Traditional Chinese medicine extract, medicine and detection method thereof

Abstract

The invention discloses a traditional Chinese medicine extract. The traditional Chinese medicine extract is prepared from the following traditional Chinese medicines in parts by weight: 1-100 parts of mangnolia officinalis, 1-100 parts of scorched betel nut, 1-100 parts of roasted tsaoko amomum fruit, 1-100 parts of ephedra, 1-100 parts of bitter apricot kernel, 1-100 parts of notopterygium root, 1-100 parts of ginger, 1-100 parts of pogostemon cablin, 1-100 parts of eupatorium, 1-100 parts of rhizoma atractylodis, 1-100 parts of poria cocos, 1-100 parts of bighead atractylodes rhizome, 1-100 parts of gypsum, 1-100 parts of scorched hawthorn fruit, 1-100 parts of scorched medicated leaven, 1-100 parts of scorched malt, 1-100 parts of earthworm, 1-100 parts of paniculate swallowwort root, 1-100 parts of rhizoma The ephedrine content of the extract is 0.5-1.5 mg / g, and the solid content transfer rate is 18-25%.

Description

Traditional Chinese medicine extract, medicine and detection method thereof Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine extract, a medicine and a detection method thereof. Background Art

[0002] Viral pneumonia is caused by a viral infection of the upper respiratory tract that spreads downward, triggering lung inflammation and impaired pulmonary ventilation. While it can occur year-round, it is most common in winter and spring, with outbreaks occurring in both epidemics and sporadic episodes. Clinical manifestations include fever, headache, body aches, dry cough, and pulmonary infiltrates. While influenza virus is the most common cause of pneumonia, other pathogens, including respiratory syncytial virus, adenovirus, and parainfluenza virus, have also garnered significant attention.

[0003] Viral infection primarily manifests as interstitial lung lesions. Initially, ciliated columnar epithelial cells are affected, followed by invasion of 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 undergo degeneration, including granular degeneration, vacuolation, cell swelling, and nuclear condensation, followed by necrosis and disintegration. Cell fragments accumulate in the airways and obstruct small airways, and airway swelling occurs. There is a pronounced inflammatory reaction in the alveolar septa, accompanied by infiltration of lymphocytes and macrophages, and occasionally plasma cell and neutrophil infiltration and edema. Fibrin thrombi with necrosis and hemorrhage may occur in the alveolar capillaries, and eosinophilic hyaline membranes may be seen in the alveoli. Severely infected patients may experience pulmonary edema, consolidation, hemorrhage, necrosis of the lung parenchyma, and atelectasis.

[0004] Influenza virus and respiratory syncytial virus are the most common viruses causing upper respiratory tract infections. Influenza viruses belong to the Orthomyxoviridae family and are RNA viruses. They are divided into three types: A, B, and C, based on the antigenicity of their nucleoprotein and matrix proteins. Influenza A virus mutates rapidly and is highly virulent. H1N1 / FM1 is a subtype A strain of influenza virus adapted to the lungs of mice, which mimics influenza A virus in producing lung infection damage. Influenza A virus initiates infection by binding to sialic acid receptors on the surface of respiratory epithelial cells via hemagglutinin. After entry, the viral genome begins transcription and replication. Large numbers of replicated progeny virus particles spread across the respiratory mucosa and begin infecting other cells, triggering a cytokine storm and leading to systemic inflammation, ultimately leading to acute respiratory distress syndrome, shock, and multiple organ failure. Respiratory syncytial virus belongs to the Paramyxoviridae family and is an RNA virus. Pathogenesis involves interactions between the virus and affected host cells, including damage, inflammation, humoral and local immune responses, and hyperresponsiveness.

[0005] Various data and clinical use have confirmed that traditional Chinese medicine (TCM) possesses significant antiviral efficacy, a broad antiviral spectrum, and minimal toxic side effects. Furthermore, many TCMs possess antipyretic and anti-inflammatory properties, while simultaneously exerting multiple effects on viral infections, such as shortening the duration of fever, controlling the spread of inflammation, and promoting its absorption. This multi-faceted, multi-faceted approach is crucial. This diverse array of TCM active ingredients makes it difficult for viruses to develop resistance, giving TCM a distinct advantage in treating viral infections and promising prospects for clinical application.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to make up for the deficiencies of the prior art and provide a traditional Chinese medicine extract with antiviral effect, characterized in that, by weight, the traditional Chinese medicine extract is made of traditional Chinese medicines including the following raw materials: 1-100 parts of Magnolia officinalis, 1-100 parts of scorched Areca catechu, 1-100 parts of stewed Amomum villosum, 1-100 parts of Ephedra, 1-100 parts of Bitter Apricot, 1-100 parts of Notopterygium wilfordii, 1-100 parts of Ginger, 1-100 parts of Patchouli, 1-100 parts of Cyperus rotundus, 1-100 parts of Atractylodes macrocephala, 1-100 parts of Poria cocos, 1-100 parts of Atractylodes macrocephala, 1-100 parts of Gypsum, 1-100 parts of scorched Crataegus pinnatifida, 1-100 parts of scorched Liu Shen Qu, 1-100 parts of scorched malt, 1-100 parts of Earthworm, 1-100 parts of Cynanchum wilfordii, 1-100 parts of Cyperus rotundus, and 1-100 parts of Trichosanthes kirilowii.

[0008] Furthermore, the Chinese medicine extract is made from Chinese medicine including the following raw materials: 1-80 parts of Magnolia officinalis, 1-80 parts of scorched Areca catechu, 1-80 parts of stewed Amomum villosum, 1-60 parts of Ephedra, 1-60 parts of Bitter Apricot, 1-80 parts of Notopterygium wilfordii, 1-60 parts of Ginger, 1-80 parts of Patchouli, 1-60 parts of Cymbopogon citratus, 1-80 parts of Atractylodes macrocephala, 1-160 parts of Poria cocos, 1-120 parts of Atractylodes macrocephala, 1-80 parts of Gypsum, 1-50 parts of scorched Crataegus pinnatifida, 1-80 parts of scorched Liu Shen Qu, 1-60 parts of scorched malt, 1-80 parts of Earthworm, 1-80 parts of Cynanchum chinense, 1-60 parts of Cyperus rotundus, and 1-80 parts of Lepidium chinense.

[0009] Furthermore, the Chinese medicine extract is made from Chinese medicine including the following raw materials: 30-50 parts of Magnolia officinalis, 20-30 parts of scorched Areca catechu, 30-50 parts of stewed Amomum villosum, 20-30 parts of Ephedra, 20-30 parts of Bitter Apricot, 30-50 parts of Notopterygium wilfordii, 30-50 parts of Ginger, 30-50 parts of Patchouli, 20-30 parts of Cyperus rotundus, 30-50 parts of Atractylodes macrocephala, 120-150 parts of Poria cocos, 80-100 parts of Atractylodes macrocephala, 30-50 parts of Gypsum, 20-30 parts of scorched Crataegus pinnatifida, 30-50 parts of scorched Liu Shenqu, 20-30 parts of scorched malt, 30-50 parts of Earthworm, 30-50 parts of Cynanchum chinense, 20-30 parts of Cyperus rotundus, and 30-50 parts of Lepidium chinense.

[0010] Furthermore, the Chinese medicine extract is made from Chinese medicine including the following raw materials: 50 parts of Magnolia officinalis, 30 parts of scorched Areca catechu, 50 parts of stewed Amomum villosum, 30 parts of Ephedra, 30 parts of Bitter Apricot, 50 parts of Notopterygium wilfordii, 50 parts of Ginger, 50 parts of Patchouli, 30 parts of Cyperus rotundus, 50 parts of Atractylodes macrocephala, 150 parts of Poria cocos, 100 parts of Atractylodes macrocephala, 50 parts of Gypsum, 30 parts of scorched Crataegus pinnatifida, 50 parts of scorched Liushenqu, 30 parts of scorched malt, 50 parts of Earthworm, 50 parts of Cynanchum chinense, 30 parts of Cyperus rotundus, and 50 parts of Lepidium chinense.

[0011] Furthermore, the ephedrine content of the aforementioned extract is 0.5-1.5 mg / g, and the solid content transfer rate is 18-25%.

[0012] Furthermore, the purpurogenol content of the aforementioned extract is 2-8 mg / g.

[0013] Furthermore, the preparation of the Chinese herbal extract includes:

[0014] Weigh magnolia bark, scorched betel nut, stewed tsaoko, ephedra, bitter almond, notopterygium wilfordii, ginger, patchouli, perilla, atractylodes, poria, atractylodes, gypsum, scorched hawthorn, scorched Liushenqu, scorched malt, earthworm, cynanchum chinense, cyperus rotundus, and scutellaria baicalensis seed, extract them twice with water, add 6 times the amount of water for the first time and extract for 1.5 hours, add 4 times the amount of water for the second time and extract for 1.0 hours, combine the extracts, filter, concentrate the filtrate to a relative density of 1.10-1.15, centrifuge and filter, and dry the filtrate; wherein the ephedrine content of the extract is 1.20-1.50 mg / g, the solid content transfer rate is 19-22%, and the peucedanum rutaecarpon content is 6-8 mg / g.

[0015] Furthermore, the preparation of the traditional Chinese medicine extract includes: weighing 15g of Magnolia officinalis, 9g of charred Areca catechu, 9g of stewed Amomum villosum, 6g of Ephedra, 9g of Bitter Apricot, 15g of Notopterygium wilfordii, 15g of Ginger, 15g of Patchouli, 9g of Cyperus rotundus, 15g of Atractylodes macrocephala, 45g of Poria cocos, 30g of Atractylodes macrocephala, 15g of Gypsum, 9g of charred Crataegus pinnatifida, 9g of charred Liushenqu, 9g of charred malt, 15g of Earthworm, 15g of Cynanchum chinense, 9g of Cynomorium mongolicum, and 15g of Lepidium tiliaceum; respectively adding water and extracting twice, adding 6 times the amount of water for the first time and extracting for 1.5h, adding 4 times the amount of water for the second time and extracting for 1.0h, combining the extracts, filtering, concentrating the filtrate to a relative density of 1.10 to 1.15, centrifuging and filtering, and drying the filtrate.

[0016] Furthermore, the similarity between the fingerprint of the Magnolia officinalis and the reference fingerprint is not less than 0.90. The reference fingerprint contains common peaks and magnolol and magnolol. The reference fingerprint can be, for example, Figure 1.

[0017] Furthermore, the similarity between the fingerprint of the burnt betel nut and the control fingerprint is not less than 0.90. The control fingerprint contains the common peaks of arecoline and arecoline. For example, the control fingerprint can be shown in Figure 2.

[0018] Furthermore, the similarity between the fingerprint of the simmered tsaoko fruit and the reference fingerprint is not less than 0.90. The reference fingerprint contains the common peak protocatechuic acid. For example, the reference fingerprint can be shown in Figure 3.

[0019] A method for preparing a Chinese herbal medicine extract, characterized by weighing Magnolia officinalis, charred Areca catechu, stewed Amomum villosum, Ephedra sinica, bitter almond, Notopterygium wilfordii, ginger, patchouli, Perilla frutescens, Atractylodes lancea, Poria cocos, Atractylodes macrocephala, gypsum, charred Crataegus pinnatifida, charred Liushenqu, charred malt, earthworm, Cynanchum chinense, Cynanchum sibiricum, and Lepidium tiliaceum fruit, extracting each extract twice with water, the first time with 6 times the amount of water for 1.5 hours, and the second time with 4 times the amount of water for 1.0 hours, combining the extracts, filtering, concentrating the filtrate to a relative density of 1.10-1.15, centrifuging, and drying the filtrate; wherein the extract has an ephedrine content of 1.20-1.50 mg / g, a solids transfer rate of 19-22%, and a peucedanum rutaecarpa glycoside content of 6-8 mg / g. The fingerprints of Magnolia officinalis, charred Areca catechu, or stewed Amomum villosum are as defined above. The present invention also proposes the use of any of the above-mentioned Chinese medicinal extracts in the preparation of a drug for treating influenza virus H1N1 or FM1, or respiratory syncytial virus.

[0020] Furthermore, the aforementioned Chinese medicine extract is made from Chinese medicine including the following raw materials: 50 parts of Magnolia officinalis, 30 parts of scorched Areca catechu, 30 parts of stewed Amomum villosum, 20 parts of Ephedra, 30 parts of bitter almond, 50 parts of Notopterygium wilfordii, 50 parts of ginger, 50 parts of Patchouli, 30 parts of Cyperus rotundus, 50 parts of Atractylodes macrocephala, 150 parts of Poria cocos, 100 parts of Atractylodes macrocephala, 50 parts of Gypsum, 30 parts of scorched Crataegus pinnatifida, 30 parts of scorched Liu Shen Qu, 30 parts of scorched malt, 50 parts of earthworm, 50 parts of Cynanchum wilfordii, 30 parts of Cyperus rotundus, and 50 parts of Lepidium chinense.

[0021] The present invention also provides a medicine, which is prepared from any of the above-mentioned Chinese medicinal extracts and pharmaceutically acceptable excipients or additives.

[0022] Specifically, the medicine is selected from decoction, granules, capsules, tablets, oral liquids, pills, soft capsules, dripping pills, tinctures, syrups, suppositories, gels, sprays, and injections.

[0023] Furthermore, the drug is a granule, and the excipients or additives are preferably dextrin and sucralose.

[0024] Furthermore, the medicine may also include antiviral drugs for upper respiratory tract infection, such as ribavirin and / or oseltamivir phosphate.

[0025] The present invention also proposes a method for detecting the fingerprint of any of the above-mentioned traditional Chinese medicine extracts or medicines, characterized in that a test solution is taken for HPLC detection, and the chromatographic conditions of the HPLC detection include: using a C18 chromatographic column, methanol as mobile phase A, and a 0.1% concentration of phosphoric acid aqueous solution as mobile phase B, and the elution in the chromatographic conditions of the HPLC detection is gradient elution, and the gradient elution program is: 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.

[0026] The test solution is a solution for detection prepared from Chinese herbal medicine extracts or drugs. The preparation method can be, for example, taking 1 g of granules, placing it in a stoppered conical flask, adding 25 ml of water, and ultrasonically treating it for 30 minutes.

[0027] Furthermore, the chromatographic conditions include: the flow rate is 1.0 mL / min, the column temperature is 30° C., and the detection wavelength is 250 nm.

[0028] Furthermore, the fingerprint is shown in FIG14 .

[0029] Specifically, in the fingerprint spectrum, 10 characteristic peaks should be presented in the characteristic spectrum of the test sample, and the peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, which should be within ±10% of the specified value, preferably within 5%. The specified values ​​of the relative retention time 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).

[0030] The present invention prepares a new traditional Chinese medicine composition, improves the quality standard of the composition, and evaluates the effectiveness of the composition particles in treating viral upper respiratory tract infections by adopting influenza virus H1N1 / FM1 strain infection models and respiratory syncytial virus infection models. It is shown that the composition can reduce the 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a comparative fingerprint of Magnolia officinalis; Peak 6: Honokiol Peak 7 (S): Magnolol;

[0032] Figure 2 is a comparative fingerprint of Areca catechu medicinal material; Peak 1: Arecoline Peak 3 (S): Arecoline;

[0033] Figure 3 is the reference fingerprint of stewed tsaoko herb; Peak 1: protocatechuic acid;

[0034] Figure 4 is a full wavelength scan 3D image of the test solution;

[0035] Figure 5 is a wavelength investigation chromatogram of the test solution;

[0036] Figure 6 is a chromatogram of the mobile phase ratio investigation;

[0037] Figure 7 is a chromatogram of the test sample preparation method;

[0038] Figure 8 is a chromatogram of durability investigation;

[0039] Figure 9 is a chromatogram of precision investigation;

[0040] Figure 10 Repeatability test chromatogram;

[0041] Figure 11 is a chromatogram of stability study;

[0042] Figure 12 Overlay of characteristic spectra of 20 batches of preparations;

[0043] Figure 13 compares characteristic patterns;

[0044] Figure 14 shows the comparison of characteristic spectra 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-bergamotol glucoside; 8-purpuroside; 9-6'-O-(trans-feruloyl)-purpuroside; 10-anisic acid 4-hydroxyphenyl ethyl ester. DETAILED DESCRIPTION

[0045] Oseltamivir is an inhibitor of the influenza virus neuraminidase and is primarily 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 is not recommended by the FDA. The present invention provides a traditional Chinese medicine composition, its preparation method, and its use. The following description uses commonly used influenza virus or RSV drugs as examples, along with experimental details.

[0046] It is particularly important to note that similar substitutions and modifications made to the present invention will be obvious to those skilled in the art and are considered to be included in the present invention. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments.

[0047] Unless otherwise specified, the present invention was carried out under conventional conditions or those recommended by the manufacturer. The raw materials or excipients, as well as the reagents or instruments used, for which the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0048] Experimental Example 1 Preparation Process Examination

[0049] 1. Investigation of extraction solvent

[0050] Weigh 15g of Magnolia officinalis, 9g of charred Areca nut, 9g of stewed Tsaoko fruit, 6g of Ephedra, 9g of Bitter Apricot, 15g of Notopterygium wilfordii, 15g of Ginger, 15g of Patchouli, 9g of Perilla frutescens, 15g of Atractylodes lancea, 45g of Poria, 30g of Atractylodes macrocephala, 15g of Gypsum, 9g of charred Crataegus pinnatifida, 9g of charred Liushenqu, 9g of charred Malt, 15g of Earthworm, 15g of Cynanchum indica, 9g of Codonopsis pilosula, and 15g of Lepidoptera: 1 portion, add water and decoct twice, extract for 1.5h for the first time and 1.0h for the second time. Combine the extracts, filter, concentrate the filtrate to a relative density of 1.10-1.15, centrifuge, vacuum dry the filtrate, spray dry and crush to obtain an intermediate composition. The test results of ephedrine transfer rate and solid content transfer rate of different extraction solvent volumes are as follows:

[0051] Calculation formula:

[0052] Among them, the total transfer rate refers to the ratio of the total amount of ephedrine hydrochloride and pseudoephedrine hydrochloride in the extract to the ephedrine hydrochloride and pseudoephedrine hydrochloride in the medicinal material.

[0053] The experimental results show that the transfer rates of ephedrine hydrochloride and pseudoephedrine hydrochloride, as well as the solid content transfer rate, increase with increasing water addition. When the solvent dosage increases from 4.3 to 6.4 times, the transfer rates of each indicator increase significantly. With further increases in water addition, the growth trend of each indicator becomes more gradual, but it actually decreases when the solvent dosage is too high. Taking into account energy efficiency and the transfer rates of the indicator components, the water dosage was determined to be 6 times the initial dosage and 4 times the initial dosage for the second addition.

[0054] 2. Soaking time investigation

[0055] Separately weigh 15g of Magnolia officinalis, 9g of charred Areca nut, 9g of stewed Tsaoko fruit, 6g of Ephedra, 9g of Bitter Apricot, 15g of Notopterygium wilfordii, 15g of Ginger, 15g of Patchouli, 9g of Perilla frutescens, 15g of Atractylodes lancea, 45g of Poria, 30g of Atractylodes macrocephala, 15g of Gypsum, 9g of charred Crataegus pinnatifida, 9g of charred Liushenqu, 9g of charred Malt, 15g of Earthworm, 15g of Cynanchum chinense, 9g of Mianma Guanzhong, and 15g of Lepidium tiliaceum. Add water and decoct twice, adding 6 times the amount of water for the first extraction and extracting for 1.5h, and adding 4 times the amount of water for the second extraction and extracting for 1.0h. The soaking time for the three extractions is 0, 30, and 60 min, respectively. Combine the extracts, filter, and concentrate the filtrate to a relative density of 1.10-1.15, centrifuge, and vacuum dry the filtrate. Spray dry and crush to obtain an intermediate composition.

[0056] The effect of soaking time on the transfer rate of ephedrine hydrochloride and pseudoephedrine hydrochloride and the transfer rate of solid content was investigated. The experimental results are as follows:

[0057] Calculation formula:

[0058] The experimental results show that the soaking time has little effect on the total transfer rate and solid content transfer rate of ephedrine hydrochloride and pseudoephedrine hydrochloride. Therefore, the extraction process is determined to be direct extraction without soaking.

[0059] 3. Extraction time investigation

[0060] Weigh 15g of Magnolia officinalis, 9g of charred Areca nut, 9g of stewed Tsaoko fruit, 6g of Ephedra, 9g of Bitter Apricot, 15g of Notopterygium wilfordii, 15g of Ginger, 15g of Patchouli, 9g of Perilla frutescens, 15g of Atractylodes macrocephala, 45g of Poria, 30g of Atractylodes macrocephala, 15g of Gypsum, 9g of charred Crataegus pinnatifida, 9g of charred Liushenqu, 9g of charred Malt, 15g of Earthworm, 15g of Cynanchum chinense, 9g of Mianma Guanzhong, and 15g of Tinglizi as one portion, for a total of 5 portions. Add water and extract twice, adding 6 times the amount of water for the first time and 4 times the amount of water for the second time. The first extraction time is 60min, 75min, 90min, 105min, and 120min, respectively, and the second extraction time is 30min, 45min, 60min, 75min, and 90min, respectively. The effect of extraction time on the transfer rate of ephedrine hydrochloride and pseudoephedrine hydrochloride and the solid content transfer rate is investigated. The experimental results are as follows:

[0061] Calculation formula:

[0062] The experimental results showed that the transfer rate of ephedrine hydrochloride and pseudoephedrine hydrochloride, as well as the solid content transfer rate, increased with extraction time. When the extraction time increased from 60, 30 minutes to 90, 60 minutes, the growth trend of each indicator was more obvious. As the extraction time continued to increase, the growth trend of each indicator gradually slowed down. Therefore, the extraction time was selected to be 90 minutes for the first extraction and 60 minutes for the second extraction.

[0063] 4. Extraction Process Validation

[0064] To validate the optimal extraction process, a scaled-up validation test was conducted based on the optimization results from the single-factor experiment. In the initial experiments, several key components of the extract fluctuated significantly, impacting the efficacy. Therefore, by strengthening control from raw materials to the preparation process, we aim to achieve an effective and controllable extraction. Weigh 15g of Magnolia officinalis, 9g of charred Areca nut, 9g of stewed Tsaoko fruit, 6g of Ephedra, 9g of Bitter Apricot, 15g of Notopterygium wilfordii, 15g of Ginger, 15g of Patchouli, 9g of Perilla frutescens, 15g of Atractylodes lancea, 45g of Poria, 30g of Atractylodes macrocephala, 15g of Gypsum, 9g of charred Crataegus pinnatifida, 9g of charred Liushenqu, 9g of charred Malt, 15g of Earthworm, 15g of Cynanchum chinense, 9g of Mianma Guanzhong, and 15g of Lepidium officinale as one portion. Weigh a total of 12 portions (some important drugs, Magnolia officinalis, Areca nut, and stewed Tsaoko fruit have established unique medicinal material and decoction piece fingerprint control standards, and the prescription medicinal materials all meet the requirements of the latest pharmacopoeia standards) and extract them with water twice, adding 6 times the amount of water for the first extraction and extracting for 1.5h, and adding 4 times the amount of water for the second extraction and extracting for 1.0h. Each 4 portions are combined as 1 group, for a total of 3 groups. The transfer rate of ephedrine hydrochloride and pseudoephedrine hydrochloride, the solid content transfer rate, and other important indicators were measured to investigate the stability and feasibility of the process. The experimental results are as follows:

[0065] The verification results showed that the preferred process was stable, feasible and had good reproducibility. Therefore, the optimal extraction process was determined to be two water extractions: the first time, 6 times the amount of water was added and the extraction was performed for 1.5 h; the second time, 4 times the amount of water was added and the extraction was performed for 1.0 h.

[0066] 5. Fingerprint control standards for Magnolia officinalis, Areca catechu, and Simmered Tsaoko fruit medicinal materials and their decoction pieces

[0067] 5.1 Control Standards for Fingerprints of Magnolia Bark and Its Decoction Pieces

[0068] The chromatographic conditions and system suitability tests were performed using octadecylsilane bonded silica gel as the filler (Waters CORTECTS C 18 Chromatographic column, 150 mm × 4.6 mm, 2.7 μm); use acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, using gradient elution as specified in the table below; column temperature 35°C; flow rate 0.8 ml / min; detection wavelength 230 nm. The theoretical plate number calculated based on the magnolol peak should be no less than 4000.

[0069] Preparation of reference solution: Take appropriate amount of magnolol and honokiol reference substances, weigh accurately, and add methanol to prepare a mixed solution containing 200 μg of each per 1 ml.

[0070] Preparation of test solution: Take 1 g of the product powder (passed through No. 3 sieve), weigh accurately, place in a stoppered conical flask, accurately add 25 ml of 50% methanol solution, shake well, stopper tightly, soak for 24 hours, filter, and take the filtrate to obtain the product.

[0071] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph and determine.

[0072] The fingerprint of the test sample should show a chromatographic peak with the same retention time as the reference substance chromatographic peak in Figure 1. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of the test sample and the reference fingerprint should be calculated, and the similarity should not be less than 0.90.

[0073] 5.2 Fingerprint Control Standards for Areca Nut and Its Decoction Pieces

[0074] Chromatographic conditions and system suitability testing were performed using a strong cation-exchange bonded silica gel as the packing (Thermo BioBasic SCX column, 25 cm length, 4.6 mm inner diameter, 5 μm particle size); a mobile phase of acetonitrile-0.2% phosphoric acid (adjusted to pH 3.8 with aqueous ammonia) (65:35); a column temperature of 35°C; a flow rate of 1.2 ml / min; and a detection wavelength of 210 nm. The number of theoretical plates, calculated based on arecoline, should be no less than 10,000.

[0075] Preparation of reference solution: Take an appropriate amount of arecoline hydrobromide reference substance, weigh it accurately, and add methanol to make a solution containing 50 μg per 1 ml.

[0076] Preparation of test solution: Take 0.5 g of the product powder (passed through No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol solution, ultrasonically treat it for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the product.

[0077] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and record the chromatogram.

[0078] The fingerprint of the test sample should show a chromatographic peak with the same retention time as the reference chromatographic peak in Figure 2. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of the test sample and the reference fingerprint should be calculated, and the similarity should not be less than 0.90.

[0079] 5.3 Fingerprint Control Standards for Simmered Tsaoko Herbs and Pieces

[0080] Chromatographic Conditions and System Suitability: Octadecylsilane bonded silica gel was used as the packing (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. The column temperature was 30°C, the flow rate was 0.8 ml / min, and the detection wavelength was 254 nm. The number of theoretical plates, calculated based on the protocatechuic acid peak, should be no less than 10,000.

[0081] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference substance, weigh accurately, and add methanol to make a solution containing 200 μg per 1 ml.

[0082] Preparation of test solution: Take 1 g of the product powder (passed through No. 2 sieve), accurately weigh, accurately add 25 ml of 50% methanol solution, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate to obtain.

[0083] Determination method: Accurately aspirate 5-10 μl of reference solution and 10 μl of test solution, inject into liquid chromatograph, determine, and record the chromatogram.

[0084] The fingerprint of the test sample should show a chromatographic peak with the same retention time as the reference chromatographic peak in Figure 3. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of the test sample and the reference fingerprint should be calculated, and the similarity should not be less than 0.90.

[0085] Experimental Example 2 Preparation of Preparation

[0086] Preparation Example 1 Chinese Medicine Compound Granules

[0087] Prescription: 50 parts of Magnolia officinalis, 30 parts of scorched Areca catechu, 30 parts of stewed Tsaoko fruit, 20 parts of Ephedra, 30 parts of Bitter Apricot, 50 parts of Notopterygium wilfordii, 50 parts of Ginger, 50 parts of Patchouli, 30 parts of Cyperus rotundus, 50 parts of Atractylodes macrocephala, 150 parts of Poria, 100 parts of Atractylodes macrocephala, 50 parts of Gypsum, 30 parts of scorched Crataegus pinnatifida, 30 parts of scorched Liushenqu, 30 parts of scorched malt, 50 parts of Earthworm, 50 parts of Cynanchum chinense, 30 parts of Cyperus rotundus, and 50 parts of Lecithin seeds.

[0088] Preparation method of the Chinese medicine composition granules:

[0089] Take the above medicinal materials, add water and reflux extract twice, add 6 times water for the first time, extract for 1.5 hours, add 4 times water for the second time, extract for 1.0 hours, 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 crush to obtain an intermediate composition, add sucralose and dextrin, mix well to obtain granules.

[0090] Preparation Example 2 Chinese Medicine Compound Capsules

[0091] Prescription: 50 parts of Magnolia officinalis, 30 parts of scorched Areca catechu, 30 parts of stewed Tsaoko fruit, 20 parts of Ephedra, 30 parts of Bitter Apricot, 50 parts of Notopterygium wilfordii, 50 parts of Ginger, 50 parts of Patchouli, 30 parts of Cyperus rotundus, 50 parts of Atractylodes macrocephala, 150 parts of Poria, 100 parts of Atractylodes macrocephala, 50 parts of Gypsum, 30 parts of scorched Crataegus pinnatifida, 30 parts of scorched Liushenqu, 30 parts of scorched malt, 50 parts of Earthworm, 50 parts of Cynanchum chinense, 30 parts of Cyperus rotundus, and 50 parts of Lecithin seeds.

[0092] Preparation method of the Chinese medicine composition capsule:

[0093] The medicinal material was added with water and refluxed for extraction twice. The first time, 6 times the amount of water was added and the extraction was performed for 1.5 h. The second time, 4 times the amount of water was added and the extraction was performed for 1.0 h. The extracts were combined and filtered. The filtrate was concentrated to a relative density of 1.10-1.15, centrifuged and filtered, the filtrate was vacuum dried, spray dried, granulated, and made into capsules.

[0094] Preparation Example 3 Chinese medicine compound tablets

[0095] Prescription: 50 parts of Magnolia officinalis, 30 parts of scorched Areca catechu, 30 parts of stewed Tsaoko fruit, 20 parts of Ephedra, 30 parts of Bitter Apricot, 50 parts of Notopterygium wilfordii, 50 parts of Ginger, 50 parts of Patchouli, 30 parts of Cyperus rotundus, 50 parts of Atractylodes macrocephala, 150 parts of Poria, 100 parts of Atractylodes macrocephala, 50 parts of Gypsum, 30 parts of scorched Crataegus pinnatifida, 30 parts of scorched Liushenqu, 30 parts of scorched malt, 50 parts of Earthworm, 50 parts of Cynanchum chinense, 30 parts of Cyperus rotundus, and 50 parts of Lecithin seeds.

[0096] Preparation method of the Chinese medicine composition tablet:

[0097] Take the medicinal material and add water to reflux extraction twice. For the first time, add 6 times water and extract for 1.5 hours. For the second time, add 4 times water and extract for 1.0 hours. 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 add microcrystalline cellulose and other auxiliary materials to obtain the extract powder to make tablets.

[0098] Experimental Example 3 Therapeutic Effect of the Composition Particles of the Present Invention on Influenza Virus H1N1 / FM1 Strain Infection

[0099] 1. Test materials

[0100] 1.1 The granules of the test drug preparation example 1 were provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0101] 1.2 Positive control drug

[0102] 1.2.1 Oseltamivir phosphate granules (Kewei): produced by Yichang East Sunshine Changjiang Pharmaceutical Co., Ltd. Ingredients: Each bag of granules contains 15 mg of oseltamivir phosphate.

[0103] 1.2.2 Ribavirin Granules: Produced by Sichuan Baili Pharmaceutical Co., Ltd. Ingredients: The main ingredient of this product is ribavirin. Specification: 50 mg.

[0104] 1.3 Reagents

[0105] 1.4 Instruments

[0106] 1.5 Experimental animals

[0107] 1.6 Virus strain

[0108] The influenza A (H1N1) virus FM1 strain was purchased from the American Type Culture Collection (ATCC), routinely passaged in our ABSL-2 laboratory, and stored at -80°C until use.

[0109] 2. Dosage design and drug preparation

[0110] 2.1 Preparation Example 1:

[0111] Preparation of drug solution: Before the experiment, take the granules and add distilled water to make up the volume. The dosage is 26.4g crude drug / kg / d, and the drug is administered by gavage at 0.2mL / 10g body weight / time, once / day for 4 consecutive days.

[0112] 2.2 Oseltamivir phosphate: The dosage for mice in the experiment was 27.5 mg / kg / d, which is equivalent to the clinical dose for humans. The dosage was administered by gavage at 0.2 mL / 10 g body weight per time, once a day for 4 consecutive days.

[0113] 2.3 Ribavirin: The dosage for mice in the experiment was 82.5 mg / kg / d, administered by oral gavage at a rate of 0.2 mL / 10 g body weight per time, once a day for 4 consecutive days.

[0114] 2.4 Composition Group 1: Preparation Example 1 13.2 g crude drug / kg / d + oseltamivir phosphate 13.7 mg / kg / d, administration method is the same as above.

[0115] 2.5 Composition Group 2: Preparation Example 1 13.2 g crude drug / kg / d + ribavirin 41.2 mg / kg / d, administration method is the same as above.

[0116] 3 Test methods

[0117] 70 ICR mice weighing 14±1g, half male and half female, were randomly divided into 7 groups according to weight, namely normal control group, model control group, oseltamivir phosphate group, ribavirin group, combination group, oseltamivir phosphate group + combination group, and ribavirin + combination group. Except for the normal control group, the mice were lightly anesthetized with isoflurane and placed in a 15 LD 50 Influenza virus fluid (H1N1 / FM1 strain) was administered intranasally (35 μL per mouse). Administration began on the day of infection, at a dose of 0.2 mL / 10 g, via gavage once daily for four consecutive days. The normal and model control groups were gavaged with distilled water under the same conditions. On day 5, mice in each group were weighed, lungs were dissected, and lung tissue was obtained for HE pathological examination. The lung index and lung index inhibition rate were calculated.

[0118] Lung index (%) = lung wet weight (g) / body weight (g) × 100

[0119] Microscopic standard:

[0120] “-”: No exudation or blood stasis was observed in the mouse lung interstitium, no enlargement of interstitial cells was observed, no inflammation was observed around the bronchioles in the lung, and the tissue structure was normal.

[0121] “+”: No obvious exudative inflammation was observed in the mouse lung tissue and alveolar interstitium, and no large-scale hemostasis was observed. There was a small amount of localized inflammation around the bronchioles in the lung, mainly in the lymph nodes.

[0122] “++”: There is no large-scale inflammation and mucus exudation in the alveolar interstitium of the mouse lung tissue, but there are localized small patches of mucus exudation. There is localized inflammation around the bronchioles of the lung, mainly lymphocytes, and there is a localized increase in endothelial cells (segmental).

[0123] "+++": Extensive inflammation and mucous exudates are present in the alveolar interstitium of the mouse lung tissue. The exudates are primarily composed of lymphocytes, with a small number of lobed cells and eosinophils. The cells are unevenly sized and aggregated in clusters, accompanied by abundant pink mucus. There is also significant perivascular inflammation, localized around the bronchioles, with endothelial cell hyperplasia and severe perivascular inflammation.

[0124] Statistical method: The results were statistically analyzed using the t-test for comparison between groups.

[0125] 4 Effects of the present invention's composite particles on lung index and lung index inhibition rate

[0126] The research results (Table 1) showed that after mice were infected with influenza A (H1N1) virus FM1 strain, the lung index of the mice in the model control group was significantly increased, which was significantly different from that in the normal control group (P<0.01); the combination particles were given for treatment for 4 days starting from the day of infection, and the lung index of the oseltamivir phosphate group, the combination group, the oseltamivir phosphate group + the combination group, and the ribavirin + the combination group was significantly reduced, which was significantly different from that in the model control group, indicating that the combination has a significant synergistic effect on antiviral drugs used for upper respiratory tract infections in fighting influenza viruses.

[0127] Table 1 Effects of the composite particles on the lung index of mice infected with influenza virus H1N1 / FM1 strain

[0128] Note: Compared with the normal control group ## P<0.01; compared with the model control group, ** P<0.01, *P<0.05.

[0129] Experimental Example 4 Therapeutic Effect of the Composition Granules of the Present Invention on Respiratory Syncytial Virus Infection

[0130] 1. Test materials

[0131] 1.1 Test Drug Preparation Example 1 Granules were provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0132] 1.2 Positive control drug

[0133] 1.2.1 Oseltamivir phosphate granules (Kewei): produced by Yichang East Sunshine Changjiang Pharmaceutical Co., Ltd. Ingredients: Each bag of granules contains 15 mg of oseltamivir phosphate.

[0134] 1.2.2 Ribavirin Granules: Produced by Sichuan Baili Pharmaceutical Co., Ltd. Ingredients: The main ingredient of this product is ribavirin. Specification: 50 mg.

[0135] 1.3 Reagents

[0136] 1.4 Instruments

[0137] 1.5 Experimental animals

[0138] 1.6 Virus strain

[0139] Respiratory syncytial virus (RSV) strains were purchased from the American Type Culture Collection (ATCC), routinely passaged in our ABSL-2 laboratory, and stored at -80°C until use.

[0140] 2. Dosage design and drug preparation

[0141] 2.1 Preparation Example 1:

[0142] Preparation of medicinal solution: Take granules, add distilled water, and make up to 40 mL. The dosage is 26.4 g crude drug / kg / d. Administer by gavage at 0.2 mL / 10 g body weight / time, once a day for 4 consecutive days.

[0143] 2.2 Oseltamivir phosphate granules: The dosage for mice in the experiment was 27.5 mg / kg / d, which is equivalent to the clinical dose for humans. The dosage was administered by gavage at a rate of 0.2 mL / 10 g body weight per time, once a day for 4 consecutive days.

[0144] 2.3 Ribavirin granules: The dosage for mice was 82.5 mg / kg / d, administered by oral gavage at a rate of 0.2 mL / 10 g body weight per time, once a day for 4 consecutive days.

[0145] 2.4 Composition Group 1: 13.2 g crude drug / kg / d of granules from Preparation Example 1 + 13.7 mg / kg / d of oseltamivir phosphate, with the same administration method as above.

[0146] 2.5 Composition Group 2: 13.2 g crude drug / kg / d of granules from Preparation Example 1 + 41.2 mg / kg / d of ribavirin, administered in the same manner as above.

[0147] 3 Test methods

[0148] 70 BALB / C mice weighing 14±1g, half male and half female, were randomly divided into 7 groups according to weight, namely normal control group, model control group, oseltamivir phosphate group, ribavirin group, combination group, oseltamivir phosphate group + combination group, and ribavirin + combination group, with 10 mice in each group. Except for the normal control group, the mice were lightly anesthetized with isoflurane and injected with 100TCID 50 RSV virus droplets were administered intranasally, with 45 μL per mouse. Dosing began on the day of infection, with 0.2 mL / 10 g administered orally once daily for four consecutive days. The normal control group and the model control group were gavaged with distilled water under the same conditions. On the fifth day, mice in each group were weighed; lungs were dissected and weighed, and lung tissue was collected for inflammatory factor testing. The lung index and lung index inhibition rate were calculated. Lung index (%) = lung wet weight (g) / body weight (g) × 100

[0149] 4 Experimental results

[0150] 4.1 Effects of composite particles on lung index and lung index inhibition rate

[0151] The research results (Table 2) showed that after mice were infected with respiratory syncytial virus (RSV) by nasal drops, the lung index of the model group mice was significantly increased, which was significantly different from that of the normal control group (P<0.01); the combination particles were given for 4 days starting from the day of infection, and the lung index of the oseltamivir phosphate group, the combination group, the oseltamivir phosphate group + the combination group, and the ribavirin + the combination group was significantly reduced, which was significantly different from that of the model control group, indicating that the combination has a certain synergistic effect on antiviral drugs used for upper respiratory tract infections in resisting respiratory syncytial virus.

[0152] Table 2 Effects of the composite particles on lung index of mice infected with respiratory syncytial virus

[0153] Note: Compared with the normal control group ## P<0.01; compared with the model control group, *P<0.05.

[0154] 4.2 Effects on IL-33 Cytokine in Lung Tissue

[0155] Lung tissue was removed from the -80°C freezer and treated with a high-efficiency protein lysis buffer (PMSF and protease inhibitors added). The lung tissue was centrifuged and the protein supernatant was collected. The protein sample was placed in an ice box and sent to Leitz Biotechnology Co., Ltd. for high-throughput liquid phase protein microarray analysis of various cytokines in mouse lung tissue. Detailed detection procedures are detailed in the appendix.

[0156] After mice were infected intranasally with respiratory syncytial virus (RSV), the IL-33 cytokine content in the lung tissue of the model group was significantly increased, showing a significant difference compared with the normal control group (P < 0.01). Treatment with the combination granules for 4 days, starting on the day of infection, showed a significant decrease in IL-33 content in the oseltamivir phosphate group, the combination group, the oseltamivir phosphate group + the combination group, and the ribavirin + the combination group, showing a significant difference compared with the model group (P < 0.01, P < 0.05). See Table 3.

[0157] Table 3 Effects of the composite particles on cytokines in lung tissue of mice infected with respiratory syncytial virus

[0158] 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.

[0159] In this study, 26.4g crude drug / kg / d (1 / 2 times) of the composition granules were administered to mice by gavage once a day for 3 to 4 consecutive days. The influenza virus H1N1 / FM1 strain infection model and the respiratory syncytial virus infection model were used to evaluate the effectiveness of the composition granules in treating viral upper respiratory tract infections. The results showed that the combination of the composition and antiviral drugs can reduce the viral load and inflammatory cytokines in lung tissue, indicating that it has a therapeutic effect on this type of common specific viral infection. The advantages of traditional Chinese medicine itself are used to further promote and consolidate the antiviral effects of other drugs, providing a basis for further clinical research.

[0160] Experimental Example 5 Study on the fingerprint of the composition of the present invention

[0161] Instruments and reagents

[0162] Instruments: Agilent 1260 high-performance liquid chromatograph with DAD ultraviolet detector; Agilent 1260 high-performance liquid chromatograph with VWD ultraviolet detector; Agilent 1290 ultrahigh-pressure liquid chromatograph with DAD ultraviolet detector, Agilent 6538 Q-TOF-MS mass spectrometer with electrospray ionization (ESI) source, all Agilent Technologies, USA; Thermo Fisher Ultimate 3000 high-performance liquid chromatograph;

[0163] Mettler Toledo XP6 electronic analytical balance, Mettler; Mettler Toledo AL204 electronic analytical balance, Mettler; KQ500DB CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; HH digital constant temperature water bath, Changzhou Guoyu Instrument Manufacturing Co., Ltd.; Milli-Q ultrapure water analyzer, Millipore Corporation, USA;

[0164] Reference substances: 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 Food and Drug Administration; 5-hydroxymethylfurfural, 95.0%, batch number 8610, Shanghai Shidande Standard Technology Co., Ltd.; magnoside A, 99.1%, Batch number 7611, Shanghai Shidande Standard Technology Co., Ltd.; bergamotol glucoside, 98%, batch number CFS202002, Wuhan Tianzhi Biotechnology Co., Ltd.; peucedanin, 99.6%, batch number 111821-201604, China Food and Drug Administration; 6'-O-(trans-feruloyl)-peucedanin, 98%, batch number CFS202002, Wuhan Tianzhi Biotechnology Co., Ltd.; 4-Hydroxyphenylethyl anisate, 99.6%, batch number A05GB156933, Shanghai Yuanye Biotechnology Co., Ltd.

[0165] Reagents: methanol (Merck Co., Ltd., Merida, USA, chromatographic grade); acetic acid (L07203503, Merck Fisher Scientific Inc.); other reagents were of analytical grade.

[0166] Samples: Granules prepared by the method of Preparation Example 1, batch numbers: 200201, 200202, 200203, 200204, 200205, 200206, 200207, 200208, 200209, 200210, 200211, 200212, 200213, 200214, 200601, 200602, 200603, of which batch 200602 was the methodological research batch, all provided by the Traditional Chinese Medicine Research and Development Department of Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0167] 1. Selection of chromatographic conditions

[0168] Take an appropriate amount of this product, grind it into powder, take about 2 g, weigh it accurately, put it into a stoppered conical flask, accurately add 50 ml of water, ultrasonically treat it for 30 minutes, shake it well, centrifuge it, and take the supernatant to obtain the test solution.

[0169] 1.1 Selection of detection wavelength

[0170] The test solution was scanned at full wavelength using DAD. The results showed that the chromatographic peak information at 250 nm was rich, the response was more uniform, and the baseline was more stable. Therefore, 250 nm was selected as the detection wavelength for the characteristic spectrum. See Figures 4 and 5.

[0171] 1.2 Selection of mobile phase

[0172] Based on the characteristics of the water extraction process of this product, a Waters Atlantis T3 (4.6×250mm, 5μm) C 18 The chromatographic column used methanol-0.1% phosphoric acid as the mobile phase system and a detection wavelength of 250 nm. The separation effects of different mobile phase gradients were examined. Gradient program IV showed good peak resolution, appropriate retention times, and a relatively stable baseline. Therefore, gradient program IV was the preferred mobile phase elution program. The gradient elution program is as follows, and the results are shown in Figure 6.

[0173] Based on the above considerations, the proposed chromatographic conditions are: 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, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in the table below; flow rate 1.0 ml / min; column temperature 30°C; detection wavelength 250 nm. The number of theoretical plates based on the purpurogenol peak should be no less than 10,000.

[0174] 2. Selection of test sample preparation method

[0175] In order to optimize a reasonable and simple method for preparing the test solution, the extraction solvent and extraction method were investigated.

[0176] Preparation of test solution: Take an appropriate amount of this product, grind it into powder, take about 1g, and weigh it accurately. Place it in a stoppered conical flask, accurately add water and 25ml of 50% methanol respectively, ultrasonically treat for 30 minutes and reflux for 30 minutes respectively, shake well, centrifuge, and take the supernatant to obtain 4 test solution.

[0177] Results: Chromatographic analysis of each sample solution under the aforementioned conditions revealed that chromatographic peaks with greater polarity before 25 minutes into the extraction process using water showed a higher response, a richer overall peak count, and a more uniform response. Considering the characteristics of the aqueous extraction process, water was the preferred extraction solvent. Chromatographic peak richness and response were not significantly different between reflux and ultrasonic extraction methods. Ultrasonic extraction was preferred for ease of use. See Figure 7.

[0178] Based on the above investigation, the proposed method for processing the test sample is: take an appropriate amount of the product, grind it into powder, take about 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of water, ultrasonically treat it (power 500W, frequency 40kHz) for 30 minutes, shake it well, centrifuge it, and take the supernatant.

[0179] 3. Durability inspection

[0180] The effects of different wavelengths (245nm, 250nm, 255nm), flow rates (0.8ml / min, 0.9ml / min, 1.0ml / min, 1.1ml / min, 1.2ml / min), column temperatures (25℃, 28℃, 30℃, 32℃, 35℃), different batches of chromatographic columns {column 1: Waters Atlantis T3 (4.6×250mm, 5μm), serial number 01813017014031; column 2: Waters Atlantis T3 (4.6×250mm, 5μm), serial number 01813017014008}, and instruments (instrument 1: Agilent 1260, instrument 2: Ultimate3000) on the separation of the selected 10 characteristic peaks were investigated.

[0181] The results showed that within the wavelength range of 250 ± 5 nm, flow rate of 0.8 ml / min to 1.2 ml / min, and column temperature of 25°C to 35°C, and using different batches of chromatographic columns and different brands of instruments, the selected characteristic peaks were well separated, demonstrating the robustness of the method (see Figure 8).

[0182] 4 Methodological validation

[0183] Precision Assessment: Test solutions were prepared as described above. Accurately pipette 10 μl of the same solution and inject six times. The relative retention times of the 10 calibrated characteristic peaks were calculated, using purpurogenol (peak 8) as the reference peak. The results showed that the RSD values ​​for the relative retention times of the 10 characteristic peaks were all less than 0.5%, indicating good instrument precision. See Table 4 and Figure 9.

[0184] Repeatability Study: Six test sample solutions were prepared in parallel according to the above method and assayed. Using purpurin (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 all 10 characteristic peaks were less than 0.5%, indicating good repeatability of the method. See Table 5 and Figure 10.

[0185] Stability Study: 10 μl of the same test solution was injected at 0, 3, 6, 9, 12, 18, 24, 30, and 36 hours. The relative retention times of the 10 characteristic peaks were calculated using purpurogenol (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 over 36 hours. See Table 6 and Figure 11.

[0186] Table 4 Calculation results of characteristic spectrum precision investigation

[0187] Table 5 Calculation results of repeatability study

[0188] Table 6 Stability investigation calculation results

[0189] 5. Multi-batch measurement and control characteristic map generation

[0190] According to the above method, 14 batches of clinical practice samples, 3 batches of pilot samples, and 3 batches of process validation samples were measured. 10 peaks were selected as characteristic peaks, and a control fingerprint was generated for 20 batches of preparations. The peak corresponding to the peak of the purpurogenol reference substance was designated as the S peak. The relative retention time of each characteristic peak was calculated. Each peak should be within ±10% of the specified value, which is: 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). The results of multiple batches are shown in Figure 12 and Table 7, and the control fingerprint is shown in Figure 13.

[0191] Table 7 Relative retention time results of 20 batches of preparations

[0192] 6 Characteristic spectrum quality standards

[0193] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the packing (Waters Atlantis T3, column length 25 cm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, using a gradient elution as specified in the table below; the flow rate was 1.0 ml / min; the column temperature was 30°C; and the detection wavelength was 250 nm. The number of theoretical plates, calculated based on the peucedanum saccharin peak, should be no less than 10,000.

[0194] Preparation of reference solution: Take an appropriate amount of purpurogenol reference substance, accurately weigh it, and add 50% methanol to make a solution containing 0.15 mg per 1 ml.

[0195] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of water, ultrasonically treat it (power 500W, frequency 40kHz) for 30 minutes, shake it well, centrifuge it, and take the supernatant.

[0196] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and record the chromatogram.

[0197] The test sample's characteristic spectrum should show 10 characteristic peaks. The peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak should be calculated 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). See Figure 14 for the reference characteristic spectrum.

[0198] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Chinese herbal medicine extract, characterized in that The Chinese medicine extract is prepared from the following Chinese medicine raw materials by weight: 1-100 parts of Magnolia officinalis, 1-100 parts of burnt betel nut, 1-100 parts of stewed tsaoko, 1-100 parts of ephedra, 1-100 parts of bitter almond, 1-100 parts of notopterygium wilfordii, 1-100 parts of ginger, 1-100 parts of patchouli, 1-100 parts of perilla, 1-100 parts of atractylodes, 1-100 parts of poria, 1-100 parts of Atractylodes macrocephala, 1-100 parts of gypsum, 1-100 parts of charred hawthorn, 1-100 parts of charred Liu Shen Qu, 1-100 parts of charred malt, 1-100 parts of earthworms, 1-100 parts of Cynanchum wilfordii, 1-100 parts of Cyperus rotundus, and 1-100 parts of Trichosanthes kirilowii seeds; wherein the ephedrine content of the extract is 0.5-1.5 mg / g, and the solid content transfer rate is 18-25%.

2. The Chinese herbal extract according to claim 1, characterized in that The Chinese medicine extract is prepared from Chinese medicines including the following raw materials: 1-80 parts of Magnolia officinalis, 1-80 parts of charred Areca catechu, 1-80 parts of stewed Amomum villosum, 1-60 parts of Ephedra, 1-60 parts of Bitter Apricot, 1-80 parts of Notopterygium wilfordii, 1-60 parts of Ginger, 1-80 parts of Patchouli, 1-60 parts of Cyperus rotundus, 1-80 parts of Atractylodes macrocephala, 1-160 parts of Poria cocos, 1-120 parts of Atractylodes macrocephala, 1-80 parts of Gypsum, 1-50 parts of charred Crataegus pinnatifida, 1-80 parts of charred Liushenqu, 1-60 parts of charred malt, 1-80 parts of Earthworm, 1-80 parts of Cynanchum chinense, 1-60 parts of Cyperus rotundus, and 1-80 parts of Lepidium tiliaceum; wherein the content of peucedanum scutellariae in the extract is 2-8 mg / g.

3. The Chinese herbal extract according to claim 1 or 2, characterized in that The Chinese medicine extract is prepared from Chinese medicines including the following raw materials: 30-50 parts of Magnolia officinalis, 20-30 parts of charred Areca catechu, 30-50 parts of stewed Amomum villosum, 20-30 parts of Ephedra sinica, 20-30 parts of Bitter Apricot, 30-50 parts of Notopterygium wilfordii, 30-50 parts of Ginger, 30-50 parts of Patchouli, 20-30 parts of Cyperus rotundus, 30-50 parts of Atractylodes macrocephala, 120-150 parts of Poria cocos, 80-100 parts of Atractylodes macrocephala, 30-50 parts of Gypsum, 20-30 parts of charred Crataegus pinnatifida, 30-50 parts of charred Liushenqu, 20-30 parts of charred malt, 30-50 parts of Earthworm, 30-50 parts of Cynanchum chinense, 20-30 parts of Cyperus rotundus, and 30-50 parts of Lepidium chinense.

4. The Chinese medicine extract according to claim 3, characterized in that The Chinese medicine extract is prepared from the following Chinese medicine raw materials: 50 parts of magnolia bark, 30 parts of charred betel nut, 30 parts of stewed tsaoko fruit, 20 parts of ephedra, 30 parts of bitter almond, 50 parts of notopterygium wilfordii, 50 parts of ginger, 50 parts of patchouli, 30 parts of perilla, 50 parts of atractylodes, 150 parts of poria, 100 parts of atractylodes, 50 parts of gypsum, 30 parts of charred hawthorn, 30 parts of charred liushenqu, 30 parts of charred malt, 50 parts of earthworm, 50 parts of cynanchum chinense, 30 parts of cyperus rotundus, and 50 parts of scutellaria baicalensis.

5. The Chinese herbal extract according to any one of claims 1 to 4, characterized in that The preparation of the Chinese herbal medicine extract comprises: Weigh Magnolia officinalis, scorched betel nut, stewed grass fruit, ephedra, bitter almond, angelica root, ginger, patchouli, perilla, atractylodes, Poria cocos, Atractylodes macrocephala, gypsum, charred hawthorn, charred Liushenqu, charred malt, earthworm, Cynanchum chinense, Cyperus rotundus, and Tripterygium wilfordii seeds are respectively extracted twice with water, with 6 times the amount of water added for the first extraction and extraction for 1.5 hours, and with 4 times the amount of water added for the second extraction and extraction for 1.0 hours. The extracts are combined and filtered, and the filtrate is concentrated to a relative density of 1.10 to 1.15, centrifuged and filtered, 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 peucedanum rutaecarpon content is 6-8 mg / g.

6. The Chinese herbal extract according to claim 5, characterized in that The preparation of the Chinese herbal medicine extract comprises: Weigh 15g of Magnolia officinalis, 9g of charred Areca nut, 9g of stewed Tsaoko fruit, 6g of Ephedra, 9g of Bitter Apricot, 15g of Notopterygium wilfordii, 15g of Ginger, 15g of Patchouli, 9g of Cyperus rotundus, 15g of Atractylodes macrocephala, 45g of Poria, 30g of Atractylodes macrocephala, 15g of Gypsum, 9g of charred Crataegus pinnatifida, 9g of charred Liushenqu, 9g of charred Malt, 15g of Earthworm, 15g of Cynanchum chinense, 9g of Cynomorium mongolicum, and 15g of Lepidium tiliaceum; extract twice with water, the first time with 6 times the amount of water and extract for 1.5h, the second time with 4 times the amount of water and extract for 1.0h, combine the extracts, filter, concentrate the filtrate to a relative density of 1.10-1.15, centrifuge and filter, and dry the filtrate.

7. Use of the Chinese medicinal extract according to any one of claims 1 to 6 in the preparation of a drug for treating influenza virus H1N1 or FM1, or respiratory syncytial virus.

8. A drug, characterized in that The medicine is prepared from the Chinese medicinal extract described in any one of items 1 to 6 and pharmaceutically acceptable excipients or additives.

9. The drug according to claim 6, characterized in that The medicine is in the form of granules, and the excipients or additives are preferably dextrin and sucralose.

10. A method for detecting the fingerprint of the Chinese herbal extract according to any one of claims 1 to 6 or the drug according to claim 6, characterized in that: The test solution was subjected to HPLC detection. The chromatographic conditions of the HPLC detection included: using a C18 chromatographic column, methanol as mobile phase A, and an aqueous solution containing 0.1% concentration of phosphoric acid as mobile phase B. The elution in the chromatographic conditions of the HPLC detection was gradient elution, and the gradient elution program was: 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.

11. The method according to claim 10, characterized in that The chromatographic conditions include: the flow rate is 1.0 mL / min, the column temperature is 30° C., and the detection wavelength is 250 nm.

12. The method according to claim 10 or 11, characterized in that In the fingerprint spectrum, the test sample should present 10 characteristic peaks in the characteristic spectrum. 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 within ±10% of the specified value. The specified value of relative retention time is: 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), 1.29 (peak 10).

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