Quality detection method of antiviral particles

Abstract

The invention provides a quality detection method of antiviral particles. The quality detection method adopts HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) to simultaneously determine the following components: forsythiaside A, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin and bergamot phenol. The detection method is a quantitative determination method for 11 active components in the antiviral particles, the detection method is short in time and low in cost, the quantitative analysis method is expected to provide reference for quality control of the antiviral particles, and the clinical curative effect and safety of the antiviral particles are guaranteed.

Description

Technical Field

[0001] The invention relates to a quality detection method for antiviral particles. Background Art

[0002] Based on the Treatise on Febrile Diseases written by Zhang Zhongjing, a famous doctor in the Eastern Han Dynasty, the anti-virus granules are improved based on the "Baihu Decoction", "Qingwen Baiduyin" and "Cangpu Yujin Decoction" in the traditional Chinese medicine prescriptions for purging fire. They are composed of Radix Isatidis, Gypsum, Phragmites australis, Rehmannia glutinosa, Curcuma, Rhizoma Anemarrhenae, Acorus calamus, Patchouli and Forsythiae. They have the effects of clearing away heat and dampness, cooling blood and detoxifying, and are used to treat wind-heat colds, upper respiratory tract infections, influenza, etc. In 2009, the anti-virus granules were approved by the State Food and Drug Administration for registration as a fourth-category traditional Chinese medicine. They are the exclusive varieties produced by Sichuan Everbright Pharmaceutical Co., Ltd. and are a large-brand traditional Chinese medicine for the treatment and prevention of viral influenza. They have been selected into the "Guidelines for the Clinical Application of Chinese Patent Medicines for the Treatment of Adult Influenza" and the "Expert Consensus on the Prevention and Treatment of Children's Influenza with Integrated Traditional Chinese and Western Medicine" of the China Association of Traditional Chinese Medicine, and are recommended for the treatment of children's influenza of dampness-restricting Weiqi type.

[0003] The prescription of antiviral granules is complex and has many ingredients. At present, the qualitative and quantitative research is mainly carried out on a few ingredients in the compound. The overall research is relatively weak and lacks systematicity. Wang Haibo, Li Yang. Determination of mangiferin in antiviral granules by HPLC [J]. Anti-infective Pharmacy, 2020, 17(2): 165-167. An HPLC determination method for mangiferin content in antiviral granules was established. Wang Jiemei, Wu Zhaoyi. Determination of forsythiaside content in antiviral granules by HPLC [J]. Chinese Modern Drug Application, 2007, 1(7): 7-8. An HPLC determination method for forsythiaside content in antiviral granules was established. Qiu Chenjin, Meng He, Xie Xiaoyan, et al. Research on the improvement of quality standards of antiviral oral liquid [J]. Chinese Journal of Hospital Drug Evaluation and Analysis, 2018, 18(7): 870-873; 875. In order to improve the quality standards of antiviral oral liquid, the qualitative fingerprints of (R, S)-gaoyichun, forsythiaside A, and forsythiaside and their content determination methods were established. He Dan, Yang Lin, Kuang Gang, et al. Simultaneous determination of the contents of three nucleosides in antiviral oral liquid by ultra-high performance liquid chromatography [J]. Journal of Chongqing Medical University, 2014, 39(4): 542-544. A HPLC method for simultaneous determination of uridine, guanosine, and adenosine in antiviral oral liquid was established. It is speculated that forsythiaside, mangoside, (R, S)-gaoyichun, forsythiaside A and other ingredients may be indicator ingredients for promoting the improvement of the quality control level of antiviral particles. (Tian Shimin, et al., Modern research progress and key issues analysis of antiviral particles, Chinese Traditional Patent Medicine, Vol. 46, No. 7, July 2024).

[0004] However, the composition of antiviral particles is complex and contains many ingredients. Single component control and network pharmacology research are not enough to objectively reflect the overall material basis of antiviral particles. Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) technology and gas chromatography-mass spectrometry (GC-MS) technology are used to quickly identify the chemical composition of antiviral particles (Tian Shimin, et al., Chemical composition analysis of antiviral particles based on UPLC-Q-Orbitrap HRMS and GC-MS technology, Chinese Journal of Hospital Pharmacy, Volume 43, Issue 2, January 2023). The UPLC detection time is 30 minutes, which is still relatively long. No quantitative detection and analysis was performed. It was only a qualitative analysis study. Summary of the invention

[0005] The present invention provides a novel quality detection method for antiviral particles, which is a method for quantitatively analyzing and evaluating screened characteristic components.

[0006] The present invention provides a method for detecting the quality of antiviral particles, which uses HPLC-MS / MS to simultaneously determine the following components:

[0007] Forsythiaside A, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin, bergamotol.

[0008] Wherein, the HPLC-MS / MS detection conditions are:

[0009] UHPLC-MS / MS analysis was performed using a Nexera UHPLC LC-30A / QTRAP 5500+MS / MS system equipped with an electrospray ionization (ESI) source (AB Sciex);

[0010] The chromatographic column was ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm; Waters, Massachusetts, USA), the temperature was 35°C, the mobile phase was 0.1% acetic acid in water (A) and acetonitrile (B), the gradient elution was: 0-1 min, 5% B; 1-2 min, 50-15% B; 2-4 min, 15-20% B; 4-12 min, 20-80% B; 12-13 min, 80% B; 13-15 min, 80-5% B; 15-16 min, 5% B, the injection volume was 5 μL, and the flow rate was 0.4 mL min -1 ;

[0011] The samples were analyzed for positive and negative ions in sMRM mode: nitrogen was used as curtain gas, nebulizer gas, and heater gas, and the pressures were set to 35, 40, and 40 psi, respectively. The optimized ion spray voltage and source temperature were 5500 V and 450 °C, respectively. The optimal sMRM parameters for 11 analytes are shown in the table. The detection window in sMRM mode was set to an expected holding time of about 90 seconds. The data were obtained by Analyst TM 1.6.3 Software collection and analysis;

[0012] Table 1 Optimal sMRM parameters for 11 analytes

[0013]

[0014]

[0015] It includes the following steps:

[0016] a. Preparation of standards and samples

[0017] Accurately weigh appropriate amounts of forsythiaside A, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin, and bergamotol, and dissolve them in methanol to make the concentrations of 0.197, 0.417, 0.426, 0.441, 0.075, 0.259, 0.102, 0.101, 0.122, 0.139, and 0.234 mg mL -1 Single reference stock solution;

[0018] Grind the antiviral particles separately, accurately weigh 2.0g of each powder into a stoppered conical flask, accurately add 50mL of 50% methanol, weigh, and ultrasonically extract for 30min; weigh after cooling, and make up the weight loss with methanol solution; filter through a 0.22μm microporous filter membrane, and take the filtrate as the UHPLC-MS / MS test solution;

[0019] b. HPLC-MS / MS detection.

[0020] Among them, the average contents of forsythiaside A, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin and bergamotol in the antiviral granules were 746.37, 37.16, 1442.19, 162.06, 32.02, 7.74, 50.04, 5.39, 165.83, 77.31 and 49.09 μg / g, respectively, with an average content fluctuation of 10%.

[0021] The detection method of the present invention is a quantitative determination method for 11 active components in antiviral particles. The detection method is short in time and low in cost. The quantitative analysis method is expected to provide a reference for the quality control of antiviral particles and ensure the clinical efficacy and safety of antiviral particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 UPLC-Q-Orbirap HRMS positive (ESI + ), negative (ESI - ) Total ion current in ion mode; Total ion current of volatile oil of antiviral particles (G) GC-MS;

[0023] Figure 2 Heat map of the docking results of potential active ingredients and NA protein molecules. Ligands represents the molecular docking results of the protein and its original ligand. The darker the blue color, the stronger the binding activity.

[0024] Figure 3 Predicted binding patterns of some active ingredients with NA protein. Green represents active compounds, translucent blue represents proteins, and dark blue represents amino acid residues;

[0025] Figure 4 NA inhibition of 20 batches of samples and active ingredients. (a) Dose-effect relationship of 11 active ingredients and positive drugs on NA inhibition. (b) IC50 values ​​of NA inhibition of active ingredients and positive drugs. (c) NA inhibition of 20 batches of samples. (d) Correlation analysis of NA inhibition of 20 batches of samples and effect component index;

[0026] Figure 5 Multiple reaction detection chromatograms of antiviral particle Q-marker standards (I) and samples (II) in positive (A) and negative (B) ion modes. DETAILED DESCRIPTION

[0027] The chemical test sources used in the present invention are as follows:

[0028] Twenty batches of antiviral particle samples were purchased from Sichuan Everbright Pharmaceutical Co., Ltd. (No. 2305444-2305463). Chemical standards Baicalin, TimosaponinA-III, Forsythiaside A,Baicalein,Gitogenin,Luteolin-7-glucuronide,Kaempferol-3-O-rutinoside,Sarsasapogenin,Rutin,Verbascoside,Quercetin,Lariciresinol-4-OD-glucoside,Pinoresinol-4-O-glucoside,Arctiin,pinoresinol,Imperatorin,Mangiferin,Philly genin,Oxypeucedanin,Matairesinol,5-methoxy-8-hydroxypsoralen,Osthol,Salidroside,Bergaptol,Deoxyvasicinone,Eremanthin,adenosine,alpha-Curcumene,Pogostone,Cyclo(-Leu-Pro),Curcumenol,beta-asarone,nootkatone,,Protocatechuic Acid, Arginine, Leucine, Adenosine, Indigotin, Indirubin were purchased from Lotte Beauty Pharmaceutical Technology Co., Ltd. (Chengdu, China); Forsythoside E, Forsythin, Salicylic acid were purchased from Chengdu Efa Biotechnology Co., Ltd. (Chengdu, China); Ursolic acid was purchased from Chengdu Kroma Biotechnology Co., Ltd. (Chengdu, China), and the purity of the above standards was not less than 98%. NA solution, NA detection buffer, and fluorescent substrate were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. (Shanghai, China). Methanol and acetonitrile (chromatographic purity) were purchased from Merck (Darmstadt, Germany). Formic acid (chromatographic purity) was purchased from Chengdu Kelong Chemical Reagent Factory (Chengdu, China).

[0029] Experimental Animals:

[0030] Twelve female SD rats, weighing 220±20g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., license number SCXK(Chuan)2020-030. The animals were housed in cages at the Experimental Animal Center of Chengdu University of Traditional Chinese Medicine, with a room temperature of 25±2℃, a relative humidity of 50%-60%, and a light-dark cycle of 12h / 12h. Rats were randomly divided into two groups (n=6): a drug-treated group (antiviral particles) and a blank group (W). Adaptive feeding for 7 days, with free drinking water during the period, and free food and drinking water during the period. Fasting for 12h before the experiment, but still free drinking water. All animal experimental procedures were approved by the Medical Ethics Committee of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine (approval number: 2021-49).

[0031] Example 1 Quality detection method of antiviral particles of the present invention

[0032] 1. Preparation of Standards and Samples

[0033] Accurately weigh appropriate amounts of forsythiaside A, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin, and bergamotol, and dissolve them in methanol to make the concentrations of 0.197, 0.417, 0.426, 0.441, 0.075, 0.259, 0.102, 0.101, 0.122, 0.139, and 0.234 mg mL -1 Single reference stock solution.

[0034] Grind each batch of antiviral particles, weigh 2.0g of each powder into a stoppered conical flask, accurately add 50mL of 50% methanol, weigh, and ultrasonically extract for 30 minutes. After cooling, weigh, and make up the weight loss with methanol solution. Filter through a 0.22μm microporous membrane, and take the filtrate as the UHPLC-MS / MS test solution.

[0035] 2. UHPLC-MS / MS Chromatographic Conditions

[0036] UHPLC-MS / MS analysis was performed using a Nexera UHPLC LC-30A / QTRAP 5500+MS / MS system (Kyoto, Japan) equipped with an electrospray ionization (ESI) source (AB Sciex).

[0037] The chromatographic column was ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm; Waters, Massachusetts, USA), the temperature was 35°C, the mobile phase was 0.1% acetic acid in water (A) and acetonitrile (B), the gradient elution was: 0-1 min, 5% B; 1-2 min, 50-15% B; 2-4 min, 15-20% B; 4-12 min, 20-80% B; 12-13 min, 80% B; 13-15 min, 80-5% B; 15-16 min, 5% B, the injection volume was 5 μL, and the flow rate was 0.4 mL min -1 .

[0038] The samples were analyzed for positive and negative ions in sMRM mode. Nitrogen was used as curtain gas, nebulizer gas, and heater gas, and the pressures were set to 35, 40, and 40 psi, respectively. The optimized ion spray voltage and source temperature were 5500 V and 450 °C, respectively. The optimal sMRM parameters for 11 analytes are shown in Table 1. Log Meaning The detection window in sMRM mode was set to an expected holding time of about 90 seconds. Data by Analyst TM 1.6.3 Software Collection and Analysis.

[0039] Table 1 Analysis parameters of antiviral particle Q-marker in multiple reaction monitoring mode

[0040]

[0041] Example 2 Determination of 11 index components in the antiviral particles of the present invention

[0042] 1. Preparation of sample solution and standard solution

[0043] Take 2 bags of samples from 5 batches and mix them. Extract volatile oil according to the volatile oil determination method of the 2020 edition of the Chinese Pharmacopoeia (General Rule 2204 Method A). Weigh 80.0g of sample and place it in a round-bottom flask, add 400mL of water, draw 2mL of cyclohexane and add it to the water surface of the volatile oil determination device, slowly heat to boiling, and keep it slightly boiling for 3h, cool, transfer the cyclohexane layer to a 2mL volumetric flask, add cyclohexane to dilute to the scale, shake well, and add an appropriate amount of anhydrous sodium sulfate for dehydration. Filter through a 0.22μm microporous membrane, and take the filtrate as the GC-MS test solution.

[0044] Take 1 bag of each sample from 5 batches, remove the packaging, grind and mix. Take about 2.0g of sample powder, accurately weigh it into a 100mL stoppered conical flask, accurately add 100mL of 70% methanol, weigh it, and extract it by ultrasonic for 30min. After cooling, weigh it and make up the weight loss with 70% methanol solution. Filter it through a 0.22μm microporous membrane and take the filtrate as the test solution for UPLC-Q-Orbitrap HRMS.

[0045] Take an appropriate amount of each reference substance and add chromatographic methanol to make a mass concentration of about 60 μg mL -1 The solution was filtered through a 0.22 μm microporous membrane and the filtrate was used as the UPLC-Q-Orbitrap HRMS standard solution.

[0046] 2. GC-MS analysis

[0047] The volatile components were analyzed using a 7890A-5975C gas chromatograph-mass spectrometer (Agilent, USA).

[0048] Agilent 19091S-433 capillary column (30 m × 250 μm × 0.25 μm), initial temperature 110 °C, maintained for 7 min, 12 °C min -1 The temperature was raised to 170°C at a rate of 20°C·min and maintained for 2 min. -1 The temperature was raised to 230°C at a rate of 1.5 °C and maintained for 2 min. The injection port temperature was 230°C, the carrier gas was helium, and the column flow rate was 1 mL min -1 ; Split ratio 20:1, injection volume 1μL. Ionization mode EI, electron energy 70eV, ion source temperature 200℃, scanning mass range 30-400, mass spectrum retrieval standard library: NIST.

[0049] 3. UPLC-Q-Orbitrap HRMS analysis

[0050] The samples were analyzed using ultra-performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry (Thermo Fisher Scientific, USA).

[0051] Chromatographic separation was performed on a Thermo Scientific AccucoreTM C18 column (3 mm × 100 mm, 2.6 μm). The mobile phase was 0.1% formic acid aqueous solution (A)-methanol (B), and the gradient elution program was as follows: 0-11 min, 5%-35% B; 11-18 min, 35%-55% B; 18-22 min, 55%-75% B; 22-24 min, 75%-90% B; 24-25 min, 90%-100% B; 25-30 min, 100% B; column temperature 50°C; flow rate 0.3 mL·min-1, injection volume 5 μL.

[0052] Electrospray ion source (ESI), positive and negative ion mode detection, spray voltage 3.5kV (+) / 3.0kV (-), auxiliary gas heating temperature 350 ° C, sheath gas flow rate 35arb, auxiliary gas flow rate 10arb, ion transfer tube temperature 320 ° C. Scanning mode is full scan of primary mass spectrometry combined with automatic triggering of secondary mass spectrometry scanning mode (Full MS / dd-MS2), primary resolution 35 000, secondary resolution 17 500, ion scanning range m / z 50-1 500, collision energy gradient 20, 40, 60eV.

[0053] The collected raw data were imported into Compound Discoverer 3.2 software. Through its wizard settings and method templates, the unknown compound identification process was established. The raw data were peak aligned and extracted. The possible molecular formulas were fitted through the extracted molecular ion chromatographic peaks and isotope peaks. The measured spectra of the secondary fragments were matched with the mzCloud network database and the local Chinese medicine component database OTCML. The filtering parameters for the matching results were set as follows: peak area threshold 80,000, primary and secondary mass deviation 5ppm, and matching score higher than 80. The filtered ions were compared with the compound information and reference substances in the database, and the chemical components were further analyzed and identified in combination with relevant literature and online databases (PubChem, Human MetabolomeDatabase, PubMed, CNKI), and the sources of each component were attributed.

[0054] 4. Study on the chemistry of antiviral particles in serum

[0055] 4.1. Preparation of oral gavage samples

[0056] The clinical oral dose of antiviral granules is 12-24 g / d. The minimum dose is 12 g / d and the rat oral dose is calculated according to the adult and rat dose conversion factor. Take an appropriate amount of antiviral granule sample and dissolve it in distilled water to make 1 g·mL -1 The intragastric dose of the rats was 10 times the equivalent daily dose of the antiviral particles, i.e. 10 g kg -1 The blank group was gavaged with an equal amount of distilled water.

[0057] 4.2. Preparation of serum samples

[0058] Each group of rats was gavaged twice a day according to the prescribed dose and drug solution for 3 consecutive days. They were fasted but not watered 12 hours before the last administration. 1 mL of blood was collected from the eye sockets at 5, 15, 30, 60, 120, 240, and 360 minutes after the last administration, and placed in an EP tube containing sodium heparin. The blood was then centrifuged at 4°C and 4000 rpm for 15 minutes, and the supernatant was collected, aliquoted, and frozen at -80°C for later use.

[0059] Before the analysis test, take 200 μL of serum from each rat at 7 time points, mix, add 3 times the amount of acetonitrile, vortex for 3 minutes, centrifuge at 4°C, 12000 rpm for 15 minutes, take the supernatant in a clean EP tube, blow dry with nitrogen at room temperature, re-dissolve the residue with 200 μL 70% methanol, vortex for 3 minutes, ultrasonicate for 5 minutes, centrifuge at 4°C, 12000 rpm for 15 minutes, transfer the supernatant to the liquid phase insert tube to obtain the serum drug chemistry test solution. Perform the detection and analysis according to the UPLC-Q-OrbitrapHRMS analysis method under 2.3.3.

[0060] 5. Molecular docking

[0061] Molecular docking technology is a research method for drug discovery and screening, and has been widely used in the research of screening active ingredients of traditional Chinese medicine. The present invention uses AutoDock Vina to evaluate the binding affinity of the serum-exposed prototype components of antiviral particles to influenza virus neuraminidase (PDB ID 3CL0; 4mwq; 4gzp; 3ti8; 3ti6; 3ckz; 2hu0), and compares it with the original ligand of the protein.

[0062] Retrieve the 3D structure of influenza virus NA-related proteins from the Protein Data Bank (PDB) http: / / www.rcsb.org / ), the bound original ligand and crystal water molecules were removed from the active site of the protein using AutoDock tools-1.5.6, and all polar hydrogen atoms and charges were added to the macromolecule. The Gasteiger charge of each atom of the receptor molecule was calculated in AutoDock 4.2, and the structures were saved as PDBQT protein receptor files. The 3D structure of the compound from PubC was imported into Open Babel-3.1.1 software and converted into PDB format. In AutoDock tools-1.5.6, the obtained 3D structure was modified by adding hydrogen and protonation. Non-polar hydrogens were incorporated into their corresponding carbon rotations, and the torsion was automatically set in the software. The structure was then saved as a PDBQT ligand file. The PDBQT structures of the receptor and ligand were imported into AutoDock tools-1.5.6 to construct the docking mating pocket. When constructing the mating box, the spacing (angstroms) was set to 1, and the DeepSite protein prediction website (https: / / playmolecule.com / deepsite / ) was used to predict the center of each protein mating box, and the number of points in the x, y, and z dimensions was set to keep the protein completely covered by the mating box. The docking calculation was completed by Autodock Vina, and the other parameters used the default settings of docking in Vina. After Vina was completed, the docking binding free energy was scored to establish the binding affinity score between the target protein receptor and the small molecule ligand. By comparing the binding energy with the original ligand of the protein, the molecule with better binding energy than the original ligand was selected as the potential active component. It is generally believed that when the conformation of the ligand and receptor binding is stable, the lower the energy, the greater the possibility of binding. The binding energy ≤-5.0kJ / mol indicates good binding activity, and the binding energy ≤-7.0kJ / mol indicates that the two have strong binding activity. Finally, the schematic diagram of the ligand interaction was visualized by PyMOL2.5.0, showing hydrogen bonds and hydrophobic interactions.

[0063] 6. In vitro NA inhibition assay

[0064] The NA inhibitor screening experiment was performed in a 96-well SpectraMax iD5 multi-function microplate reader (Molecular Corporation, USA). The experimental design was based on the instructions provided by the manufacturer of the kit (Shanghai Biotech Biotechnology Co., Ltd., Shanghai, China). The NA inhibitor screening experiment of active ingredients of traditional Chinese medicine has been reported. A reaction mixture of 70 μL of reaction buffer, 10 μL of NA and 10 μL of standard sample was added to each well of a black 96-well plate. The sample was dissolved in DMSO, the concentration of the standard stock solution was 10 mM, and the stock solution was diluted 2, 10, 100, and 1000 times. The concentration of the antiviral particle sample solution was 0.04 g mL -1. Vibrate and mix for about 1 minute and incubate at 37°C for 30 minutes to allow NA and standard samples to fully interact. Then add 10 μL of fluorescent substrate, and the reaction mixture is 100 μL in total. The entire mixture is fully mixed by vibration for about 1 minute, and the plate is incubated at 37°C for 30 minutes. The sample fluorescence is obtained by a multifunctional microplate reader, with an excitation wavelength of 322 nm and an emission wavelength of 450 nm. The blank control well contains the same reaction mixture and the same volume of dissolved DMSO as the sample to be tested. Three replicate wells are set up for each experiment, and the results are averaged to calculate the inhibition rate of the sample on NA activity. Then, the data is analyzed using GraphPad Prism 9.3 software (GraphPad software Inc., La Jolla, CA, USA) to obtain the maximum inhibitory concentration (IC50) for inhibiting NA activity. The inhibition rate is calculated by formula (1):

[0065] NA inhibition rate (%) = [1-(F s -F0) / (F m -F0)]×100%(1)

[0066] F s : fluorescence intensity of the object to be tested; F0: background fluorescence intensity; F m :Blank control fluorescence intensity

[0067] 7. Construction of the effect component index

[0068] 7.1. Calculation of the effect component index

[0069] According to the results of the NA inhibitor screening experiment, the inverse of the IC50 value of each component was normalized and the efficacy weight of each component was calculated. The calculation method is shown in formula (2).

[0070]

[0071] Where W i is the efficacy weight of each component, IC 50i is the IC50 value of the active ingredient, i is the ingredient name, and n is the number of ingredients.

[0072] The “effect component index” is reflected through the comprehensive index evaluation method, and the calculation method is shown in formula (3).

[0073]

[0074] Where Z m is the “effect component index” of different batches of samples, W i is the efficacy weight of each component, X i is the amount of each component in the sample.

[0075] 7.2. Validation of the Antiviral Particle Effect Component Index

[0076] The NA inhibitory activity of 20 batches of antiviral granule samples was tested, and the relative inhibition rate of each batch of antiviral granule samples was calculated with the inhibitory activity of the same concentration of forsythiaside A as a reference. In order to explore the correlation between the effect component index and the efficacy, the Pearson's correlation coefficient (R) was used to calculate the correlation between the effect component index and the content of each component and the relative inhibition rate.

[0077] 8. Results

[0078] 8.1. Analysis of the components of antiviral particles in vivo and in vitro

[0079] UPLC-Q-Orbitrap HRMS and GC-MS techniques were used to analyze the in vitro components of antiviral particle samples. The relevant chromatograms are shown in Figure 1 . According to the accurate molecular weight, retention time, mass spectrometry data information library and standard material information of the compounds, the identification of 241 components was completed, of which 209 were identified by UPLC-Q-Orbi trap HRMS and 32 were identified by GC-MS. UPLC-Q-Orbitrap HRMS was used to identify 89 prototype components and 19 metabolites from the serum of rats in the antiviral particle administration group (Table 2). The prototype components include organic acids, alkaloids, nucleosides, coumarins, terpenes, phenylethanol glycosides, etc. The metabolic components are mainly products obtained by hydrolysis, dehydrogenation, methylation, hydroxylation and other reactions of the in vitro components. The medicinal material sources, mass spectrometry behaviors, chemical structure types and possible metabolic pathways of the metabolic components of the prototype components are shown in Table 2. The relevant chromatograms of serum pharmacological chemistry are shown in Figure 1 .

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] 8.2. Molecular docking results

[0087] In order to investigate the direct interaction between antiviral particles and NA, molecular docking modeling was used to evaluate the binding affinity of the prototype components of antiviral particles entering the blood with related proteins, and compared with the original ligand of the protein, and the components with binding activity better than or equal to the original ligand were screened as potential active ingredients. The results showed that among the 89 prototype components entering the blood, 35 compounds such as Anemarrhena saponin A-Ⅲ, baicalin, and Forsythiaside A had binding activities that were at least better than the binding activity of one of the proteins with its original ligand, and the binding activities of these active ingredients were lower than -5.0 kJ / mol ( Figure 2 ).

[0088] Molecular docking showed that the binding activity trends of the blood-entering components with different proteins were well consistent, and most potential active components could bind well to different proteins. The PyMOL software was used to visualize and analyze some of the docking results, showing that Baicalin could hydrogen bond with the ASN-296, ARG-294, ASN-348, and G39-513 amino acid residues of the 4mwq protein; TimosaponinA-III could hydrogen bond with the TYR-100, VAL-136, ARG-172, GLU-174, and LYS-206 amino acid residues of the 2hu0 protein; ForsythiasideA could hydrogen bond with the TYR-169A, ASN-170, ARG-1 Kaempferol-3-O-rutinoside can hydrogen bond with GLU-276, TYR-374, ARG-224, ARG-292, ASN-294, GLU-119, THR-439, ARG-156, TRP-178 amino acid residues of 2hu0 protein; Gitogenin can hydrogen bond with LYS-431, ARG-430, ARG-118 amino acid residues of 4gzp protein. Among them, a computer simulation experiment of NA inhibitors showed that the active sites ARG118 and ARG292 of pH1N1NA protein can form hydrogen bonds and electrostatic interactions with ligand molecules. Vavricka et al. found that the NA inhibition mechanism of Laninamivir may be related to the hydrogen bond network that the drug can form with the Glu276 and Arg224 residues of the NA protein. These studies are in good agreement with the results of our molecular docking experiments, indicating that the potential active ingredients obtained may interact with proteins and inhibit NA activity. The predicted binding modes of the potential active ingredients with significant binding activity to the five NA proteins are as follows: Figure 3 shown.

[0089] 3.3. NA inhibitor screening experiment

[0090] The NA inhibitor screening kit was used to determine the NA inhibitory activity of 35 compounds that showed potential activity through molecular docking and the positive control oseltamivir acid. The results showed that ( Figure 4 aandb), the IC50 of the positive control was 1.461mM. The IC50 of the test samples forsythiaside A, baicalein, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin, and bergamotol were 0.594, 0.802, 1.063, 1.215, 1.331, 1.487, 1.515, 1.526, 1.599, 1.773, 2.657, and 3.408mM, respectively. The inhibition rate of the remaining compounds at a concentration of 10mM did not reach 50%, and their IC50 could not be calculated.

[0091] 9. Quantitative analysis of antiviral particle Q-marker based on UHPLC-MS / MS

[0092] Optimization of UHPLC-MS / MS analysis conditions

[0093] In order to obtain the best resolution chromatogram in a short time, the mass spectrometry parameters and chromatographic conditions of the target compound were optimized in the early stage of the present invention. According to the characteristics of the compound, suitable precursor ions (Q1) and product ions (Q3) are selected, and the collision energy (CE) and clustering voltage (DP) parameters are further optimized according to the response value of the target ion. Detailed information is shown in Table 1. However, when conducting methodological investigations, it was found that when baicalein was used as a potential Q-marker for content determination, its content in the sample was extremely low, and the sample recovery rate and quantitative limit could not meet the quantitative requirements. Considering that the NA inhibitory effect has a clear dose-effect relationship, the extremely low content of baicalein has almost no effect on the overall inhibitory effect of the sample. At the same time, combined with the measurability requirements of Q-marker, it was determined that 11 Q-markers other than baicalein were accurately quantified. In addition, there is isomerism in the target compound, and the detection of the above-mentioned target compound can be achieved by separating the chromatographic column in combination with the relationship of retention time. Therefore, in order to enhance the separation process within a reasonable running time, different mobile phases (water / acetonitrile, water / methanol, 0.1% formic acid water / acetonitrile, 0.1% acetic acid water / acetonitrile) were used for analysis. The final elution condition was determined to be water / acetonitrile with an acetic acid concentration of 0.1% and a flow rate of 0.4 mL min -1 Gradient elution. At the same time, combined with the adjustment of reasonable gradient elution time, each target compound has a better separation effect, symmetrical peak shape and ideal response, and ensures a shorter running time. Figure 5 UHPLC-MS / MS chromatograms of standard solutions and samples are shown.

[0094] 9.2. Methodological validation

[0095] After optimizing the detection conditions, the linearity, detection limit, quantification limit, precision, repeatability, stability and recovery of the UHPLC-MS / MS method were first verified. The correlation coefficients of the chemical components to be tested were all above 0.997 within the corresponding concentration range, and the linear relationship was good. After 6 consecutive injections, the RSD of the peak area of ​​each component was 1.16% to 3.96%, indicating that the precision of the instrument was good. A certain amount of reference substance was added to the sample, and the recovery of each component was 96.66% to 105.04%, indicating that the sample preparation method was good. Six samples were prepared in parallel, and the RSD of the peak area of ​​each component was 0.42% to 3.90%, indicating that the preparation method had good repeatability. The samples were measured at 0, 4, 8, 12 and 24h after preparation, and the RSD of the peak area of ​​each component was 2.33% to 4.61%, indicating that the stability of each component was good (see Table 3). The above methodological investigation results are in line with the provisions of the Chinese Pharmacopoeia "Guidelines for Validation of Analytical Methods", indicating that this method is sensitive and reliable and can simultaneously determine the above 11 compounds.

[0096] Table 3 Linear regression data, LOD, LOQ of antiviral particle Q-marker

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[0099] Sample analysis

[0100] The present invention uses UHPLC-MS / MS to quantitatively analyze 11 Q-markers on 20 batches of antiviral granules (sugar-free type) samples. The analysis results show (Table 4) that the contents of forsythiaside A and verbascoside in the samples are the highest, reaching 562.97-1829.26 μg·g -1. Forsythiaside A is derived from the main herb of the prescription, Forsythia suspensa, and the content of this ingredient obtained similar results in a quantitative analysis of Forsythia suspensa. Verbascoside is derived from both Rehmannia glutinosa and Patchouli sinensis, which may be one of the reasons for the high content of this ingredient. In addition, the sample also contains relatively high levels of mangiferin and baicalin, among which mangiferin is the main active ingredient of Anemarrhena asphodeloides and baicalin is the active ingredient in Forsythia suspensa. The contents of bergamot and kaempferol-3-O-rutinoside were the lowest in the samples tested. These results show that the contents of different Q-markers vary greatly in the samples, which in turn leads to different effects on their inhibitory effects on NA activity. At the same time, the same ingredient in the antiviral particles also varies between different batches, suggesting the necessity of improving its quality control level. Therefore, this quantitative analysis method is expected to provide a reference for the quality control of antiviral particles and ensure the clinical efficacy and safety of antiviral particles. However, the contribution of these ingredients to the inhibitory effect of NA needs further study. The multiple reaction detection chromatograms of standards and samples are shown in Figure 5 .

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[0103] 10. Construction of effect component index

[0104] The IC50 values ​​of the NA inhibition rates of the 11 Q-markers in the antiviral particles were normalized and the efficacy weights were calculated. The effect weight of forsythiaside A was the largest, i.e., the best activity; while the effect weight of bergamotol was the smallest, i.e., the weakest activity. Combined with the content of Q-markers in each batch of samples, the effect component index was calculated according to formula (3). The results are shown in Table 4, where the maximum ECI value was 460.84 (sample S4) and the minimum ECI value was 256.81 (sample S16). Correlation analysis showed that the r value of the correlation analysis between ECI and bioavailability was 0.81, and the NA inhibitory activity of each batch of samples was significantly correlated with the calculation results of the "effect component index", which was a positive correlation, that is, the larger the "effect component index", the stronger the NA inhibitory activity of the antiviral particles. In addition, the R values ​​of the correlation analysis between the content of each component and the NA inhibitory activity were all smaller than the R values ​​between the NA inhibitory activity and the effect component index, and were 0.69 to 0.39. Therefore, compared with using the ingredient content as an indicator, the "effect ingredient index" can more scientifically and reasonably characterize the pharmacological activity of antiviral particles.

[0105] 11. Summary

[0106] Antiviral granules are a traditional Chinese medicine compound preparation that has been on the market for many years and has a proven efficacy in treating influenza, but its active ingredients and mechanism of action are still unclear, which limits the quality evaluation level. Q-marker is the main active ingredient that is closely related to efficacy. Scientific selection and evaluation of Q-marker can ensure the quality control and clinical use of drugs.

[0107] The present invention firstly characterizes the components of antiviral particles by high-throughput technology to clarify their material basis; further serum pharmacological chemistry research is carried out to reveal the blood-entering components of antiviral particles, providing a basis for further screening of effective components; then, potential Q-markers for treating influenza with antiviral particles are confirmed step by step through molecular docking virtual screening and in vitro evaluation of NA inhibition by a kit; the measurability of potential Q-markers is then investigated by UHPLC-MS / MS to confirm the final Q-marker and carry out content determination; finally, the effect component index of the antiviral particles is calculated based on the dose-effect relationship of the Q-marker and its rationality is successfully verified. In summary, based on the characteristics of Q-marker in terms of testability, mass transferability, traceability, effectiveness, and compatibility contribution of traditional Chinese medicine compound, the present invention found that Forsythiaside A, Pogostone, Verbascoside, Mangiferin, 5-methoxy-8-hydroxypsoralen, Curcumenol, Luteolin-7-glucuronide, Kaempferol-3-O-rutinoside, Baicalin, Rutin, and Bergaptol are Q-markers for antiviral particles to treat influenza.

[0108] The present invention is the first to establish a method for evaluating the quality of a Chinese medicine compound by using Q-marker and effect component index to corroborate each other. It can not only calculate its effect component index by screening Q-marker, but also reversely prove the rationality of Q-marker confirmation by effect component index. However, the in vivo exposure and in vivo effects of each Q-marker in the present invention are still unclear, and further research will be carried out in the future through network pharmacology, serum pharmacokinetics, and in vivo efficacy experiments.

Claims

1. A method for detecting the quality of antiviral particles, characterized in that: It uses HPLC-MS / MS to simultaneously determine the following components: Forsythiaside A, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin, bergamotol.

2. The method for detecting the quality of antiviral particles according to claim 1, characterized in that: The HPLC-MS / MS detection conditions are: UHPLC-MS / MS analysis was performed using a Nexera UHPLC LC-30A / QTRAP 5500+MS / MS system equipped with an electrospray ionization (ESI) source (AB Sciex); The chromatographic column was ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm; Waters, Massachusetts, USA), the temperature was 35°C, the mobile phase was 0.1% acetic acid in water (A) and acetonitrile (B), the gradient elution was: 0-1 min, 5% B; 1-2 min, 50-15% B; 2-4 min, 15-20% B; 4-12 min, 20-80% B; 12-13 min, 80% B; 13-15 min, 80-5% B; 15-16 min, 5% B, the injection volume was 5 μL, and the flow rate was 0.4 mL min -1 ; The samples were analyzed for positive and negative ions in sMRM mode: nitrogen was used as curtain gas, nebulizer gas, and heater gas, and the pressures were set to 35, 40, and 40 psi, respectively. The optimized ion spray voltage and source temperature were 5500 V and 450 °C, respectively. The optimal sMRM parameters for 11 analytes are shown in the table. The detection window in sMRM mode was set to an expected holding time of about 90 seconds. The data were obtained by Analyst TM 1.6.3 Software collection and analysis; Optimal sMRM parameters for 11 analytes 3. The method for quality detection of antiviral particles according to claim 2, characterized in that: It includes the following steps: a. Preparation of standards and samples Accurately weigh appropriate amounts of forsythiaside A, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin, and bergamotol, and dissolve them in methanol to make the concentrations of 0.197, 0.417, 0.426, 0.441, 0.075, 0.259, 0.102, 0.101, 0.122, 0.139, and 0.234 mg mL -1 Single reference stock solution; Grind the antiviral particles separately, accurately weigh 2.0g of each powder into a stoppered conical flask, accurately add 50mL of 50% methanol, weigh, and ultrasonically extract for 30min; weigh after cooling, and make up the weight loss with methanol solution; filter through a 0.22μm microporous filter membrane, and take the filtrate as the UHPLC-MS / MS test solution; b. HPLC-MS / MS detection.

4. The method for detecting the quality of antiviral particles according to claim 4, characterized in that: The average contents of forsythiaside A, patchouli ketone, verbascoside, mangiferin, 5-methoxy-8-hydroxypsoralen, curcumenol, luteolin-7-glucuronide, kaempferol-3-O-rutinoside, baicalin, rutin and bergamotol in the antiviral granules were 746.37, 37.16, 1442.19, 162.06, 32.02, 7.74, 50.04, 5.39, 165.83, 77.31 and 49.09 μg / g, respectively, with an average content fluctuation of 10%.

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