A traditional Chinese medicine extract, drug and its detection method

Abstract

The application discloses a traditional Chinese medicine extract, which is made of traditional Chinese medicines including the following raw materials: Magnolia officinalis 1-100 parts, Strychnos ignatii 1-100 parts, Toddalia asiatica 1-100 parts, Ephedra 1-100 parts, Bitter almond 1-100 parts, Notopterygium 1-100 parts, Ginger 1-100 parts, Pogostemonis 1-100 parts, Pterocephalus 1-100 parts, Atractylodes 1-100 parts, Poria cocos 1-100 parts, Atractylodes 1-100 parts, Gypsum fibrosum 1-100 parts, Fritillaria cirrhosa 1-100 parts, Ficus 1-100 parts, Fagopyrum 1-100 parts, Earthworm 1-100 parts, Radix et Rhizoma Cynanchi 1-100 parts, Equisetum ramosissimum 1-100 parts, Lepidium apetalum 1-100 parts, wherein the extract contains ephedrine with a content of 0.5-1.5 mg / g, and the solid content transfer rate is 18-25%.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine extract, a drug, and a method for detecting it. Background Technology

[0002] Viral pneumonia is a disease caused by upper respiratory tract viral infection that spreads downwards, leading to inflammation of the lungs and impaired pulmonary gas exchange. It can occur year-round, but is most common in winter and spring, and can occur in outbreaks or sporadically. Clinical manifestations mainly include fever, headache, body aches, dry cough, and pulmonary infiltration. Influenza virus-induced pneumonia is the most common, but other pathogens, including respiratory syncytial virus, adenovirus, and parainfluenza virus, have also attracted widespread attention.

[0003] Viral infection primarily manifests as interstitial lung disease. It initially affects ciliated columnar epithelial cells, then invades other respiratory cells, including alveolar cells, mucous gland cells, and macrophages. The virus replicates within the cells and then releases infectious virus to infect adjacent cells. Infected ciliated cells may exhibit degenerative changes, including granular degeneration, vacuolation, cell swelling, and nuclear pyknosis, followed by necrosis and disintegration. Cellular debris accumulates in the airways and obstructs small airways, causing respiratory tract swelling. Significant inflammatory reactions are observed in the alveolar septa, accompanied by infiltration of lymphocytes and macrophages, and occasionally plasma cells and neutrophils with edema. Fibrin thrombi with necrosis and hemorrhage may appear in the alveolar capillaries, and eosinophilic hyaline membranes may be visible in the alveoli. Severe infections may present with pulmonary edema, consolidation, hemorrhage, pulmonary parenchymal necrosis, and atelectasis.

[0004] Influenza virus and respiratory syncytial virus (RSV) are the most common viruses causing upper respiratory tract infections. Influenza virus belongs to the Orthomyxoviridae family and is an RNA virus. Based on the antigenicity of its nucleoprotein and matrix protein, it is classified into three types: A, B, and C. Type A influenza virus mutates rapidly and is highly virulent. H1N1 / FM1 is a mouse lung-adapted strain of influenza A virus, designed to mimic influenza A virus infection and damage to the lungs. Influenza A virus initiates infection by binding to the surface of respiratory epithelial cells containing sialic acid receptors via hemagglutinin. After entering the cell, the viral genome begins transcription and replication. The large number of progeny viral particles replicated spread through the respiratory mucosa and begin infecting other cells, inducing a cytokine storm, leading to a systemic inflammatory response, and subsequently acute respiratory distress syndrome (ARDS), shock, and multiple organ failure. Respiratory syncytial virus (RSV) belongs to the Paramyxoviridae family and is an RNA virus. Its pathogenesis involves interactions between the virus and the host's affected cells, inflammation, humoral and local immune responses, and hyperreactivity.

[0005] Current data and clinical use confirm that traditional Chinese medicine (TCM) has significant antiviral efficacy, a broad antiviral spectrum, and minimal side effects. Furthermore, many TCM herbs also possess antipyretic and anti-inflammatory properties, exhibiting multiple effects against viral infections, such as shortening fever duration, controlling the spread of inflammation, and promoting inflammation absorption—acting through multiple pathways and in multiple ways. It is precisely because of the diversity of effective components in TCM during treatment that viruses are less likely to develop drug resistance, giving TCM a significant advantage in treating viral infectious diseases and promising a bright future for clinical application. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a traditional Chinese medicine extract with antiviral effects. The extract is characterized by being made from the following traditional Chinese medicine ingredients by weight: 1-100 parts Magnolia officinalis, 1-100 parts Areca catechu (roasted), 1-100 parts Amomum villosum (roasted), 1-100 parts Ephedra sinica, 1-100 parts Prunus armeniaca (bitter), 1-100 parts Notopterygium incisum, 1-100 parts Zingiber officinale (fresh), 1-100 parts Pogostemon cablin, and 1-10 parts Eupatorium fortunei. 0 parts, Atractylodes lancea 1-100 parts, Poria cocos 1-100 parts, Atractylodes macrocephala 1-100 parts, Gypsum 1-100 parts, Crataegus pinnatifida 1-100 parts, Massa fermentata 1-100 parts, Malt 1-100 parts, Earthworm 1-100 parts, Cynanchum paniculatum 1-100 parts, Dryopteris crassirhizoma 1-100 parts, Lepidium apetalum 1-100 parts; wherein, the ephedrine content of the extract is 0.5-1.5 mg / g, and the solids transfer rate is 18-25%.

[0007] Furthermore, the herbal extract is made from the following raw materials: 1-80 parts Magnolia officinalis, 1-80 parts Areca catechu (roasted), 1-80 parts Amomum villosum (roasted), 1-60 parts Ephedra sinica, 1-60 parts Prunus armeniaca (bitter), 1-800 parts Notopterygium incisum, 1-60 parts fresh ginger, 1-80 parts Pogostemon cablin, 1-60 parts Eupatorium fortunei, 1-80 parts Atractylodes lancea, 1-160 parts Poria cocos, 1-120 parts Atractylodes macrocephala, 1-80 parts gypsum, 1-50 parts Crataegus pinnatifida (roasted), 1-80 parts Massa fermentata (roasted), 1-60 parts Hordeum vulgare (roasted), 1-80 parts Pheretima aspergillum, 1-80 parts Cynanchum paniculatum, 1-60 parts Dryopteris crassirhizoma, and 1-80 parts Lepidium apetalum; wherein the content of purslane in the extract is 2-8 mg / g.

[0008] Furthermore, the herbal extract is made from the following ingredients: 50 parts Magnolia officinalis, 30 parts Areca catechu (roasted), 50 parts Amomum villosum (roasted), 30 parts Ephedra sinica, 30 parts Prunus armeniaca (bitter), 50 parts Notopterygium incisum, 50 parts fresh ginger, 50 parts Pogostemon cablin, 30 parts Eupatorium fortunei, 50 parts Atractylodes lancea, 150 parts Poria cocos, 100 parts Atractylodes macrocephala, 50 parts gypsum, 30 parts Crataegus pinnatifida (roasted), 50 parts Massa fermentata (roasted), 30 parts Hordeum vulgare (roasted), 50 parts Pheretima aspergillum, 50 parts Cynanchum paniculatum, 30 parts Dryopteris crassirhizoma, and 50 parts Lepidium apetalum.

[0009] Furthermore, the preparation of the traditional Chinese medicine extract includes:

[0010] Weigh out the following ingredients: Magnolia officinalis, Areca catechu (roasted), Amomum villosum (roasted), Ephedra sinica, Prunus armeniaca (bitter), Notopterygium incisum, Zingiber officinale, Pogostemon cablin, Eupatorium fortunei, Atractylodes lancea, Poria cocos, Atractylodes macrocephala, Gypsum fibrosum, Crataegus pinnatifida (roasted), Massa fermentata (roasted), Malt (roasted), Pheretima aspergillum, Cynanchum paniculatum, Dryopteris crassirhizoma, and Lepidium apetalum. Extract each ingredient twice with water. For the first extraction, add 6 times the amount of water and extract for 1.5 hours. For the second extraction, add 4 times the amount of water and extract for 1.0 hour. Combine the extracts, filter, concentrate the filtrate to a relative density of 1.10–1.15, centrifuge, and dry the filtrate. The extract contains 1.20–1.50 mg / g of ephedrine, has a solids transfer rate of 19–22%, and contains 6–8 mg / g of purslane.

[0011] Furthermore, the similarity between the fingerprint spectrum of Magnolia officinalis and the control fingerprint spectrum must not be less than 0.90. The control fingerprint spectrum includes common peaks and magnolol and honokiol. The control fingerprint spectrum may be, for example, a... Figure 1 .

[0012] Furthermore, the similarity between the fingerprint spectrum of the charred areca nut and the control fingerprint spectrum must not be less than 0.90. The control fingerprint spectrum contains common peaks of arecoline and arecoline. The control fingerprint spectrum may, for example, be... Figure 2 .

[0013] Furthermore, the similarity between the fingerprint spectrum of the prepared licorice root and the control fingerprint spectrum must not be less than 0.90. The control fingerprint spectrum contains a common peak of protocatechuic acid. The control fingerprint spectrum may, for example, be... Figure 3 .

[0014] A method for preparing a traditional Chinese medicine extract, characterized by: weighing Magnolia officinalis, Areca catechu (roasted), Amomum villosum (baked), Ephedra sinica, Prunus armeniaca (bitter), Notopterygium incisum, Zingiber officinale, Pogostemon cablin, Eupatorium fortunei, Atractylodes lancea, Poria cocos, Atractylodes macrocephala, Gypsum fibrosum, Crataegus pinnatifida (roasted), Massa fermentata (roasted), Hordeum vulgare (roasted), Pheretima aspergillum, Cynanchum paniculatum, Dryopteris crassirhizoma, and Lepidium apetalum, and extracting each twice with water. The first extraction is with 6 times the amount of water for 1.5 hours, and the second extraction is with 4 times the amount of water for 1 hour. The extracts are combined, filtered, and the filtrate is concentrated to a relative density of 1.10–1.15. The filtrate is then centrifuged and dried. The extract contains 1.20–1.50 mg / g of ephedrine, has a solids transfer rate of 19–22%, and contains 6–8 mg / g of purslane. The fingerprints of Magnolia officinalis, Areca catechu (roasted), or Amomum villosum (baked) are as defined previously. The present invention also proposes the use of the Chinese herbal extracts as described above in the preparation of drugs for treating influenza viruses H1N1 or FM1, or respiratory syncytial virus.

[0015] The present invention also proposes a medicament prepared from a traditional Chinese medicine extract as described above and pharmaceutically acceptable excipients or additives.

[0016] Specifically, the drug is selected from decoctions, granules, capsules, tablets, oral liquids, pills, soft capsules, drop pills, tinctures, syrups, suppositories, gels, sprays, and injections.

[0017] Furthermore, the drug is in granule form, and the excipients or additives are preferably dextrin and sucralose.

[0018] Furthermore, the drug may also include antiviral drugs for upper respiratory tract infections, such as ribavirin and / or oseltamivir phosphate.

[0019] This invention also proposes a method for detecting the fingerprint chromatogram of the Chinese herbal extract or drug as described above, characterized in that the test sample solution is subjected to HPLC detection, and the chromatographic conditions for HPLC detection include: using a C18 column, using methanol as mobile phase A, and using an aqueous solution containing 0.1% phosphoric acid as mobile phase B, and the elution in the HPLC detection chromatographic conditions is gradient elution, the gradient elution program being: 0-5 min, 0% A; 5-25 min, 0%-15% A; 25-60 min, 15%-55% A; 60-75 min, 55%-100% A; 75-80 min, 100% A.

[0020] The test solution is a solution prepared from traditional Chinese medicine extracts or drugs for detection. For example, the preparation method can be to take 1g of granules, place them in a stoppered conical flask, add 25ml of water, and sonicate for 30 minutes to obtain the solution.

[0021] Furthermore, the chromatographic conditions include: a flow rate of 1.0 mL / min, a column temperature of 30 °C, and a detection wavelength of 250 nm.

[0022] Furthermore, the fingerprint spectrum is as follows: Figure 14 As shown.

[0023] Specifically, in the fingerprint spectrum, the sample should exhibit 10 characteristic peaks, and the peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak should be calculated and should be within ±10% of the specified value. The specified relative retention time values ​​are: 0.11 (peak 1), 0.27 (peak 2), 0.36 (peak 3), 0.38 (peak 4), 0.41 (peak 5), 0.84 (peak 6), 0.92 (peak 7), 1.00 [peak 8 (S)], 1.19 (peak 9), and 1.29 (peak 10).

[0024] This invention prepares a novel traditional Chinese medicine composition, improves the quality standard of the composition, and evaluates the effectiveness of the composition granules in treating viral upper respiratory tract infections by using influenza virus H1N1 / FM1 strain infection model and respiratory syncytial virus infection model. The results show that the composition can reduce viral load and inflammatory cytokines in lung tissue, indicating that it has an enhanced therapeutic effect on this type of common specific viral infection, providing a basis for further clinical research. Attached Figure Description

[0025] Figure 1 Fingerprint chromatogram of Magnolia officinalis (Honorophylla) as a reference; Peak 6: and magnolol peak 7(S): magnolol;

[0026] Figure 2 Fingerprint spectrum of areca nut medicinal material; Peak 1: arecoline; Peak 3 (S): arecoline;

[0027] Figure 3 Fingerprint spectrum of roasted licorice root (for comparison); Peak 1: protocatechuic acid;

[0028] Figure 4 This is a 3D image of the test solution across all wavelengths.

[0029] Figure 5 To investigate the wavelength of the test sample solution, a chromatogram was prepared.

[0030] Figure 6 To investigate the chromatogram for mobile phase ratio;

[0031] Figure 7 To investigate the chromatogram of the test sample preparation method;

[0032] Figure 8 Chromatograms for durability testing;

[0033] Figure 9 To determine the precision of the chromatogram;

[0034] Figure 10 Repeatability testing of chromatograms;

[0035] Figure 11 Stability study chromatogram;

[0036] Figure 12 Overlay of characteristic chromatograms of 20 batches of formulations;

[0037] Figure 13 Compare with the characteristic maps;

[0038] Figure 14Compare the characteristic chromatograms and the identification results of each characteristic peak; among them, 1-adenine; 2-uridine; 3-guanosine; 4-inosine; 5-5-hydroxymethylfurfural; 6-magnoside A; 7-bergamot glucoside; 8-imperatorin; 9-6'-O-(trans-feruloyl)-imperatorin; 10-anisolic acid p-hydroxyphenylethyl ester. Detailed Implementation

[0039] Oseltamivir is an inhibitor of neuraminidase in influenza viruses and is mainly used clinically to treat influenza A and B. Ribavirin is a broad-spectrum antiviral drug that can be used clinically to treat respiratory syncytial virus (RSV), influenza A, and influenza B, but it is not highly recommended by the FDA. This invention aims to provide a traditional Chinese medicine composition, its preparation method, and its application. The following will use commonly used influenza virus or RSV drugs as examples, combined with experimental details, for specific description.

[0040] It is particularly important to note that similar substitutions and modifications made to this invention are obvious to those skilled in the art, and they are all considered to be included in this invention. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0041] Unless otherwise specified, this invention is carried out under conventional conditions or conditions recommended by the manufacturer. The raw materials or excipients used, as well as the reagents or instruments used, whose manufacturers are not specified, are all conventional products that can be obtained commercially.

[0042] Experiment Example 1: Examination of Preparation Process

[0043] 1. Investigation of extraction solvent

[0044] Weigh out 15g of Magnolia officinalis, 9g of Areca catechu (roasted), 9g of Amomum villosum (roasted), 6g of Ephedra sinica, 9g of Prunus armeniaca (bitter), 15g of Notopterygium incisum, 15g of Zingiber officinale (fresh), 15g of Pogostemon cablin, 9g of Eupatorium fortunei, 15g of Atractylodes lancea, 45g of Poria cocos, 30g of Atractylodes macrocephala, 15g of Gypsum fibrosum, 9g of Crataegus pinnatifida (roasted), 9g of Massa fermentata (roasted), 9g of Hordeum vulgare (roasted), 15g of Pheretima aspergillum, 15g of Cynanchum paniculatum, 9g of Dryopteris crassirhizoma, and 15g of Lepidium apetalum as one part. Add water and decoct twice, the first extraction for 1.5 hours and the second for 1.0 hour. Combine the extracts, filter, concentrate the filtrate to a relative density of 1.10–1.15, centrifuge, filter again, vacuum dry the filtrate, spray dry and pulverize to obtain the intermediate composition. The results of the ephedrine transfer rate and solid content transfer rate with different extraction solvent volumes are as follows:

[0045]

[0046] Calculation formula:

[0047]

[0048]

[0049] The total transfer rate refers to the ratio of the total amount of ephedrine hydrochloride and pseudoephedrine hydrochloride in the extract to the total amount of ephedrine hydrochloride and pseudoephedrine hydrochloride in the medicinal material.

[0050] Experimental results showed that the transfer rates of ephedrine hydrochloride and pseudoephedrine hydrochloride, as well as the transfer rate of solids, increased with increasing water volume. When the solvent volume increased from 4.3 times to 6.4 times, the transfer rates of each indicator increased significantly. As the water volume continued to increase, the growth trend of each indicator slowed down, but it even decreased when the solvent volume was too high. Considering both energy efficiency and the transfer rate of the indicator components, the recommended water volume was 6 times the volume for the first addition and 4 times the volume for the second addition.

[0051] 2. Soaking time assessment

[0052] Another formula was prepared by taking 15g of Magnolia officinalis, 9g of Areca catechu (roasted), 9g of Amomum villosum (roasted), 6g of Ephedra sinica, 9g of Prunus armeniaca (bitter), 15g of Notopterygium incisum, 15g of Zingiber officinale (fresh), 15g of Pogostemon cablin, 9g of Eupatorium fortunei, 15g of Atractylodes lancea, 45g of Poria cocos, 30g of Atractylodes macrocephala, 15g of Gypsum fibrosum, 9g of Crataegus pinnatifida (roasted), 9g of Massa fermentata (roasted), 9g of Hordeum vulgare (roasted), 15g of Pheretima aspergillum, 15g of Cynanchum paniculatum, 9g of Dryopteris crassirhizoma, and 15g of Lepidium apetalum, and making a total of 3 portions. These were decocted twice with water. The first decoction was performed with 6 times the amount of water for 1.5 hours, and the second decoction was performed with 4 times the amount of water for 1 hour. The soaking times for the three portions were 0, 30, and 60 minutes, respectively. The extracts were combined, filtered, and the filtrate was concentrated to a relative density of 1.10–1.15. The filtrate was centrifuged, vacuum dried, spray-dried, and pulverized to obtain the intermediate composition.

[0053] The effects of soaking time on the transfer rates of ephedrine hydrochloride and pseudoephedrine hydrochloride, as well as the transfer rate of solids, were investigated. The experimental results are as follows:

[0054]

[0055] Calculation formula:

[0056]

[0057]

[0058] Experimental results showed that soaking time had little effect on the total transfer rate of ephedrine hydrochloride and pseudoephedrine hydrochloride, as well as the transfer rate of solids. Therefore, the extraction process was determined to be direct extraction without soaking.

[0059] 3. Examination of extraction time

[0060] Five portions were prepared, each containing 15g of Magnolia officinalis, 9g of Areca catechu (roasted), 9g of Amomum villosum (roasted), 6g of Ephedra sinica, 9g of Prunus armeniaca (bitter), 15g of Notopterygium incisum, 15g of Zingiber officinale (fresh), 15g of Pogostemon cablin, 9g of Eupatorium fortunei, 15g of Atractylodes lancea, 45g of Poria cocos, 30g of Atractylodes macrocephala, 15g of Gypsum fibrosum, 9g of Crataegus pinnatifida (roasted), 9g of Massa fermentata (roasted), 9g of Hordeum vulgare (roasted), 15g of Pheretima aspergillum, 15g of Cynanchum paniculatum, 9g of Dryopteris crassirhizoma, and 15g of Lepidium apetalum. Each portion was extracted twice with water. The first extraction used 6 times the amount of water, and the second extraction used 4 times the amount of water. The extraction times for the first extraction were 60 min, 75 min, 90 min, 105 min, and 120 min, respectively. The extraction times for the second extraction were 30 min, 45 min, 60 min, 75 min, and 90 min, respectively. The effects of extraction time on the transfer rates of ephedrine hydrochloride and pseudoephedrine hydrochloride, as well as the transfer rate of solids, were investigated. The experimental results are as follows:

[0061]

[0062] Calculation formula:

[0063]

[0064]

[0065] Experimental results showed that the transfer rates of ephedrine hydrochloride and pseudoephedrine hydrochloride, as well as the transfer rate of solids, increased with increasing extraction time. The increase was more pronounced when the extraction time increased from 60 and 30 minutes to 90 and 60 minutes, but the increase gradually leveled off with further increases in extraction time. Therefore, the optimal extraction time was 90 minutes for the first extraction and 60 minutes for the second.

[0066] 4. Validation of extraction process

[0067] To verify the optimized extraction process, a scale-up verification experiment was conducted based on the results of the single-factor experiments. In the preliminary experiments, the main components of the extract fluctuated significantly, which affected the therapeutic effect. Therefore, by strengthening the control from raw materials to the preparation process, the aim was to achieve effective and controllable extraction of the extract. Weigh out 15g of Magnolia officinalis, 9g of Areca catechu (roasted), 9g of Amomum villosum (baked), 6g of Ephedra sinica, 9g of Prunus armeniaca (bitter), 15g of Notopterygium incisum, 15g of Zingiber officinale (fresh), 15g of Pogostemon cablin, 9g of Eupatorium fortunei, 15g of Atractylodes lancea, 45g of Poria cocos, 30g of Atractylodes macrocephala, 15g of Gypsum fibrosum, 9g of Crataegus pinnatifida (roasted), 9g of Massa fermentata (roasted), 9g of Hordeum vulgare (roasted), 15g of Pheretima aspergillum, 15g of Cynanchum paniculatum, 9g of Dryopteris crassirhizoma, and 15g of Lepidium apetalum as one portion, and weigh out 12 portions in total (for some important drugs, Magnolia officinalis, Areca catechu, and Amomum villosum (baked), unique fingerprint chromatogram control standards have been established for medicinal materials and processed medicinal pieces, and all prescription medicinal materials comply with the latest pharmacopoeia standards). Extract each portion twice with water. The first extraction uses 6 times the amount of water and lasts for 1.5 hours, while the second extraction uses 4 times the amount of water and lasts for 1 hour. Combine 4 portions into 3 groups. The transfer rates of ephedrine hydrochloride and pseudoephedrine hydrochloride, the transfer rate of solids, and other important indicators were determined to investigate the stability and feasibility of the process. The experimental results are as follows:

[0068]

[0069] The verification results show that the optimized process is stable, feasible, and has good reproducibility. Therefore, the optimal extraction process is determined to be two extractions with water. The first extraction is with 6 times the amount of water for 1.5 hours, and the second extraction is with 4 times the amount of water for 1.0 hour.

[0070] 5. Fingerprint spectral control standards for Magnolia officinalis, Areca catechu, and Amomum villosum var. sarcodactylis and processed medicinal materials.

[0071] 5.1 Fingerprint control standards for Magnolia officinalis medicinal materials and processed slices

[0072] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase (Waters CORTECTSC). 18 The chromatographic column (150 mm × 4.6 mm, 2.7 μm) was used; acetonitrile was used as mobile phase A, and 0.1% acetic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the column temperature was 35℃; the flow rate was 0.8 ml / min; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the magnolol peak, should not be less than 4000.

[0073]

[0074] Preparation of reference solution: Take appropriate amounts of magnolol and magnolol reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 200 μg of each per ml.

[0075] Preparation of the test solution: Weigh 1g of the powder (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, add 25ml of 50% methanol solution accurately, shake well, seal tightly, soak for 24 hours, filter, and collect the filtrate to obtain the test solution.

[0076] For the assay, precisely pipette 10 μl each of the reference solution and the test solution and inject them into the liquid chromatograph for determination.

[0077] The fingerprint chromatogram of the test sample should show the same chromatographic peaks as the reference sample. Figure 1 Chromatographic peaks with the same retention time. According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram shall not be less than 0.90.

[0078] 5.2 Fingerprint Spectrum Control Standards for Areca Nut Medicinal Materials and Processed Slices

[0079] Chromatographic conditions and system suitability tests were conducted using strongly cation-exchange bonded silica gel as the packing material (ThermoBioBasicSCX column, 25 cm long, 4.6 mm inner diameter, 5 μm particle size); acetonitrile-0.2% phosphoric acid solution (adjusted to pH 3.8 with ammonia) (65:35) as the mobile phase; column temperature 35℃; flow rate 1.2 mL / min; detection wavelength 210 nm. The theoretical plate number, calculated based on arecoline, should be no less than 10,000.

[0080] Preparation of the reference solution: Take an appropriate amount of arecoline hydrobromide reference standard, accurately weigh it, and add methanol to prepare a solution containing 50 μg per ml.

[0081] Preparation of the test solution: Weigh 0.5 g of the powder (passed through a No. 2 sieve) accurately, place it in a stoppered conical flask, add 50 ml of 50% methanol solution accurately, sonicate for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0082] For the assay, accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, measure and record the chromatograms to obtain the results.

[0083] The fingerprint chromatogram of the test sample should show the same chromatographic peaks as the reference sample. Figure 2 Chromatographic peaks with the same retention time. According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram shall not be less than 0.90.

[0084] 5.3 Fingerprint Spectrum Control Standards for Prepared Amomum villosum Medicinal Materials and Slices

[0085] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase (Waters Cortects T3 column, 15 cm length, 4.6 mm inner diameter, 2.7 μm particle size); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution as specified in the table below; column temperature was 30℃; flow rate was 0.8 mL / min; detection wavelength was 254 nm. The theoretical plate number, calculated based on the protocatechuic acid peak, should be no less than 10,000.

[0086]

[0087]

[0088] Preparation of the reference solution: Take an appropriate amount of protocatechuic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 200 μg per ml.

[0089] Preparation of the test solution: Weigh 1g of the powder (passed through a No. 2 sieve) accurately, add 25ml of 50% methanol solution accurately, sonicate for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0090] For the assay, accurately pipette 5–10 μl of the reference solution and 10 μl of the test solution, inject them into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0091] The fingerprint chromatogram of the test sample should show the same chromatographic peaks as the reference sample. Figure 3 Chromatographic peaks with the same retention time. According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram shall not be less than 0.90.

[0092] Preparation of the formulation in Experimental Example 2

[0093] Preparation Example 1: Traditional Chinese Medicine Compound Granules

[0094] Formula: Magnolia officinalis 50 parts, Areca catechu 30 parts, Amomum villosum 50 parts, Ephedra sinica 30 parts, Prunus armeniaca 30 parts, Notopterygium incisum 50 parts, Zingiber officinale 50 parts, Pogostemon cablin 50 parts, Eupatorium fortunei 30 parts, Atractylodes lancea 50 parts, Poria cocos 150 parts, Atractylodes macrocephala 100 parts, Gypsum 50 parts, Crataegus pinnatifida 30 parts, Massa fermentata 50 parts, Hordeum vulgare 30 parts, Pheretima aspergillum 50 parts, Cynanchum paniculatum 50 parts, Dryopteris crassirhizoma 30 parts, Lepidium apetalum 50 parts.

[0095] Preparation method of the granules of this traditional Chinese medicine composition:

[0096] Take the above-mentioned medicinal materials, add water and reflux extract twice. The first time, add 6 times the amount of water and extract for 1.5 hours. The second time, add 4 times the amount of water and extract for 1.0 hour. Combine the extracts, filter, concentrate the filtrate to a relative density of 1.10-1.15, centrifuge and filter, vacuum dry the filtrate, spray dry and pulverize to obtain an intermediate composition. Add sucralose and dextrin, mix well, and obtain granules.

[0097] Preparation Example 2: Traditional Chinese Medicine Compound Capsules

[0098] Formula: Magnolia officinalis 50 parts, Areca catechu 30 parts, Amomum villosum 50 parts, Ephedra sinica 30 parts, Prunus armeniaca 30 parts, Notopterygium incisum 50 parts, Zingiber officinale 50 parts, Pogostemon cablin 50 parts, Eupatorium fortunei 30 parts, Atractylodes lancea 50 parts, Poria cocos 150 parts, Atractylodes macrocephala 100 parts, Gypsum 50 parts, Crataegus pinnatifida 30 parts, Massa fermentata 50 parts, Hordeum vulgare 30 parts, Pheretima aspergillum 50 parts, Cynanchum paniculatum 50 parts, Dryopteris crassirhizoma 30 parts, Lepidium apetalum 50 parts.

[0099] Preparation method of the capsule of this traditional Chinese medicine composition:

[0100] The medicinal materials were extracted twice by reflux with water. The first extraction was carried out with 6 times the amount of water for 1.5 hours, and the second extraction was carried out with 4 times the amount of water for 1 hour. The extracts were combined, filtered, and the filtrate was concentrated to a relative density of 1.10-1.15. The filtrate was then centrifuged, vacuum dried, spray dried, granulated, and made into capsules.

[0101] Preparation Example 3: Traditional Chinese Medicine Compound Tablets

[0102] Formula: Magnolia officinalis 50 parts, Areca catechu 30 parts, Amomum villosum 50 parts, Ephedra sinica 30 parts, Prunus armeniaca 30 parts, Notopterygium incisum 50 parts, Zingiber officinale 50 parts, Pogostemon cablin 50 parts, Eupatorium fortunei 30 parts, Atractylodes lancea 50 parts, Poria cocos 150 parts, Atractylodes macrocephala 100 parts, Gypsum 50 parts, Crataegus pinnatifida 30 parts, Massa fermentata 50 parts, Hordeum vulgare 30 parts, Pheretima aspergillum 50 parts, Cynanchum paniculatum 50 parts, Dryopteris crassirhizoma 30 parts, Lepidium apetalum 50 parts.

[0103] Preparation method of this traditional Chinese medicine composition tablet:

[0104] The medicinal materials were extracted twice by reflux with water. The first extraction was carried out with 6 times the amount of water for 1.5 hours, and the second extraction was carried out with 4 times the amount of water for 1 hour. The extracts were combined, filtered, and the filtrate was concentrated to a relative density of 1.10-1.15. The filtrate was centrifuged, dried under vacuum, and spray-dried to obtain an extract powder. Microcrystalline cellulose and other excipients were added to make tablets.

[0105] Experimental Example 3: Therapeutic effect of the composition particles of the present invention on influenza virus H1N1 / FM1 strain infection. 1. Experimental materials

[0106] 1.1 Test drug preparation Example 1: Particles provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0107] 1.2 Positive control drug

[0108] 1.2.1 Oseltamivir Phosphate Granules (Kewei): Produced by Yichang Dongyangguang Changjiang Pharmaceutical Co., Ltd. Ingredients: Each sachet of granules contains 15 mg of oseltamivir phosphate.

[0109] 1.2.2 Ribavirin Granules: Produced by Sichuan Baili Pharmaceutical Co., Ltd. Ingredients: The main ingredient of this product is ribavirin. Specification: 50mg.

[0110] 1.3 Reagents

[0111]

[0112] 1.4 Instruments

[0113]

[0114]

[0115] 1.5 Experimental Animals

[0116]

[0117] 1.6 virus strain

[0118] The H1N1 influenza virus FM1 strain was purchased from the American Center for Standard Biological Collections (ATCC). It was routinely passaged in our ABSL-2 laboratory and stored at -80℃ for future use.

[0119] 2 Dosage design and drug preparation

[0120] 2.1 Preparation Example 1:

[0121] Drug preparation: Before the experiment, take the granules and add distilled water to make up the volume. The dosage is 26.4g crude drug / kg / day. Administer the drug by gavage at a dose of 0.2mL / 10g body weight / time, once a day for 4 consecutive days.

[0122] 2.2 Oseltamivir phosphate: The dosage for mice in the experiment was 27.5 mg / kg / day, which is equivalent to the same clinical dose in humans. The medication was administered by gavage at a dose of 0.2 mL / 10 g body weight once a day for 4 consecutive days.

[0123] 2.3 Ribavirin: The dosage for mice in the experiment was 82.5 mg / kg / day. The administration was 0.2 mL / 10 g body weight / time by gavage, once a day for 4 consecutive days.

[0124] 2.4 Composition Group 1: Preparation Example 1 13.2g crude drug / kg / d + oseltamivir phosphate 13.7mg / kg / d, administration method as above.

[0125] 2.5 Composition Group 2: namely, Preparation Example 1 13.2g crude drug / kg / d + ribavirin 41.2mg / kg / d, administered in the same manner as above.

[0126] 3. Test Methods

[0127] Seventy ICR mice, weighing 14±1g, with half males and half females, were randomly divided into 7 groups according to weight class: normal control group, model control group, oseltamivir phosphate group, ribavirin group, combination group, oseltamivir phosphate group + combination group, and ribavirin group + combination group. Except for the normal control group, the mice were lightly anesthetized with isoflurane at 15 LD50. 50 Influenza virus fluid (H1N1 / FM1 strain) was administered via nasal drops to each mouse, 35 μL per mouse. Administration began on the day of infection, with 0.2 mL / 10 g administered via gavage once daily for 4 consecutive days. The normal control and model control groups were administered distilled water via gavage under the same conditions. On day 5, the mice in each group were weighed; lung weight was measured upon dissection, and lung tissue was collected for HE pathological examination. The lung index and lung index inhibition rate were calculated.

[0128] Lung Index (%) = Lung wet weight (g) / Body weight (g) × 100

[0129]

[0130] Microscopic standards:

[0131] "-": No exudation or congestion was observed in the interstitial lungs of the mice. No enlargement of interstitial cells was observed. No inflammation was observed around the bronchioles in the lungs. The tissue structure was normal.

[0132] "+": No obvious exudative inflammation was observed in the lung tissue and alveolar interstitium of mice, and no large-area congestion was observed. There was a small amount of localized inflammation around the bronchioles in the lungs, mainly lymphatic.

[0133] "++": No large-scale inflammation and mucus exudation were observed in the alveolar interstitium of mouse lung tissue, but there were localized small patches of mucus exudation. There was localized inflammation around the bronchioles of the lungs, mainly lymphatic, and localized increase of endothelial cells (segmental).

[0134] "+++": The mouse lung tissue showed extensive inflammation and mucus exudation in the alveolar interstitium. The exudative inflammation was mainly composed of lymphocytes, with a small number of segmented nuclei and eosinophils. The cells were of uneven size, clustered together, and contained a large amount of pink mucus. Perivascular inflammation was significant, with localized inflammation around the bronchioles in the lungs. There was proliferation of endovascular cells, and the perivascular inflammation was relatively severe.

[0135] Statistical methods: The results were statistically analyzed using t-tests for intergroup comparisons.

[0136] 4. Effect of the particles of the composition of the present invention on the lung index and the lung index inhibition rate

[0137] The results (Table 1) showed that after mice were infected with the H1N1 influenza virus FM1 strain, the lung index of the model control group mice was significantly increased, which was significantly different from that of the normal control group (P<0.01). After 4 days of treatment with the combination granules starting from the day of infection, the lung index of the oseltamivir phosphate group, the combination group, the oseltamivir phosphate + combination group, and the ribavirin + combination group was significantly reduced, which was significantly different from that of the model control group. This indicates that the combination has a significant synergistic effect with antiviral drugs used for upper respiratory tract infections in the fight against influenza virus.

[0138] Table 1. Effects of the composite particles on the lung index of mice infected with influenza virus H1N1 / FM1 strain.

[0139]

[0140] Note: Compared with the normal control group ## P<0.01; compared with the model control group, ** P<0.01, *P<0.05.

[0141] Experimental Example 4: Therapeutic effect of the particles of the composition of the present invention on respiratory syncytial virus infection

[0142] 1. Experimental Materials

[0143] 1.1 Preparation of test drug Example 1: Granules, provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0144] 1.2 Positive control drug

[0145] 1.2.1 Oseltamivir Phosphate Granules (Kewei): Produced by Yichang Dongyangguang Changjiang Pharmaceutical Co., Ltd. Ingredients: Each sachet of granules contains 15 mg of oseltamivir phosphate.

[0146] 1.2.2 Ribavirin Granules: Produced by Sichuan Baili Pharmaceutical Co., Ltd. Ingredients: The main ingredient of this product is ribavirin. Specification: 50mg.

[0147] 1.3 Reagents

[0148] Reagent Name batch number Manufacturer Reagent Uses Isoflurane S200106 Shanghai Yuyan Scientific Instruments Co., Ltd. Mouse anesthesia IL-4 239645-001 Thermo Fisher Lung tissue factor detection IL-33 230852-006 Thermo Fisher Lung tissue factor detection

[0149] 1.4 Instruments

[0150]

[0151] 1.5 Experimental Animals

[0152]

[0153] 1.6 virus strain

[0154] The respiratory syncytial virus (RSV) strain was purchased from the American Center for Standard Biological Collections (ATCC). It was routinely passaged in our ABSL-2 laboratory and stored at -80°C for future use.

[0155] 2 Dosage design and drug preparation

[0156] 2.1 Preparation Example 1:

[0157] Preparation of the drug solution: Take the granules, add distilled water, and make up to 40 mL. The dosage is 26.4 g crude drug / kg / day. When administering the drug, administer 0.2 mL / 10 g body weight / time by gavage, once a day, for 4 consecutive days.

[0158] 2.2 Oseltamivir phosphate granules: The dosage for mice in the experiment was 27.5 mg / kg / d, which is equivalent to the same clinical dose for humans. The medication was administered by gavage at a dose of 0.2 mL / 10 g body weight once a day for 4 consecutive days.

[0159] 2.3 Ribavirin Granules: The dosage for mice in the experiment was 82.5 mg / kg / day. The administration was 0.2 mL / 10 g body weight / time by gavage, once a day for 4 consecutive days.

[0160] 2.4 Composition Group 1: namely, 13.2g crude drug / kg / d of granules from Preparation Example 1 + 13.7mg / kg / d of oseltamivir phosphate, administered in the same manner as above.

[0161] 2.5 Composition Group 2: namely, 13.2g crude drug / kg / d of granules from Preparation Example 1 + 41.2mg / kg / d of ribavirin, administered in the same manner as above.

[0162] 3. Test Methods

[0163] Seventy BALB / c mice, weighing 14±1g, with half males and half females, were randomly divided into seven groups according to weight class: normal control group, model control group, oseltamivir phosphate group, ribavirin group, combination group, oseltamivir phosphate group + combination group, and ribavirin group + combination group, with 10 mice in each group. Except for the normal control group, the mice were lightly anesthetized with isoflurane and injected with 100 TCID45. 50 RSV virus fluid was administered via nasal instillation to each mouse, 45 μL per mouse. Administration began on the day of infection, with 0.2 mL / 10 g administered via gavage once daily for 4 consecutive days. The normal control and model control groups were administered distilled water via gavage under the same conditions. On day 5, the mice in each group were weighed; lung weight was measured upon dissection, and lung tissue was collected for inflammatory factor detection, lung index, and lung index inhibition rate were calculated.

[0164] Lung Index (%) = Lung wet weight (g) / Body weight (g) × 100

[0165]

[0166] 4 Experimental Results

[0167] 4.1 Effect of the composition particles on the lung index and lung index inhibition rate

[0168] The results (Table 2) showed that after mice were infected with respiratory syncytial virus (RSV) via nasal drops, the lung index of the model group mice was significantly increased, showing a significant difference compared with the normal control group (P<0.01). After 4 days of treatment with the combined granules starting on the day of infection, the lung index of the oseltamivir phosphate group, the combined granules group, the oseltamivir phosphate + combined granules group, and the ribavirin + combined granules group was significantly reduced, showing a significant difference compared with the model control group. This indicates that the combined granules have a certain synergistic effect with antiviral drugs used for upper respiratory tract infections in the fight against respiratory syncytial virus.

[0169] Table 2. Effects of the composite particles on the lung index of mice infected with respiratory syncytial virus.

[0170]

[0171] Note: Compared with the normal control group ## P<0.01; compared with the model control group, *P<0.05.

[0172] 4.2 Effects on the cytokine IL-33 in lung tissue

[0173] Lung tissue was removed from a -80°C freezer, treated with high-efficiency protein lysis buffer (with added PMSF and protease inhibitors), and centrifuged to collect the protein supernatant. The protein samples were placed in an ice box and sent to Leeds Biotechnology Co., Ltd. for high-throughput liquid chromatography-protein microarray analysis to detect the levels of various cytokines in mouse lung tissue.

[0174] After mice were infected with respiratory syncytial virus (RSV) via nasal drops, the level of the cytokine IL-33 in the lung tissue of the model group mice was significantly increased, showing a significant difference compared with the normal control group (P<0.01). After 4 days of treatment with the combined granules starting on the day of infection, the IL-33 levels in the oseltamivir phosphate group, the combined granules group, the oseltamivir phosphate + combined granules group, and the ribavirin + combined granules group were significantly decreased, showing significant differences compared with the model group (P<0.01, P<0.05). See Table 3.

[0175] Table 3. Effects of the composite particles on cytokines in lung tissue of mice infected with respiratory syncytial virus.

[0176]

[0177] Note: Compared with the normal control group ## P<0.01, # P<0.05; compared with the cold-dampness epidemic pneumonia model group, **P<0.01.

[0178] In this study, mice were administered the combined granules at a dose of 26.4 g crude drug / kg / d (1 / 2 times) via gavage once daily for 3–4 consecutive days. Influenza virus H1N1 / FM1 infection model and respiratory syncytial virus infection model were used to evaluate the efficacy of the combined granules in treating viral upper respiratory tract infections. The results showed that the combination of the combined granules with antiviral drugs could reduce viral load and inflammatory cytokines in lung tissue, indicating a therapeutic effect on this common type of specific viral infection. This study utilized the advantages of traditional Chinese medicine to further promote and consolidate the antiviral effects of other drugs, providing a basis for further clinical research.

[0179] Experimental Example 5: Fingerprint Analysis of the Composition of the Invention

[0180] Instruments and reagents

[0181] Instruments: Agilent 1260 HPLC system with DAD UV detector; Agilent 1260 HPLC system with VWD UV detector; Agilent 1290 UHPLC system with DAD UV detector, Agilent 6538Q-TOF-MS mass spectrometer with electrospray ionization (ESI) source (Agilent Technologies, Inc.); Thermo Fisher Ultimate 3000 HPLC system;

[0182] Mettler Toledo XP6 electronic analytical balance, Mettler Toledo; Mettler Toledo AL204 electronic analytical balance, Mettler Toledo; KQ500DB CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; HH digital display constant temperature water bath, Changzhou Guoyu Instrument Manufacturing Co., Ltd.; Milli-Q ultrapure water system, Millipore Corporation, USA;

[0183] Reference standards: Adenine, 99.4%, batch number B0002908, Beijing Manhag Biotechnology Co., Ltd.; Uridine, 99.4%, batch number B0008580, Beijing Manhag Biotechnology Co., Ltd.; Guanosine, 96.1%, batch number B0006767, Beijing Manhag Biotechnology Co., Ltd.; Inosine, 99.2%, batch number 140669-202007, China National Institutes for Food and Drug Control; 5-Hydroxymethylfurfural, 95.0%, batch number 8610, Shanghai Shidander Standard Technology Co., Ltd.; Magnolia officinalis A, 99.1%. Batch No. 7611, Shanghai Shidander Standard Technology Co., Ltd.; Bergamot Glucoside, 98%, Batch No. CFS202002, Wuhan Tianzhi Biotechnology Co., Ltd.; Imperatorin, 99.6%, Batch No. 111821-201604, China National Institutes for Food and Drug Control; 6'-O-(trans-feruloyl)-Imperatorin, 98%, Batch No. CFS202002, Wuhan Tianzhi Biotechnology Co., Ltd.; Anisolic Acid p-hydroxyphenylethyl ester, 99.6%, Batch No. A05GB156933, Shanghai Yuanye Biotechnology Co., Ltd.

[0184] Reagents: Methanol (Merck, Inc., Meridian, chromatographic grade); acetic acid (L07203503, Merck Fessil Technologies, Inc.); other reagents were of analytical grade.

[0185] Samples: Granules prepared by the method in Preparation Example 1, batch numbers: 200201, 200202, 200203, 200204, 200205, 200206, 200207, 200208, 200209, 200210, 200211, 200212, 200213, 200214, 200601, 200602, and 200603, of which batch 200602 was a methodological research batch. All samples were provided by the Traditional Chinese Medicine R&D Department of Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0186] 1. Selection of chromatographic conditions

[0187] Take an appropriate amount of this product, grind it into a fine powder, weigh about 2g accurately, place it in a stoppered conical flask, add 50ml of water accurately, sonicate for 30 minutes, shake well, centrifuge, and take the supernatant to obtain the test solution.

[0188] 1.1 Selection of detection wavelength

[0189] The test solution was taken and scanned at full wavelength using a DAD. The results showed that the chromatographic peak information was richer, the response was more uniform, and the baseline was more stable at 250 nm. Therefore, 250 nm was selected as the detection wavelength for the characteristic spectrum. (See attached image) Figure 4 , Figure 5 .

[0190] 1.2 Selection of mobile phase

[0191] Based on the characteristics of the water extraction process for this product, WatersAtlantis T3 (4.6×250mm, 5μm) C, which has a good separation effect on water-soluble components, was used. 18 The chromatographic column used a methanol-0.1% phosphoric acid mobile phase system with a detection wavelength of 250 nm. The separation effects of different mobile phase gradients were investigated. The results showed that gradient program IV exhibited better peak resolution, appropriate retention times, and a more stable baseline. Therefore, gradient program IV was selected as the preferred mobile phase elution program. The gradient elution program is as follows, and the results are attached. Figure 6 .

[0192]

[0193]

[0194] Based on the above considerations, the proposed chromatographic conditions are as follows: Octadecylsilane-bonded silica gel as the packing material (Waters Atlantis T3, column length 25 cm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, and 0.1% phosphoric acid as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 250 nm. The theoretical plate number, calculated based on the purslane peak, should not be less than 10,000.

[0195]

[0196] 2. Selection of test sample preparation method

[0197] To optimize the preparation method of the test sample solution, the extraction solvent and extraction method were investigated.

[0198] Preparation of test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 1g, make 4 portions, weigh them accurately, place them in stoppered conical flasks, add water and 25ml of 50% methanol accurately to each, sonicate for 30 minutes and reflux for 30 minutes respectively, shake well, centrifuge, and take the supernatant to obtain 4 portions of test solution.

[0199] Results: Each test solution was analyzed under the chromatographic conditions described above. The results showed that the chromatogram obtained using water as the extraction solvent exhibited higher peak responses (more polar peaks before 25 minutes), with a richer overall peak count and more uniform response. Considering the characteristics of the water extraction process, water was the preferred extraction solvent. There was no significant difference in peak richness and response between reflux and ultrasonic extraction methods. For ease of operation, ultrasonic extraction was the preferred method. (See attached image) Figure 7 .

[0200] Based on the above investigation, the proposed method for processing the test sample is as follows: Take an appropriate amount of this product, grind it into a fine powder, take about 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of water, sonicate (power 500W, frequency 40kHz) for 30 minutes, shake well, centrifuge, and take the supernatant to obtain the sample.

[0201] 3 Durability Test

[0202] The effects of different wavelengths (245 nm, 250 nm, 255 nm), flow rates (0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min, 1.2 ml / min), column temperatures (25℃, 28℃, 30℃, 32℃, 35℃), different column batches {Column 1: Waters Atlantis T3 (4.6 × 250 mm, 5 μm), serial number 01813017014031; Column 2: Waters Atlantis T3 (4.6 × 250 mm, 5 μm), serial number 01813017014008}, and instruments (Instrument 1: Agilent 1260, Instrument 2: Ultimate 3000) on the separation of 10 selected characteristic peaks were investigated.

[0203] The results showed that within a wavelength range of 250±5 nm, a flow rate of 0.8 ml / min to 1.2 ml / min, and a column temperature range of 25℃ to 35℃, and using different batches of chromatographic columns and different brands of instruments, the selected characteristic peaks could be separated well, indicating that the method has good robustness. (See attached image) Figure 8 .

[0204] 4. Methodological Validation

[0205] Precision test: The test solution was prepared according to the above method. 10 μl of the same test solution was accurately pipetted and injected six times consecutively. The relative retention times of the 10 characteristic peaks were calculated, using imperatorin (peak 8) as the reference peak. The results showed that the RSD values ​​of the relative retention times of the 10 characteristic peaks were all less than 0.5%, indicating good instrument precision. See Table 4 and Appendix. Figure 9 .

[0206] Repeatability test: Six parallel test solutions were prepared according to the above method and measured. Using imperatorin (peak 8) as the reference peak, the relative retention times of the 10 calibrated characteristic peaks were calculated. The results showed that the RSD values ​​of the relative retention times of the 10 characteristic peaks were all less than 0.5%, indicating good repeatability of the method. See Table 5. Figure 10 .

[0207] Stability study: The same test solution was injected at 0h, 3h, 6h, 9h, 12h, 18h, 24h, 30h, and 36h, with 10μl injected each time. The relative retention times of the 10 characteristic peaks were calculated, using purslane (peak 8) as the reference peak. The results showed that the RSD values ​​of the relative retention times of the 10 characteristic peaks were all less than 0.5%, indicating that the test solution had good stability within 36 hours. See Table 6. Figure 11 .

[0208] Table 4. Calculation results of precision evaluation of feature maps

[0209]

[0210] Table 5. Results of Repeatability Tests

[0211]

[0212] Table 6. Stability assessment calculation results

[0213]

[0214] 5. Generation of multiple batches of determination and control characteristic spectra

[0215] Following the above method, 14 batches of clinical practice samples, 3 batches of pilot-scale samples, and 3 batches of process validation samples were analyzed. Ten peaks were selected as characteristic peaks. A control fingerprint spectrum was generated using 20 batches of formulations. The corresponding peak of the imperatorin reference peak was used as the S peak. The relative retention times of each characteristic peak were calculated. Each peak should be within ±10% of the specified value. The specified values ​​are: 0.11 (peak 1), 0.27 (peak 2), 0.36 (peak 3), 0.38 (peak 4), 0.41 (peak 5), 0.84 (peak 6), 0.92 (peak 7), 1.00 (peak 8), 1.19 (peak 9), and 1.29 (peak 10). Results for multiple batches are attached. Figure 12 Table 7, and the corresponding characteristic maps are attached. Figure 13 .

[0216] Table 7. Relative retention times of 20 batches of formulations

[0217]

[0218]

[0219] 6. Feature Map Quality Standards

[0220] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase (Waters Atlantis T3, column length 25 cm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.1% phosphoric acid as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 250 nm. The theoretical plate number, calculated based on the purslane peak, should not be less than 10,000.

[0221]

[0222] Preparation of the reference solution: Take an appropriate amount of imperatorin reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 0.15 mg per ml.

[0223] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, weigh about 1g accurately, place it in a stoppered conical flask, accurately add 25ml of water, sonicate (power 500W, frequency 40kHz) for 30 minutes, shake well, centrifuge, and take the supernatant to obtain the test solution.

[0224] For the assay, accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, measure and record the chromatograms to obtain the results.

[0225] The characteristic chromatogram of the test sample should show 10 characteristic peaks. The peak corresponding to the reference peak is designated as the S peak. The relative retention time of each characteristic peak should be calculated and should be within ±10% of the specified value. The specified relative retention times are: 0.11 (peak 1), 0.27 (peak 2), 0.36 (peak 3), 0.38 (peak 4), 0.41 (peak 5), 0.84 (peak 6), 0.92 (peak 7), 1.00 [peak 8 (S)], 1.19 (peak 9), and 1.29 (peak 10). See the attached characteristic chromatogram for reference. Figure 14 .

[0226] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a traditional Chinese medicine extract in the preparation of a medicament for treating respiratory syncytial virus infection, characterized in that, The traditional Chinese medicine extract is made of the following traditional Chinese medicines by weight ratio: Magnolia officinalis 50 parts, Strychnos ignatii 30 parts, Toddalia asiatica 50 parts, Ephedra sinica 30 parts, Bitter almond 30 parts, Notopterygium 50 parts, Zingiber officinale 50 parts, Pogostemonis 50 parts, Pterocephalus 30 parts, Atractylodes 50 parts, Poria cocos 150 parts, Atractylodes 100 parts, Gypsum fibrosum 50 parts, Fructus forsythiae 30 parts, Fructus forsythiae 50 parts, Fructus forsythiae 30 parts, Earthworm 50 parts, Radix valerianae 50 parts, Lepidogrammitis 30 parts, Semen Lepidii 50 parts; wherein the ephedrine content of the extract is 0.5-1.5 mg / g, the solid content transfer rate is 18-25%, and the decursin content is 2-8 mg / g.

2. Use of a traditional Chinese medicine extract in the preparation of a medicament for treating respiratory syncytial virus infection, characterized in that, The preparation of the traditional Chinese medicine extract comprises: The Magnolia officinalis, Strychnos ignatii, Toddalia asiatica, Ephedra sinica, Bitter almond, Notopterygium, Zingiber officinale, Pogostemonis, Pterocephalus, Atractylodes, Poria cocos, Atractylodes, Gypsum fibrosum, Fructus forsythiae, Fructus forsythiae, Fructus forsythiae, Earthworm, Radix valerianae, Lepidogrammitis and Semen Lepidii are weighed and extracted with water twice, 6 times the amount of water is added in the first extraction and 4 times the amount of water is added in the second extraction, the extraction time is 1.5 h and 1.0 h respectively, the extraction liquid is combined, filtered, concentrated to a relative density of 1.10-1.15, centrifuged, and the filtrate is dried; wherein the ephedrine content of the extract is 1.20-1.50 mg / g, the solid content transfer rate is 19-22%, and the decursin content is 6-8 mg / g; wherein in the preparation of the traditional Chinese medicine extract, the weighed raw materials are as follows: The Magnolia officinalis, Strychnos ignatii, Toddalia asiatica, Ephedra sinica, Bitter almond, Notopterygium, Zingiber officinale, Pogostemonis, Pterocephalus, Atractylodes, Poria cocos, Atractylodes, Gypsum fibrosum, Fructus forsythiae, Fructus forsythiae, Fructus forsythiae, Earthworm, Radix valerianae, Lepidogrammitis and Semen Lepidii are weighed and extracted with water twice, 6 times the amount of water is added in the first extraction and 4 times the amount of water is added in the second extraction, the extraction time is 1.5 h and 1.0 h respectively, the extraction liquid is combined, filtered, concentrated to a relative density of 1.10-1.15, centrifuged, and the filtrate is dried; wherein the ephedrine content of the extract is 1.20-1.50 mg / g, the solid content transfer rate is 19-22%, and the decursin content is 6-8 mg / g; wherein in the preparation of the traditional Chinese medicine extract, the weighed raw materials are as follows:

3. Use of a traditional Chinese medicine extract in the preparation of a medicament for treating respiratory syncytial virus infection, characterized in that, The Magnolia officinalis, Strychnos ignatii, Toddalia asiatica, Ephedra sinica, Bitter almond, Notopterygium, Zingiber officinale, Pogostemonis, Pterocephalus, Atractylodes, Poria cocos, Atractylodes, Gypsum fibrosum, Fructus forsythiae, Fructus forsythiae, Fructus forsythiae, Earthworm, Radix valerianae, Lepidogrammitis and Semen Lepidii are weighed and extracted with water twice, 6 times the amount of water is added in the first extraction and 4 times the amount of water is added in the second extraction, the extraction time is 1.5 h and 1.0 h respectively, the extraction liquid is combined, filtered, concentrated to a relative density of 1.10-1.15, centrifuged, and the filtrate is dried; wherein the ephedrine content of the extract is 1.20-1.50 mg / g, the solid content transfer rate is 19-22%, and the decursin content is 6-8 mg / g; wherein in the preparation of the traditional Chinese medicine extract, the weighed raw materials are as follows: The Magnolia officinalis, Strychnos ignatii, Toddalia asiatica, Ephedra sinica, Bitter almond, Notopterygium, Zingiber officinale, Pogostemonis, Pterocephalus, Atractylodes, Poria cocos, Atractylodes, Gypsum fibrosum, Fructus forsythiae, Fructus forsythiae, Fructus forsythiae, Earthworm, Radix valerianae, Lepidogrammitis and Semen Lepidii are weighed and extracted with water twice, 6 times the amount of water is added in the first extraction and 4 times the amount of water is added in the second extraction, the extraction time is 1.5 h and 1.0 h respectively, the extraction liquid is combined, filtered, concentrated to a relative density of 1.10-1.15, centrifuged, and the filtrate is dried; wherein the ephedrine content of the extract is 1.20-1.50 mg / g, the solid content transfer rate is 19-22%, and the decursin content is 6-8 mg / g; wherein in the preparation of the traditional Chinese medicine extract, the weighed raw materials are as follows: ​

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