignans from forsythia fresh leaves, characterized by comprising the following steps:

[0009] Step one: extract: after the forsythia fresh leaf sample is vacuum freeze-dried and crushed, the sample powder is weighed and sieved, 75% ethanol is added, and reflux extraction is performed to obtain an extract, and after recovering the solvent, a sample extract is obtained, which is dissolved in an appropriate amount of methanol, centrifuged to obtain the supernatant, and then filtered to obtain the filtrate;

[0010] Step two: enrichment: the filtrate is recovered to obtain an extract, the extract is mixed with MCI resin at a mass ratio of 1:10-15, and then column packed, and gradient elution is performed with pure water, 20% ethanol / water, 60% ethanol / water and 95% ethanol / water, respectively, and the elution fractions of 20% ethanol / water and 60% ethanol / water are collected, respectively, and the solvent is recovered, and then redissolved with methanol to obtain an enriched liquid;

[0011] Step three: liquid chromatography-mass spectrometry analysis: the enriched liquid is subjected to chromatographic separation, the stationary phase is a Waters ACQUITY UPLC HSS T3 C18 chromatographic column, the mobile phase is a solution containing 0.2% formic acid / water A-acetonitrile B, and gradient elution is performed; mass spectrometry conditions: electrospray ion source ESI, positive and negative ion mode full scan, MSe acquisition mode, capillary voltage 2.5kV(-) / 3.0kV(+); ion source temperature 120℃; desolvation gas temperature 450℃; cone hole voltage 30V; cone hole backflush gas flow rate (N2) 50L·h-1; desolvation gas is nitrogen, flow rate 1000L·h-1; mass scan range m / z 50-1500; collision gas is argon, low-energy scan voltage 6eV, high-energy scan voltage 20-40eV; when the scanning mode is MS / MS mode, the collision energy is 20eV, and the parent ion mass-to-charge ratio is input according to the MSe data;

[0012] Step four: data analysis and processing: full mass scan analysis is performed on the UPLC-Q-TOF-MS mass spectrum, MSe acquisition mode, data acquisition is performed by using UNIFYTM1.9.4.053 software, LC-HR-MSe and HDMS (High Definition Mass Spectrometry) data sets are obtained, characteristic neutral loss / ion filtering parameters are set, 5-point mass loss filtering MDF parameters are set, and phenylethanoid glycosides and lignans are identified.

[0013] Furthermore, in step one, 75% ethanol is added at a volume ratio of 1:8-10, and the mixture is refluxed and extracted twice, 40 min each time. The extracts are combined to obtain the total extract. After the solvent is recovered, the sample extract is obtained. The total extract is dissolved in an appropriate amount of methanol, centrifuged at 14000 rpm for 10 min, and the supernatant is collected. The supernatant is filtered through a 0.22 μm microporous membrane, and the filtrate is collected.

[0014] Furthermore, in step three, the chromatographic parameters for the elution fraction of 20% ethanol / water are as follows: elution gradient: 0-10 min, 10%-15% B; 3-18 min, 18% B; 12-20 min, 15%-16% B; 20-22 min, 16%-55% B; 22-23 min, 55%-75% B; 23-25 ​​min, 75%-5% B; column temperature 35℃, flow rate 0.3 mL·min⁻¹, and detection wavelength 254 nm.

[0015] Chromatographic parameters for the elution fraction of 60% ethanol: Elution gradient: 0-3 min, 10%-18% B; 3-10 min, 18%-23% B; 10-17 min, 23%-35% B; 17-22 min, 35%-50% B; 22-25 min, 50%-75% B; 25-28 min, 75%-95% B; Column temperature 35℃, flow rate 0.3 mL·min⁻¹, detection wavelength 254 nm.

[0016] Furthermore, in step four, the neutral loss / ion filtering parameters are as follows: In negative ion mode, 20% of the MCI resin eluent is analyzed using liquid chromatography-mass spectrometry (LC-MS). Neutral loss parameters for phenylethanol glycosides are set to 162.05 Da (neutral loss of glucose glu), 146.06 Da (neutral loss of rhamnose rha), 132.0423 Da (neutral loss of apiose api), 152.07 Da (neutral loss of xylose xyl), and 150.05 Da (neutral loss of arabinose ara). The characteristic ion is set to m / z. 179.04(caff), 161.02(caffeoyl-H2O), 135.04(caffeoyl-CO2), 175.04(feruloylH2O), 193.05(feru loyl), 163.04coumaroyl), 149.06(feruloyl-CO2), 147.05coumaroyl-H2O), 119.05(coumaroyl-CO2);

[0017] In positive ion mode, 60% of the MCI resin eluent was analyzed by liquid chromatography-mass spectrometry (LC-MS). The neutral loss of lignan compound structures was set to 162.0528 Da (Glucose), 18.0106 Da (H2O), and 32.0262 Da (CO), and the characteristic ions were set to 139.04 (C7H7O3+), 139.05 (C8H11O2+), 125.06 (C7H9O2+), and 111.04 (C6H7O2+).

[0018] Furthermore, in step four, the mass defect filtering (MDF) parameters are selected as follows: the compound characteristics of the five selected points are as follows: point a is the compound with the smallest molecular weight; point b is the compound with a small core and the smallest fractional part; point c is determined based on the smallest slope of bc; point e is the compound with a small core and a positive mass defect; point d is the compound with many glycosidic groups attached to the substituents of the core, resulting in the largest slope connecting points ed; the parameter range for the precursor ions in forsythoside phenylethanol glycosides and lignans is as follows:

[0019]

[0020] Furthermore, the fraction eluted with 20% ethanol in step two is a mixture containing phenylethanol glycosides.

[0021] Furthermore, the stream eluted with 60% ethanol in step two is a mixture containing lignan compounds.

[0022] The compound has applications in the preparation of drugs against human coronavirus HCoV-229E.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects:

[0024] 1. This invention provides a method for enriching phenylethanoid glycosides and lignans from fresh leaves of Forsythia suspensa. The pharmacodynamic material basis of the enriched substances is demonstrated through pharmacodynamic screening. At the same time, unknown components are separated, purified and structurally identified by chromatographic separation and mass spectrometry.

[0025] 2. The method for extracting and enriching phenylethanol glycosides and lignans from fresh leaves of Forsythia suspensa, the enriched products and their uses, can be used as drugs against human coronavirus HCoV-229E and have good pharmaceutical application prospects. Attached Figure Description

[0026] Figure 1 This is the structural formula of the basic parent nucleus of the phenylethanoid glycosides in Forsythia suspensa in this invention;

[0027] Figure 2 This is the structural formula of the substituents in the phenylethanol glycoside compounds in Forsythia suspensa of this invention;

[0028] Figure 3 This is the structural formula of the characteristic ionic fragments of the phenylethanoid glycosides in Forsythia suspensa of the present invention;

[0029] Figure 4 This is the structural formula of the basic parent nucleus of the lignan compounds in Forsythia suspensa of this invention;

[0030] Figure 5 This is the structural formula of the characteristic ionic fragments of the lignan compounds in Forsythia suspensa of the present invention;

[0031] Figure 6 This is a model diagram of the parameters established for screening compounds using the MDF method of this invention;

[0032] Figure 7 This is a graph showing the number of detectable chromatographic peaks for different extraction methods of the samples in this invention;

[0033] Figure 8 This is the MRM detection peak diagram of the present invention;

[0034] Figure 9 This is a graph showing the analysis results of each component after enrichment with the D101 type resin of this invention;

[0035] Figure 10 This is a graph showing the analysis results of each component after enrichment with the MCI-type resin of this invention;

[0036] Figure 11 This is a graph showing the gradient elution results of the reverse-phase silica column of the present invention;

[0037] Figure 12 This is an LC-MS result diagram of the MCI resin enriched components of this invention;

[0038] Figure 13 This is an LC-MS chromatogram of the drug-active components enriched by the D101 resin separation method of this invention;

[0039] Figure 14 This is a graph showing the results of screening target compounds using the MDF method of this invention;

[0040] Figure 15 This is a data graph of the target compounds finally screened by the MDF in this invention;

[0041] Figure 16 This is the MS of the present invention. e The results of total ion chromatogram analysis of 20% of the active pharmaceutical components in negative ion mode;

[0042] Figure 17 This is the MS of the present invention. e Figure showing the total ion chromatogram analysis results of 60% of the pharmacologically active components in positive ion mode;

[0043] Figure 18This is a schematic diagram of the structural formula of phenylethanol glycosides in the 20% elution fraction enriched from fresh Forsythia leaves of this invention.

[0044] Figure 19 This is a schematic diagram of the structural formula of the lignan compounds enriched in 60% of the eluted fraction from fresh Forsythia leaves of this invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0046] This invention provides a method for extracting and enriching phenylethanoid glycosides and lignans from fresh forsythia leaves, comprising the following steps:

[0047] Step 1 Extraction: After vacuum freeze-drying, the fresh leaves of Forsythia are crushed, the sample powder is weighed and sieved, 75% ethanol is added, and the mixture is refluxed to obtain the extract. After recovering the solvent, the sample extract is obtained, dissolved in an appropriate amount of methanol, centrifuged to obtain the supernatant, and filtered to obtain the filtrate.

[0048] Step 2 Enrichment: The solvent was recovered from the filtrate to obtain the extract. The extract was mixed with MCI resin at a mass ratio of 1:10-15 and packed into a column. Gradient elution was performed with pure water, 20% ethanol / water, 60% ethanol / water and 95% ethanol / water, respectively. The elution fractions of 20% ethanol / water and 60% ethanol were collected, the solvent was recovered, and the solution was redissolved with methanol to obtain the enriched solution.

[0049] Step 3: Liquid chromatography-mass spectrometry (LC-MS): The enriched solutions were separated by chromatography. The stationary phase was a Waters ACQUITYUPLC HSST3 C18 column, and the mobile phase was A-acetonitrile B containing 0.2% formic acid / water solution, with gradient elution. Mass spectrometry conditions: electrospray ionization (ESI), full scan in both positive and negative ion modes, MS. e Acquisition mode: capillary voltage 2.5kV(-) / 3.0kV(+); ion source temperature 120℃; desolvation gas temperature 450℃; cone voltage 30V; cone backflush gas flow rate (N2) 50L·h -1 The solvent removal gas is nitrogen, with a flow rate of 1000 L·h. -1； Mass scan range m / z 50–1500; collision gas is argon; low-energy scan voltage 6 eV; high-energy scan voltage 20–40 eV; when scanning in MS / MS mode, the collision energy is 20 eV; the parent ion mass-to-charge ratio is determined according to MS... e Data input.

[0050] Step 4: Data Analysis and Processing: Perform a full mass scan analysis on the UPLC-Q-TOF-MS mass spectrometer. e Acquisition mode, using UNIFY TM Data acquisition was performed using software 1.9.4.053 to obtain LC-HR-MS data. e Using the HDMS (High Definition Mass Spectrometry) dataset, feature neutral loss / ion filtering parameters and 5-point mass defect filtering (MDF) parameters were set to identify phenylethanol glycosides and lignans.

[0051] Example 1

[0052] Examination of extraction methods

[0053] This invention compares the following three extraction methods, and the experimental methods are as follows:

[0054] Method 1: Reflux Extraction: Fresh Forsythia leaves were freeze-dried under vacuum and then crushed. 50g of the sample powder (passed through a No. 5 sieve) was accurately weighed and placed in a 500mL round-bottom flask. 300mL of 75% or 95% ethanol was added, and the mixture was refluxed twice, 40min each time. The extracts were combined to obtain the total extract. After solvent recovery, the sample paste was obtained. The total extract was dissolved in an appropriate amount of methanol and centrifuged at 14000rpm for 10min. The supernatant was collected and filtered through a 0.22μm microporous membrane. The filtrate was then collected as the final product.

[0055] Method 2: Ultrasonic Extraction. Accurately weigh 10g of the above-mentioned fresh Forsythia leaf sample and place it in a 100mL stoppered conical flask. Add 100mL of 75% methanol or 95% methanol / water and sonicate for 20min to obtain the extract. After recovering the solvent, dissolve the extract in an appropriate amount of methanol and centrifuge at 14000rpm for 10min. Take the supernatant and filter it through a 0.22μm microporous membrane. Collect the filtrate to obtain the extract.

[0056] Method 3: Water decoction extraction. Accurately weigh 100g of the above-mentioned fresh Forsythia leaf sample and place it in an HDZ20 intelligent decoction pot. Add 1000mL of deionized water and decoct for 30 minutes. Filter to obtain the decoction. Add 800mL of water to the residue and decoct for 20 minutes. Filter again and combine the two decoctions to obtain the total extract. Concentrate the extract to obtain the total extract paste. Dissolve the total extract in an appropriate amount of methanol and centrifuge at 14000rpm for 10 minutes. Collect the supernatant and filter it through a 0.22μm microporous membrane. Collect the filtrate to obtain the final extract.

[0057] This invention also compares methods for enriching different types of medicinal components from fresh Forsythia suspensa leaves, as follows:

[0058] (1) D101 macroporous resin method

[0059] Pretreatment of D101 macroporous resin (25g) was carried out in a 50mL beaker. 5g of lyophilized Forsythia suspensa leaf extract was dissolved in methanol and then mixed with the macroporous resin. The mixture was packed into a column and then eluted with a gradient of pure water, 20% ethanol / water, 40% ethanol / water, 60% ethanol / water and 95% ethanol / water, respectively. The solvents were recovered, and the mixture was reconstituted with methanol and injected into a high-performance liquid chromatograph for analysis.

[0060] (2) MCI resin method

[0061] Pretreatment of 10g of MCI resin was performed and placed in a 50mL beaker. 5g of lyophilized Forsythia suspensa leaf extract was dissolved in methanol and then mixed with the MCI resin at a mass ratio of 1:3. Gradient elution was performed at room temperature using pure water, 20% ethanol / water, 40% ethanol / water, 60% ethanol / water, and 95% ethanol / water, respectively, at a flow rate of 5 mL / min, with 8–10 column volumes for each gradient. The eluent was recovered, reconstituted with methanol, and injected into the high-performance liquid chromatograph for analysis.

[0062] (3) Reversed-phase chromatography

[0063] Weigh 1.5g of freeze-dried powder extracted from fresh Forsythia suspensa leaves, mix with an appropriate amount of ODS packing material using a dry mixing method, and after the solvent has evaporated, pack the sample into a 20g pre-packed ODS column. Using a Biotage preparative chromatograph, elute with gradients of 20%, 40%, 60%, 80%, and 100% ethanol / water solutions until the UV absorption baseline stabilizes, yielding different fractions. Recover the solvent, reconstitute with methanol, and inject the solutions into a high-performance liquid chromatograph for analysis.

[0064] Chromatographic conditions

[0065] Chromatographic parameters for the elution fraction of MCI resin with 20% ethanol: Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm), mobile phase: 0.2% formic acid / water solution (A) - acetonitrile (B), gradient elution: 0–10 min, 10%–15% B; 3–18 min, 18% B; 12–20 min, 15%–16% B; 20–22 min, 16%–55% B; 22–23 min, 55%–75% B; 23–25 min, 75%–95% B; column temperature: 35 °C, flow rate: 0.3 mL / min. -1 The injection volume was 2 μL, and the detection wavelength was 254 nm.

[0066] Chromatographic parameters for the elution fraction of MCI resin with 60% ethanol: Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm), mobile phase: 0.2% formic acid / water solution (A) - acetonitrile (B), gradient elution: 0–3 min, 10%–18% B; 3–10 min, 18%–23% B; 10–17 min, 23%–35% B; 17–22 min, 35%–50% B; 22–25 min, 50%–75% B; 25–28 min, 75%–95% B; column temperature: 35 °C, flow rate: 0.3 mL / min. -1 The injection volume was 2 μL, and the detection wavelength was 254 nm.

[0067] Mass spectrometry conditions

[0068] Electrospray ionization (ESI) source, full scan in both positive and negative ion modes, MS e Acquisition mode: capillary voltage 2.5kV(-) / 3.0kV(+); ion source temperature 120℃; desolvation gas temperature 450℃; cone voltage 30V; cone backflush gas flow rate (N2) 50L·h -1 The solvent removal gas is nitrogen, with a flow rate of 1000 L·h. -1； Mass scan range m / z 50–1500; collision gas is argon; low-energy scan voltage 6 eV; high-energy scan voltage 20–40 eV; when scanning in MS / MS mode, the collision energy is 20 eV; the parent ion mass-to-charge ratio is determined according to MS... e Data Input. When quantification is required, Multiple Reaction Monitoring (MRM) mode is used. Accurate mass values ​​are obtained using leucine enkephalin as a calibration solution (negative ion m / z 554.2620, positive ion m / z 556.2766), and data is acquired using UNIFY™ 1.9.4.053 software.

[0069] Establishment of analytical methods: UPLC-Q-TOF-MS n Mass spectrometry coupled with multiple data processing methods was used to analyze the medicinal components in fresh Forsythia suspensa leaves. First, a full mass scan analysis was performed using UPLC-Q-TOF-MS, and LC-HR-MS data were obtained. e The dataset includes HDMS (High Definition Mass Spectrometry); secondly, in subsequent post-acquisition data mining, characteristic ion filtering and conventional MDF methods are used in combination to identify compounds.

[0070] Characteristic neutral loss / ion filtering parameter settings

[0071] Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using an electrospray ionization (ESI) source, with both positive and negative ion modes for a full scan of the two pharmacodynamic components. e Acquisition mode. Using UNIFY. TM The 1.9.4.053 software is used for data acquisition.

[0072] (1) The elution fraction of MCI resin with 20% ethanol was analyzed by liquid chromatography-mass spectrometry (LC-MS) in negative ion mode. Based on the phenylethanol glycoside class, the basic parent nucleus is as follows: Figure 1 The diagram shows the structure of a phenylethanol glycoside with β-glucose as its structural center. Glucosides often have various sugar groups, caffeoyl, coumaroyl, or feruloyl substitutions. Figure 2 When phenylethanol glycosides are subjected to suitable collision energies, they typically lose neutral fragments to produce characteristic ionic fragments. Therefore, the neutral loss values ​​are set at 162.05 Da (neutral loss of glucose glu), 146.06 Da (neutral loss of rhamnose rha), 132.0423 Da (neutral loss of apiose api), 152.07 Da (neutral loss of xylose xyl), and 150.05 Da (neutral loss of arabinose ara). Characteristic ions are set at m / z 179.04 (caff), 161.02 (caff-H2O), 135.04 (caff-CO2), 175.04 (feul-H2O), 193.05 (feul), 163.04 (coum), 149.06 (feul-CO2), 147.05 (coum-H2O), and 119.05 (coum-CO2), as shown below. Figure 3 During the identification process, setting the characteristic neutral fragment loss or ion fragmentation can quickly and accurately identify the range.

[0073] (2) The elution fraction of MCI resin with 60% ethanol was analyzed by liquid chromatography-mass spectrometry (LC-MS) in positive ion mode. Lignans are the main active components of Forsythia suspensa, and various types of lignans have been isolated from Forsythia suspensa. The main types of lignan compounds in Forsythia suspensa are bis(tetrahydrofuran), dibenzylbutyrolactone, and arylnaphthalene, with common core structures being... Figure 4As shown, the substituents are mostly glucoside, methoxy, or hydroxyl. Based on the cleavage patterns of lignan compounds in Forsythia suspensa, the three types of lignans typically undergo ring cleavage due to the loss of glucoside and methyl groups or water molecules, followed by RDA cleavage to form characteristic fragment ions; or they directly undergo single bond cleavage between the benzene ring and the furan ring. Therefore, in positive ion mode, the neutral loss thresholds for screening lignan compound structures were set to 162.0528 Da (Glucose), 18.0106 Da (H2O), and 32.0262 Da (CO), and the characteristic ion was set to 139.04 Da (C7H7O3). + ), 139.05 (C8H 11 O2 + ), 125.06(C7H9O2) + ), 111.04(C6H7O2) + ()( Figure 5 ).

[0074] Setting the Quality Loss Filtering (MDF) parameters

[0075] Using the five-point MDF technique, the cores of phenylethanoid glycosides and bis(tetrahydrofuran), benzylbutyrolactone, and arylnaphthalene lignans from Forsythia suspensa were selected as template compounds. The minimum and maximum substituent numbers for mass deficit of various substituents were comprehensively considered, and appropriate mass deficit limits were set. The extreme values ​​were connected to form corresponding rectangular filter regions. All compounds whose precise relative mass is located within the filter region were identified and analyzed.

[0076] First, a database was established based on the phenylethanol glycosides and lignans isolated from Forsythia suspensa as reported in the literature. The steps were as follows: (1) Structural analysis was performed on the phenylethanol glycosides and lignans reported in Forsythia suspensa; (2) The reported phenylethanol glycosides and lignans were decomposed into aglycones and basic substituent building units according to their structures, as shown in Table 3-1; (3) The aglycones and ligand substituents obtained in the second step were associated with the parent nucleus, and finally the compound list in Table S1 and Table S2 of the appendix was obtained.

[0077] The five selected points are characterized as follows: point a is the compound with the smallest molecular weight; point b is the compound with a small core and the smallest fractional part; point c is determined by the smallest slope between points b and c; point e is the compound with a small core and a positive mass defect; and point d is the compound with many glycosidic groups attached to the core substituents, resulting in the largest slope connecting points ed and e. Based on the established pentagon formed by these five points, the points within the shape are considered potential target compounds.

[0078] Table 1. Parameter range for the five-point screening method for rapid screening of precursor ions in forsythosides phenylethanoids and lignans.

[0079]

[0080] a) MDF parameter settings for phenylethanol glycosides

[0081] Forsythia suspensa phenylethanoid glycosides typically share a common structure, usually centered around β-glucose and composed of phenylethanoid groups and glycosyl groups or dimers. Based on the predicted types and numbers of substituents, and using the precise molecular weights and predicted compounds from 260 reported phenylethanoid glycosides (see Appendix Table S1 for a detailed list), a two-dimensional distribution map (x = Mass Range, y = Mass Defect) was constructed using Microsoft Office Excel software. A five-point MDF method was then applied. Figures 3-6 To screen for phenylethanoid glycosides in Forsythia suspensa, five suitable points were selected and connected to each other based on calculated and predicted structures. Following the principles of the five-point screening technique, the sequential connection of these five points determined the screening range and boundary slope for phenylethanoid glycosides. The final selection of the five points and the information from the equations are as follows:

[0082] Point a is C 13 H 17 O5 (m / z 253.1081), the molecular formula at point b is C. 13 H 15 O8(m / z299.0772), point c is C 37 H 35 O 19 (m / z 783.1778), the molecular formula at point d is C 54 H 71 O 29 (m / z 1183.4086), point e is C 37 H 35 O 19 (m / z549.2141), establish the five-sided equation using Microsoft Excel ( Figures 3-6 ), respectively: y ab = -0.0007x + 0.2781(253) <X ab <299);

[0083] y bc =0.0002x-0.0151(299) <X bc <783;y cd :0.0006x-0.2740(783 <X cd <1183);

[0084] y ae =0.0004x + 0.0004(253) <X ae<549); y ed =0.0003x + 0.0830(549) <X ed <1183).

[0085] b) MDF parameter settings for lignans

[0086] The core structures of forsythoside lignans are mostly derived from the oxidation of phenylpropanoids, and some components can form glycosides with sugars. Based on the characteristics of 110 reported lignan compounds and predicted substituents, a list of specific compounds is provided in Appendix Table S2. A five-point MDF method was established to screen lignan compounds in forsythoside. Five suitable points were selected and connected to each other based on the calculated and predicted lignan structures. Following the principles of the five-point screening technique, the final five points were sequentially connected to determine the screening range and boundary slope of the lignan compounds. A five-sided equation was established using Microsoft Excel. Figure 6 The information is as follows:

[0087] Point a is C 15 H 21 O2 (m / z 233.1536), the molecular formula at point b is C. 17 H 15 O5(m / z299.0914), point c is C 19 H 17 O7 (m / z 357.0969), the molecular formula at point d is C 32 H 43 O 18 (m / z 715.2444), point e is C 22 H 31 O6 (m / z 391.2115);

[0088] y ab =-0.0009x+0.3485(233) <X ab <299); y bc =0.0001x + 0.0630(299) <X bc <715),

[0089] y cd =0.0004x-0.0502(357) <X cd <715); y ae =0.0005x-0.0778(233) <X ae <391);

[0090] y ed =0.0001x + 0.1718(391) <X ed <715).

[0091] Example 2

[0092] Experimental results

[0093] (1) Use the “Peakpicking” function in QI software to perform chromatographic peak identification data analysis on samples obtained by different extraction methods.

[0094] The optimal extraction method was selected based on the number of chromatographic peaks for each sample. (See...) Figure 7 As shown, A: water decoction extraction; B: 75% ethanol ultrasonic extraction; C: 95% ethanol / water ultrasonic extraction; D: 75% ethanol reflux extraction; E: 95% ethanol / water reflux extraction. Among these, the sample obtained by water decoction extraction had 4416 identifiable chromatographic peaks (A); the sample obtained by 75% ethanol / water ultrasonic extraction had 2538 identifiable chromatographic peaks (B); the sample obtained by 95% ethanol / water ultrasonic extraction had 2301 identifiable chromatographic peaks (C); the sample obtained by 75% ethanol / water reflux extraction had 3702 identifiable chromatographic peaks (D); and the sample obtained by 95% ethanol / water reflux extraction had 3022 identifiable chromatographic peaks (E).

[0095] Therefore, the ethanol / water reflux extraction method can detect more chromatographic peaks than the ultrasonic extraction method, and the peaks of the sample extracted by 75% ethanol / water reflux are stronger than those of the sample extracted by 95% ethanol / water reflux. Therefore, the 75% ethanol / water reflux extraction method is selected.

[0096] (2) The extraction method was examined based on the content of the indicator components.

[0097] According to the 2020 edition of the Chinese Pharmacopoeia, the contents of forsythoside A and forsythoside are used as indicator components for evaluating extraction methods. Based on UPLC-Q-TOF-MS technology, in positive and negative ion detection modes, a specific set of multiple reaction monitoring (MRM) acquisitions were used to analyze the characteristic ion pairs of the two compounds (forsythoside A: 623.20-461.1 and forsythoside: 557.20-355.16) in the sample, resulting in the MRM mode multi-peak chromatogram of the sample, as shown below. Figure 8 As shown.

[0098] Table 2 MRM Parameter Settings

[0099]

[0100] Table 3 shows the peak area under MRM mode for three extraction methods.

[0101]

[0102] Based on the above verification of extraction methods, it can be concluded that 75% ethanol / water reflux is more suitable for the extraction of fresh forsythia leaves compared with other extraction methods.

[0103] Samples prepared by different chromatographic methods in Example 1 were analyzed by high performance liquid chromatography, wherein: A: 20% elution fraction; B: 40% elution fraction; C: 60% elution fraction; D: 95% elution fraction.

[0104] The separation of Forsythia suspensa fresh leaf extract using D101 resin showed that different polar components were not well separated, indicating that the preparation results of the D101 resin column were not ideal. The results are as follows... Figure 9 As shown in Figure 0, the application of MCI-type resin to separate ethanol-eluted fractions with different proportions effectively separates components from fresh Forsythia suspensa leaves. Furthermore, the 20% ethanol and 40% ethanol eluates can be distinguished, which is superior to the separation effect with resin D101. In the preparation of various components using low-pressure reversed-phase preparative chromatography, the separation effect of reversed-phase chromatography is not significant (see Figure 0). Figure 11 Furthermore, considering the applicability of resin fillers, MCI resin filler was selected for the preparation of effective components from fresh Forsythia suspensa leaves.

[0105] Based on the above experimental results, the MCI resin separation method is the most effective for enriching different types of medicinal components in Forsythia suspensa.

[0106] The pharmacodynamic components separated by different resins were analyzed by liquid chromatography-mass spectrometry (LC-MS). LC-MS also demonstrated the advantages of the MCI resin separation method for preparing pharmacodynamic components. The LC-MS results of the MCI resin-enriched components are shown in [Figure number missing]. Figure 12 The LC-MS chromatogram of the drug active ingredient enriched by the D101 resin separation method is shown below. Figure 13 .

[0107] The elution results of MCI were analyzed under both positive and negative ion modes. Analysis revealed that, according to experimental results "2.3.1.2" and "2.3.1.3", based on the reference standards, their mass spectrometric fragmentation patterns, and the polarity characteristics of the compounds, the retention times of forsythoside A, forsythoside B, and verbascoside were 8.9 min, 8.38 min, and 7.45 min, respectively, all of which were identified in 20% of the MCI resin eluent. Figure 12 A) The fraction eluted with 20% ethanol was a mixture containing phenylethyl glycosides. The retention times of lignans such as forsythoside, (+)-pinoresinin-β-D-glucopyranoside, podophyllin, arctiin, and forsythoside were 15.35 min, 10.38 min, 15.17 min, 15.60 min, and 16.30 min, respectively, all identified in the 60% eluent of the MCI resin eluent. Figure 12B). Therefore, it is inferred that phenylethyl alcohol-like components are mainly concentrated in the elution fraction of MCI resin with 20% ethanol, and the elution fraction with 20% ethanol is a mixture containing phenylethyl alcohol-like compounds. Meanwhile, lignan-like components are mainly concentrated in the elution fraction of MCI resin with 60% ethanol, and the elution fraction with 60% ethanol is a mixture containing lignan-like compounds.

[0108] Qualitative analysis of the active components in fresh Forsythia suspensa leaves based on the MDF method

[0109] After optimizing the chromatographic conditions, chemical composition mass spectrometry full scan data were acquired for different components of fresh Forsythia suspensa leaves under both positive and negative ion modes. Data extraction was then performed using Progenesis QI software, yielding lists of 3641 ions in the 20% elution fraction and 2652 ions in the 60% fraction, with retention times and neutral mass included in the lists. Since MDF is a self-built data screening method, further data filtering was performed on the data obtained from Progenesis QI software, ultimately resulting in lists of 2604 ions in the 20% fraction and 1083 ions in the 60% fraction.

[0110] The constructed pentagonal MDF can filter out most of the components other than the target compound, such as... Figure 14 As shown, good selectivity can be obtained, thus demonstrating the advantages of the multilateral MDF method in targeted analysis and characterization.

[0111] Multiple data processing techniques were applied to thoroughly identify the compound data within the pentagonal frame, ultimately confirming the identification of 53 phenylethanoid glycosides and 26 lignans. A: Phenylethanoid glycosides; B: Lignans, such as... Figure 15 Identify peak labels in Figure 16 and Figure 17 The identification results are shown in Tables 4 and 5. The structural diagrams of the identified compounds are shown below. Figure 18 and 19 .

[0112] Table 4. Identification of phenylethanol glycosides after enrichment of Forsythia suspensa

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Table 5. Identification of lignan glycosides after enrichment of Forsythia suspensa

[0119]

[0120]

[0121]

[0122]

[0123] Two types of medicinal components from fresh Forsythia suspensa leaves were prepared by MCI chromatography and analyzed by HPLC and UPLC-MS. UPLC-QTOF-HDMS was used to perform HDMS analysis on the 20% ethanol / water, 60% ethanol / water, and 90% ethanol / water fractions eluted by MCI. e Analysis and data acquisition clarified the relative mass numbers, core structures, and various substituents of phenylethanoid glycosides and lignans, and established a compound mass spectrometry database based on structural characteristics. Diagnostic fragment ion and neutral loss filtering methods were applied to mine similar compounds, and the fragment ion formation process was inferred by combining the similar mass spectrometric fragmentation patterns of similar compounds, enabling the identification and structural prediction of target components. Using multiple mass spectrometry analysis methods, 53 phenylethanoid glycosides and 25 lignans were ultimately identified, such as... Figure 18 , 19 .

[0124] Example 3

[0125] In vitro efficacy experiments against human coronavirus HCoV-229E using fractions eluted with 20% ethanol and 60% ethanol enriched by this patent.

[0126] The experimental materials were as follows: Cell line: Beas-2b, purchased from Beijing Beina Chuanglian Biotechnology Research Institute, passaged in our laboratory, and stored in liquid nitrogen for later use. Human coronavirus 229E (HCoV-229E) was purchased from the American Center for Standard Biological Collections (ATCC). It was passaged in our laboratory and stored at -80℃ for later use.

[0127] Test drugs: The fraction eluted with 20% ethanol yielded a 20% crude sample (5 mg); the fraction eluted with 60% ethanol yielded a 60% crude sample (5.3 mg); and the fraction eluted with 95% ethanol yielded a 95% crude sample (6 mg). The eluents were concentrated under reduced pressure to obtain a gray or brown powder. Forsythoside A (1.1 mg), forsythoside B (1.5 mg), forsythoside (1 mg), arctiin (1 mg), and pinoresinin (1.1 mg) were also gray or brown powders.

[0128] 1. Cell Culture

[0129] Beas-2b cells were digested with 0.25% trypsin according to standard methods, centrifuged at 1000 rpm for 5 min. The trypsin was discarded, and 10 mL of DMEM complete cell culture medium was added. The cells were then thoroughly mixed by pipetting and seeded into 96-well cell culture plates at 100 μL / well. The culture plates were incubated at 37°C in a 5% CO2 incubator. After 24 h, a cell monolayer was formed for experimental use.

[0130] 2. Drug preparation: For 20% crude sample, take 5 mg and add 10 mL of maintenance solution to obtain 20% crude sample stock solution with a concentration of 500 μg / mL; for 60% crude sample, take 5.3 mg and add 10.6 mL of maintenance solution to obtain 60% crude sample stock solution with a concentration of 500 μg / mL; for 95% crude sample, take 6 mg and add 12 mL of maintenance solution to obtain 95% crude sample stock solution with a concentration of 500 μg / mL.

[0131] Forsythoside A has a molecular weight of 624.59. 1.1 mg was added to 3.44 mL of maintenance solution to obtain a stock solution of forsythoside A with a concentration of 512 μM. Forsythoside B has a molecular weight of 756.70. 1.5 mg was added to 3.88 mL of maintenance solution to obtain a stock solution of forsythoside B with a concentration of 512 μM. Forsythoside has a molecular weight of 534.56. 1 mg was added to 3.64 mL of maintenance solution to obtain a stock solution of forsythoside with a concentration of 512 μM. Arctiin has a molecular weight of 534.55. 1 mg was added to 3.64 mL of maintenance solution to obtain a stock solution of arctiin with a concentration of 512 μM. Pinoresinin has a molecular weight of 358.39. 1.1 mg was added to 5.99 mL of maintenance solution to obtain a stock solution of pinoresinin with a concentration of 512 μM.

[0132] 3. In vitro cultured Beas-2b cytotoxicity test

[0133] Eight serial dilutions (20%, 60%, and 95% crude samples) were prepared using 500 μg / mL as the initial concentration, and forsythoside A, forsythoside B, forsythoside A, arctiin, and pinoresinin were prepared using 512 μM as the initial concentration. These were serially diluted 2-fold with cell maintenance medium, resulting in a total of eight dilutions. Each dilution was added to a 96-well Beas-2b cell culture plate (100 μL / well), with four replicates for each concentration. A normal cell control was also included. The culture plates were incubated at 37°C in a 5% CO2 incubator. Cell pathogenesis was observed daily under an inverted microscope. After 48 hours, the lowest concentration (maximum non-toxic concentration TC0) at which no significant cytotoxicity was observed was determined, and the 50% cytotoxicity concentration (TC0) was calculated using the Reed-Muench method. 50 ).

[0134] Cellular lesions are judged according to a 6-grade standard:

[0135] -: Cell growth is normal, with no lesions appearing; ±: Cell lesions are less than 10% of the entire monolayer; +: Cell lesions account for less than 25% of the entire monolayer cells; ++: Cell lesions account for less than 50% of the entire monolayer cells; +++: Cell lesions account for less than 75% of the entire monolayer cells; ++++: Cell lesions account for more than 75% of the entire monolayer cells.

[0136] Table 6. Toxicity of 20%, 60%, and 95% crude samples on cultured Beas-2b cells.

[0137]

[0138]

[0139] Note: Cytopathic effect scoring: "+" = 1, "++" = 2, "+++" = 3, "++++" = 4, with scores assigned to each of the four replicate wells; TC 50 TC0 unit: μg / mL.

[0140] The results showed that the maximum non-toxic concentration (TC0) of the 20% crude sample was 31.25 μg / mL after a 16-fold dilution of the mother liquor. 50 The value was 44.56 μg / mL; the maximum non-toxic concentration (TC0) of the 60% crude sample was obtained by diluting the mother liquor 8 times, resulting in a drug concentration of 62.50 μg / mL. 50 The value was 88.91 μg / mL; the maximum non-toxic concentration (TC0) of the 95% crude sample was obtained by diluting the mother liquor 8 times, resulting in a drug concentration of 62.50 μg / mL. 50 The value was 88.91 μg / mL.

[0141] Table 7. Toxicity of forsythoside A, forsythoside B, forsythoside, arctiin, and pinoresinin on cultured Beas-2b cells in vitro.

[0142]

[0143]

[0144] Note: Cytopathic effect scoring: "+" = 1, "++" = 2, "+++" = 3, "++++" = 4, with scores assigned to each of the four replicate wells; TC 50 TC0 unit: μM.

[0145] The results showed that the maximum non-toxic concentration (TC0) of forsythoside A was 128 μM after a 4-fold dilution of the mother liquor. 50The value was 181.76 μM; the maximum non-toxic concentration (TC0) of forsythoside B was 16 μM after a 32-fold dilution of the stock solution. 50 The value was 11.47 μM; the maximum non-toxic concentration (TC0) of forsythoside was 64 μM after an 8-fold dilution of the stock solution. 50 The value was 91.14 μM; the maximum non-toxic concentration (TC0) of arctiin was 32 μM after a 16-fold dilution of the stock solution. 50 The value was 45.57 μM; the maximum non-toxic concentration (TC0) of pinoresinin was obtained by diluting the stock solution 8 times, resulting in a drug concentration of 64 μM and a TC0 of 45.57 μM. 50 The value is 91.14 μM.

[0146] Inhibitory effect of human coronavirus 229E virus on in vitro cultured Beas-2b cell cytopathic effect

[0147] Take a 96-well plate of Beas-2b cells that have grown into a monolayer, discard the culture medium, and seed with 100 TCID50. 50 HCoV-229E virus, 100 μL / well, was incubated at 37℃ in a 5% CO2 incubator for 2 hours for adsorption. The virus solution was then discarded, and the cells were washed once with maintenance solution. Five dilutions of the drug solution below the maximum non-toxic concentration were added sequentially, 100 μL / well. A normal control group and a virus control group were also included. The cells were incubated at 37℃ in a 5% CO2 incubator. Cellular pathogenesis was observed daily under an inverted microscope. After 48 hours, when the cytopathic effect in the virus control group reached +++ to ++++, the experimental results were recorded. The 50% inhibitory concentration (IC50) was calculated according to Reed-Muench. 50 ) and the therapeutic index (TI), TI = TC 50 / IC 50 .

[0148] Cellular lesions are judged according to a 6-grade standard:

[0149] -: Cell growth is normal, with no lesions appearing; ±: Cell lesions are less than 10% of the entire monolayer; +: Cell lesions account for less than 25% of the entire monolayer cells; ++: Cell lesions account for less than 50% of the entire monolayer cells; +++: Cell lesions account for less than 75% of the entire monolayer cells; ++++: Cell lesions account for more than 75% of the entire monolayer cells.

[0150] Table 8. Inhibitory effects of 20%, 60%, and 95% crude samples on HCoV-229E virus in cultured Beas-2b cells.

[0151]

[0152] Note: Cytopathic effect scoring: "+" = 1, "++" = 2, "+++" = 3, score each of the 4 replicates; IC 50 Unit: μg / mL.

[0153] The results showed that the crude sample had an IC50 value for HCoV-229E. 50 The concentration was 11.2 μg / mL, and the therapeutic index (TI) was 3.98, indicating that the 20% crude sample had an inhibitory effect on HCoV-229E; the IC50 of the 60% crude sample against HCoV-229E was... 50 The concentration was 11.2 μg / mL, and the therapeutic index (TI) was 7.95, indicating that 60% of the crude sample had an inhibitory effect on HCoV-229E; the IC50 of 95% of the crude sample against HCoV-229E was [missing value]. 50 The concentration was 50.1 μg / mL, and the therapeutic index (TI) was 1.77, indicating that 95% of the crude sample had an inhibitory effect on HCoV-229E.

[0154] Table 9. Inhibitory effects of forsythoside A, forsythoside B, forsythoside, arctiin, and pinoresinin on HCoV-229E virus in cultured Beas-2b cells.

[0155]

[0156]

[0157] Note: Cytopathic effect scoring: "+" = 1, "++" = 2, "+++" = 3, score each of the 4 replicates; IC 50 Unit: μM.

[0158] The results showed that forsythoside A had an inhibitory effect on the IC50 of HCoV-229E. 50 The concentration of forsythoside A was 51.2 μM, and the therapeutic index (TI) was 3.55, indicating that forsythoside A has an inhibitory effect on HCoV-229E. The IC50 of forsythoside B against HCoV-229E was 6.4 μM, and the therapeutic index (TI) was 1.79, indicating that forsythoside B has an inhibitory effect on HCoV-229E. The IC50 of forsythoside A against HCoV-229E was... 50 The concentration was 25.7 μM, and the therapeutic index (TI) was 3.55, suggesting that forsythoside has an inhibitory effect on HCoV-229E; arctiin has an IC50 value of 25.7 μM for HCoV-229E. 50 The concentration was 12.9 μM, and the therapeutic index (TI) was 3.53, suggesting that forsythoside has an inhibitory effect on HCoV-229E; pinoresinin has an IC50 value of 12.9 μM for HCoV-229E. 50 The concentration was 51.2 μM, and the therapeutic index (TI) was 1.78, suggesting that forsythoside has an inhibitory effect on HCoV-229E.

[0159] By establishing an LPS-induced BEAS-2B inflammation model, it was found that the main active site of the anti-inflammatory effect of Forsythia suspensa extract is the polar site of phenylethanoid glycosides, and its anti-inflammatory active component may be forsythoside A. Forsythoside A may exert its anti-inflammatory effect by inhibiting the activation of MAPK and NF-κB signaling pathways. The structures of 68 metabolites of fresh Forsythia suspensa leaves in rat plasma were deduced using UPLC-Q-TOF-MS, indicating that the metabolic pathway of fresh Forsythia suspensa leaves in vivo involves phase I and phase II metabolic reactions, including oxidation, demethylation, sulfation, and glucuronidation, with redox products being the predominant metabolites.

[0160] Therefore, the mixture containing phenylethanoid glycosides extracted and enriched by the method of this patent, eluted with 20% ethanol, and the mixture containing lignans eluted with 60% ethanol, have applications in the preparation of drugs against human coronavirus HCoV-229E.

[0161] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for extracting and enriching phenylethanoid glycosides and lignans from fresh Forsythia leaves, characterized by, Comprising the following steps: Step one extraction: the fresh leaf sample of Forsythia suspensa was vacuum freeze-dried, crushed, and sieved. 75% ethanol was added, and the sample was extracted by refluxing. The extract was obtained by recovering the solvent. The sample extract was dissolved in an appropriate amount of methanol, centrifuged, and the supernatant was collected. The filtrate was collected by filtration. Step two enrichment: the filtrate was recovered to obtain the extract, which was mixed with MCI resin at a mass ratio of 1:10-15, and then loaded into a column. The column was eluted with pure water, 20% ethanol / water, 60% ethanol / water, and 95% ethanol / water, respectively. The 20% ethanol / water elution fraction and the 60% ethanol elution fraction were collected, and the solvent was recovered. The enriched liquid was obtained by redissolving with methanol. Step three liquid chromatography-mass spectrometry analysis: the enrichment liquid was separated by chromatography, the stationary phase was Waters ACQUITY UPLCHSS T3 C18 column, the mobile phase was 0.2% formic acid / water solution A-acetonitrile B, gradient elution; mass spectrometry conditions: electrospray ion source ESI, positive and negative ion mode full scan, MS e Acquisition mode, capillary voltage 2.5kV(-) / 3.0kV(+); ion source temperature 120℃; desolvation gas temperature 450℃; cone hole voltage 30V; cone hole backflush gas flow (N2) 50L·h -1 ; desolvation gas is nitrogen, flow rate 1000L·h -1 ; mass scan range m / z 50~1500; collision gas is argon, low energy scan voltage 6eV, high energy scan voltage 20~40eV; when the scanning mode is MS / MS mode, the collision energy is 20eV, the parent ion mass-to-charge ratio is input according to MS e data; Step four data analysis processing: UPLC-Q-TOF-MS mass spectrum was analyzed by full mass scan, MS e Acquisition mode, data acquisition was performed by UNIFY TM 1.9.4.053 software, LC-HR-MS e and HDMS (High Definition Mass Spectrometry) data sets were obtained, the characteristic neutral loss / ion filter parameters were set, the 5-point mass deficiency filter MDF parameters were set, and the phenylethanoid glycosides and lignans were identified.

2. The extraction enrichment method according to claim 1, characterized by, In step one, 75% ethanol was added at a volume ratio of 1:8-10, and the sample was extracted by refluxing twice, each for 40 min. The total extract was obtained by recovering the solvent. The total extract was dissolved in an appropriate amount of methanol, centrifuged at 14000 rpm for 10 min, and the supernatant was collected. The filtrate was filtered through a 0.22 μm microporous filter.

3. The extraction enrichment method according to claim 1, characterized by, In step three, the chromatographic parameters of the elution fraction of 20% ethanol / water: elution gradient: 0-10 min, 10%-15% B; 3-18 min, 18% B; 12-20 min, 15%-16% B; 20-22 min, 16%-55% B; 22-23 min, 55%-75% B; 23-25 min, 75%-5% B; column temperature 35°C, flow rate 0.3 mL·min -1 , detection wavelength 254 nm. Chromatographic parameters of the elution fraction of 60% ethanol: elution gradient: 0-3 min, 10%-18% B; 3-10 min, 18%-23% B; 10-17 min, 23%-35% B; 17-22 min, 35%-50% B; 22-25 min, 50%-75% B; 25-28 min, 75%-95% B; column temperature 35°C, flow rate 0.3 mL·min -1 , detection wavelength 254 nm.

4. The extraction enrichment method according to claim 1, characterized by, In step four, the neutral loss / ion filter parameters: in negative ion mode, the MCI resin eluate 20% component was analyzed by liquid chromatography-mass spectrometry. The neutral loss of phenylethanoid glycosides was set to 162.05 Da (neutral loss of glucose glu), 146.06 Da (neutral loss of rhamnose rha), 132.0423 Da (neutral loss of api), 152.07 (neutral loss of xylose xyl), and 150.05 (neutral loss of arabinose ara). The characteristic ions were set to m / z 179.04 (caff), 161.02 (caffeoyl-H2O), 135.04 (caffeoyl-CO2), 175.04 (feruloyl H2O), 193.05 (feruloyl), 163.04 coumaroyl), 149.06 (feruloyl-CO2), 147.05 coumaroyl-H2O), 119.05 (coumaroyl-CO2); In positive ion mode, the MCI resin eluate 60% fraction was analyzed by liquid chromatography-mass spectrometry, with neutral loss set to 162.0528 Da (Glucose), 18.0106 Da (H20) and 32.0262 Da (CO) for screening lignan compound structure types, and characteristic ions set to 139.04 (C7H703 + ), 139.05 (C8H 11 O2 +) , 125.06 (C7H902 + ), 111.04 (C6H702 + ).

5. The extraction enrichment method according to claim 1, characterized by, In step four, the mass deficiency filter (MDF) parameters: the selected five points of the compound characteristics are: a point is the smallest molecular weight compound; b point is the smallest compound with smaller parent nucleus and smaller decimal part, c point is determined according to the smallest slope of bc; e point is the compound with smaller parent nucleus and positive mass deficiency; d point is the compound with more sugar ligands connected to the substituent of the parent nucleus, so that the slope of ed is the largest; the setting parameters of the precursor ions in forsythia phenylethanoid glycosides and lignans:

6. An enrichment enriched according to the method of claims 1-5, characterized in that, The 20% ethanol elution fraction in step two is a mixture containing phenylethanoid glycosides.

7. The enrichment of the method according to claims 1-5, characterized in that, The 60% ethanol elution fraction in step two is a mixture containing lignans.

8. The compound of claim 6 has application in the preparation of an anti-human coronavirus HCoV-229E drug.

9. The compound of claim 7 has application in the preparation of an anti-human coronavirus HCoV-229E drug.

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