Traditional chinese medicine composition and use thereof

Abstract

Provided are a traditional Chinese medicine composition and a method for preparing same. The composition is prepared from the following raw materials and optionally a pharmaceutically acceptable excipient. The raw materials include: Astragali Radix, Lonicerae Flos, Saposhnikoviae Radix, Cinnamomi Ramulus, Paeoniae Alba Radix, Forsythiae Fructus, Atractylodis Macrocephalae Rhizoma, Platycodonis Radix, Zingiberis Rhizoma, Jujubae Fructus, Citri Reticulatae Pericarpium, Isatidis Radix, and Glycyrrhizae Radix et Rhizoma. The composition can be used for preparing a medicament for treating common cold, viral infection, fever, inflammation, or cough, for relieving phlegm, for relieving pain, for immunoregulation, or for protecting influenza A close contacts.

Description

Traditional Chinese medicine compositions and their uses

[0001] This application claims priority to Chinese patent application No. 202411909632X, filed on December 24, 2024. Technical Field

[0002] This application relates to a traditional Chinese medicine composition and its uses. Background Technology

[0003] Chinese physicians throughout history have accumulated rich clinical experience in treating exogenous diseases. During the Spring and Autumn and Warring States periods, the *Huangdi Neijing* (Yellow Emperor's Inner Classic) first proposed the pathogenesis of "exogenous pathogens entering the body due to deficiency" and the transmission pattern of "collaterals-meridians-collaterals." Zhang Zhongjing of the Eastern Han Dynasty, inheriting the principles of the *Huangdi Neijing*, established the "Six Channels Differentiation" theoretical system, elucidating the transmission pathways, pathogenesis, and treatment of exogenous diseases, and creating the classic formula Guizhi Tang (Cinnamon Twig Decoction). Physicians from the Sui, Tang, Song, Ming, and Qing dynasties further interpreted the transmission pattern of exogenous pathogens from the exterior to the interior in cases of deficiency, pointing out the recurrent nature of colds due to deficiency. Colds due to deficiency are further subdivided into Qi deficiency and Yin deficiency types. Patients with Qi deficiency colds exhibit symptoms of wind-cold colds such as fever, dizziness, headache, limb aches, nasal congestion, and runny nose, as well as manifestations of spleen and lung Qi deficiency such as fatigue, weakness, and reluctance to speak. The classic formula Yupingfeng San (Jade Screen Powder), which tonifies Qi and consolidates the exterior, is a commonly used prescription for treating colds due to Qi deficiency in later generations.

[0004] Currently, there are six representative commercially available traditional Chinese medicines that can treat colds caused by physical weakness. Specific information is as follows:

[0005] Shensu Oral Liquid / Tablets: National Drug Approval Numbers Z20060317 and Z50020140, etc. It is composed of Codonopsis pilosula, Perilla frutescens leaf, Pueraria lobata, Peucedanum praeruptorum, Poria cocos, Pinellia ternata (processed), Citrus reticulata peel, Citrus aurantium (fried), Platycodon grandiflorus, Glycyrrhiza uralensis, Aucklandia lappa, Zingiber officinale, and Ziziphus jujuba. It has the effects of invigorating qi and relieving exterior syndromes, dispelling wind and cold, resolving phlegm and relieving cough. It is used for colds caused by wind-cold due to physical weakness, with symptoms such as chills and fever, headache and nasal congestion, cough with excessive phlegm, chest tightness and nausea, fatigue and shortness of breath.

[0006] Yu Ping Feng Granules / Oral Liquid / Capsules: National Drug Approval Numbers Z10930036, Z65020123, Z10980026, etc. Composed of Astragalus membranaceus, Saposhnikovia divaricata, and Atractylodes macrocephala (fried). It has the effects of invigorating qi, strengthening the exterior, and stopping sweating. Used for exterior deficiency with spontaneous sweating, aversion to wind, and pale complexion. Those who are pale or have a weak constitution and are easily susceptible to wind-evil.

[0007] Danxi Yupingfeng Granules: National Medicine Approval Number Z53021556. Composed of Astragalus membranaceus, Saposhnikovia divaricata, and Atractylodes macrocephala (stir-fried with wheat bran). It has the effects of invigorating qi, strengthening the exterior, and stopping sweating. Used for exterior deficiency with spontaneous sweating and aversion to wind, and pale complexion. Those who are pale or have a weak constitution and are easily susceptible to wind-evil.

[0008] Formula for Colds Due to Weakness: National Medicine Approval Number B20020235. Composed of Astragalus membranaceus, Scutellaria baicalensis, Lonicera japonica, Atractylodes macrocephala, Saposhnikovia divaricata, Isatis indigotica, Scrophularia ningpoensis, Ophiopogon japonicus, Phragmites communis, and Platycodon grandiflorus. It has the effects of tonifying Qi and nourishing Yin, relieving exterior symptoms and dispelling pathogens. Suitable for colds due to weakness, fatigue, nasal congestion, and runny nose.

[0009] Pingfeng Shengmai Capsules: National Medicine Approval Number Z61020270, etc. Composed of Astragalus membranaceus, Saposhnikovia divaricata, Ginseng, Ophiopogon japonicus, Schisandra chinensis, Prepared Aconitum carmichaelii, and Atractylodes macrocephala (stir-fried with soil). It has the effects of invigorating Qi, supporting Yang, and consolidating the exterior. Used for shortness of breath and palpitations, spontaneous sweating due to exterior deficiency, fatigue and dizziness, and susceptibility to wind-cold.

[0010] Xinqin Tablets: National Medicine Approval Number Z20050204. Composed of Asarum, Scutellaria baicalensis, Schizonepeta tenuifolia, Saposhnikovia divaricata, Angelica dahurica, Xanthium sibiricum, Astragalus membranaceus, Atractylodes macrocephala, Cinnamomum cassia, and Acorus tatarinowii. It has the effects of tonifying Qi and strengthening the exterior, dispelling wind and clearing the nasal passages. It is used for nasal congestion, lung Qi deficiency, exogenous wind-evil syndrome, aversion to wind and spontaneous sweating, runny nose with clear discharge, nasal congestion, and weak and floating pulse; also for allergic rhinitis with the above symptoms.

[0011] In summary, currently available traditional Chinese medicine (TCM) products mainly target symptoms such as superficial weakness and susceptibility to wind-cold, qi and yin deficiency, external pathogenic factors, and qi deficiency with external wind-cold. They often employ qi-tonifying and exterior-strengthening methods, lacking the combination of exterior-releasing and pathogen-dispelling drugs, or using exterior-releasing drugs in addition to qi-tonifying and exterior-strengthening formulas. There is a lack of TCM products specifically targeting qi-deficiency colds, wind-cold invasion, and simultaneously addressing lung and spleen deficiencies, capable of tonifying qi, strengthening the exterior, harmonizing the nutritive and defensive qi, and dispelling pathogenic factors. Currently, there are no marketed products with a clear indication for qi-deficiency colds. Summary of the Invention

[0012] The inventors of this application have conducted in-depth research on Qi deficiency-related colds and discovered that stagnation of the lung collaterals is the root cause of the disease, invasion of wind pathogens is the manifestation of the disease, and disharmony between Ying and Wei (nutritive and defensive Qi) is the key pathogenesis. In patients with Qi deficiency, the lung collaterals are deficient, and the defensive functions of Qi and Wei Qi are impaired. External pathogens take advantage of this weakness and enter through the skin, mouth, and nose, initially attacking the Yang collaterals on the body surface. This leads to stagnation of the Yang collaterals, stagnation of the lung collaterals, and disharmony between Ying and Wei, resulting in the disease. Based on the above pathogenesis, the treatment principle of "tonifying Qi and strengthening Wei, dispelling wind and detoxifying" is established.

[0013] This application provides a traditional Chinese medicine composition with the effects of "tonifying qi and strengthening the body, dispelling wind and detoxifying". The raw materials used to prepare the composition include Astragalus membranaceus, Lonicera japonica, Saposhnikovia divaricata, Cinnamomum cassia, Paeonia lactiflora, Forsythia suspensa, Atractylodes macrocephala, Platycodon grandiflorus, Zingiber officinale, Ziziphus jujuba, Citrus reticulata, Isatis indigotica, Glycyrrhiza uralensis, or the raw materials are composed of these drugs.

[0014] The herbal composition of this application uses Astragalus membranaceus and Lonicera japonica as the principal herbs. Astragalus membranaceus can greatly tonify the Qi of the lungs and spleen, strengthen the exterior and replenish the Wei Qi to clear the deficiency of the collaterals. Lonicera japonica clears heat and detoxifies, clearing heat on the exterior. The combination of the two herbs addresses both the root cause and the symptoms, eliminating pathogens and regulating the body's resistance. Saposhnikovia divaricata, Cinnamomum cassia, Paeonia lactiflora, and Forsythia suspensa are the assistant herbs. Saposhnikovia divaricata dispels wind and releases the exterior. When combined with the principal herb Astragalus membranaceus, it allows Astragalus membranaceus to strengthen the exterior without retaining pathogens, and Saposhnikovia divaricata to expel pathogens without harming the body's resistance. This combination embodies the principle of tonifying while dispersing, and dispersing while tonifying. Cinnamomum cassia assists the Wei Qi, clears the meridians, releases the exterior and eliminates pathogens on the surface. Paeonia lactiflora nourishes Yin and astringes the Ying Qi, consolidating the outward-leaking Ying Qi. The combination of the two herbs works together to harmonize the Ying and Wei Qi. Forsythia suspensa disperses wind-heat, clears heat and detoxifies. When combined with Lonicera japonica, it enhances the overall efficacy of the formula in eliminating pathogens and also moderates the pungent and warm herbs, taking into account the characteristic of external pathogens accumulating and transforming into heat. The formula consists of Atractylodes macrocephala, Platycodon grandiflorus, dried ginger, jujube, tangerine peel, and Isatis indigotica. Atractylodes macrocephala invigorates qi and strengthens the spleen. Combined with the principal herb Astragalus membranaceus and the assistant herb Saposhnikovia divaricata, it strengthens qi and strengthens the exterior, preventing sweating and making it difficult for external pathogens to invade. Platycodon grandiflorus promotes lung qi, dispels pathogens, and soothes the throat. Dried ginger warms and invigorates spleen yang. Combined with the guiding herb Glycyrrhiza uralensis, its pungent and sweet properties restore yang, allowing the middle yang to recover and the spleen qi to function properly. Tangerine peel regulates qi and strengthens the spleen. Combined with Atractylodes macrocephala and jujube, it invigorates spleen qi. Combined with Astragalus membranaceus, it raises spleen yang, making the spleen earth strong and the lung metal sufficient. At the same time, it dries dampness and invigorates the spleen, allowing qi to flow smoothly and pathogens to be expelled. Isatis indigotica clears heat and detoxifies, dispels pathogens, and soothes the throat. All the exterior-releasing herbs in the formula exert their effects of dispelling wind and detoxifying. Licorice is used as the guiding herb, combined with cinnamon twig to nourish yang and strengthen wei qi, and white peony root to transform yin and harmonize ying qi. It enters the spleen and stomach and is combined with tangerine peel to invigorate qi and strengthen the spleen to harmonize the middle jiao. When all the herbs are used together, they can dispel wind and detoxify externally, invigorate qi and strengthen the exterior internally, and replenish the deficiency of collaterals. They treat both the root cause and the symptoms, and preventive measures are included in the treatment. By combining prevention and treatment, all symptoms will disappear.

[0015] This application is significantly innovative in its understanding of pathogenesis, treatment principles, and formulation of prescriptions and drugs. Attached Figure Description

[0016] Figure 1 shows a pathological section of lung tissue from Experiment 1 of Example 3 (HE, ×200), where A is the normal control group, B is the model control group, C is the ribavirin granule group, D is the low-dose test substance group, E is the medium-dose test substance group, and F is the high-dose test substance group.

[0017] Figure 2 shows the effect of the test substance on the immune barrier of rhinovirus mouse lung tissue in Experiment 1 of Example 3 (immunohistochemical staining, ×200), where (1) A1~F1 is E-cadherin, A2~F2 is Occludin, and A3~F3 is TLR7; (2) A is the normal control group, B is the model control group, C is the ribavirin granule group, D is the low-dose test substance group, E is the medium-dose test substance group, and F is the high-dose test substance group.

[0018] Figure 3 shows the lung tissue pathological sections (HE, ×200) of Experiment 2 in Example 3, where A is the normal control group, B is the model control group, C is the oseltamivir phosphate granule group, D is the low-dose test substance group, E is the medium-dose test substance group, and F is the high-dose test substance group.

[0019] Figure 4 shows the effect of the test substance on the immune barrier of mouse lung tissue of H1N1 influenza A virus in Experiment 2 of Example 3 (immunohistochemical staining, ×200). Among them, (1) A1~F1 is E-cadherin, A2~F2 is Occludin, and A3~F3 is TLR7; (2) A is the normal control group, B is the model control group, C is the oseltamivir phosphate granule group, D is the low-dose test substance group, E is the medium-dose test substance group, and F is the high-dose test substance group.

[0020] Figure 5 shows a lung tissue pathological section (HE, ×200) from Example 4, where A is the normal control group, B is the model control group, C is the ribavirin granule group, D is the low-dose test substance group, E is the medium-dose test substance group, and F is the high-dose test substance group.

[0021] Figure 6 shows pathological sections of Experiment 1 in Example 5. Figure 6-1 shows pathological sections of BALB / c mouse tissue (HE, ×200), where A is the normal control group, B is group 1 of close contact transmission, and C is group 2 of close contact transmission (A1, B1, C1 are nasal tissues; A2, B2, C2 are trachea; A3, B3, C3 are lung tissues). Figure 6-2 shows pathological sections of Hartley guinea pig tissue (HE, ×200), where A is the normal control group, B is group 1 of close contact transmission, and C is group 2 of close contact transmission (A1, B1, C1 are nasal tissues; A2, B2, C2 are trachea; A3, B3, C3 are lung tissues). Figure 6-3 shows pathological sections of the trachea and bronchi (HE, ×200). A is the normal control group, B is the close contact transmission-model control group, C is the close contact transmission-oseltamivir phosphate granule group, D is the close contact transmission-low dose group of the test substance, E is the close contact transmission-medium dose group of the test substance, and F is the close contact transmission-high dose group of the test substance. Figure 6-4 shows pathological sections of lung tissue (HE, ×200). A is the normal control group, B is the close contact transmission-model control group, C is the close contact transmission-oseltamivir phosphate granule group, D is the close contact transmission-low dose group of the test substance, E is the close contact transmission-medium dose group of the test substance, and F is the close contact transmission-high dose group of the test substance.

[0022] Figure 7 shows the pathological sections of Experiment 2 in Example 5. Figure 7-1 shows the pathological sections of the trachea and bronchi (HE, ×200). A is the normal control group, B is the close contact transmission-model control group, C is the close contact transmission-oseltamivir phosphate granule group, D is the close contact transmission-low dose group of the test substance, E is the close contact transmission-medium dose group of the test substance, and F is the close contact transmission-high dose group of the test substance. Figure 7-2 shows the pathological sections of lung tissue (HE, ×200). A is the normal control group, B is the close contact transmission-model control group, C is the close contact transmission-oseltamivir phosphate granule group, D is the close contact transmission-low dose group of the test substance, E is the close contact transmission-medium dose group of the test substance, and F is the close contact transmission-high dose group of the test substance. Detailed Implementation

[0023] In this application, unless otherwise specified, "parts" means "parts by weight" and "about" means "±10%".

[0024] The raw materials used to prepare the traditional Chinese medicine composition of this application include: Astragalus membranaceus, Lonicera japonica, Saposhnikovia divaricata, Cinnamomum cassia, Paeonia lactiflora, Forsythia suspensa, Atractylodes macrocephala, Platycodon grandiflorus, Zingiber officinale, Ziziphus jujuba, Citrus reticulata, Isatis indigotica, and Glycyrrhiza uralensis, or the raw materials are composed of these drugs.

[0025] In a specific embodiment, the raw materials used to prepare the traditional Chinese medicine composition of this application include or are composed of the following drugs: 250-500 parts of Astragalus membranaceus, 250-450 parts of Lonicera japonica, 120-220 parts of Saposhnikovia divaricata, 80-150 parts of Cinnamomum cassia, 100-180 parts of Paeonia lactiflora, 100-180 parts of Forsythia suspensa, 120-220 parts of Atractylodes macrocephala, 100-180 parts of Platycodon grandiflorus, 40-80 parts of Zingiber officinale (dried), 40-80 parts of Ziziphus jujuba, 100-180 parts of Citrus reticulata, 250-450 parts of Isatis indigotica, and 40-80 parts of Glycyrrhiza uralensis.

[0026] In a more specific embodiment, the raw materials used to prepare the traditional Chinese medicine composition of this application include or are composed of the following drugs: Astragalus membranaceus 300-480 parts, Lonicera japonica 300-440 parts, Saposhnikovia divaricata 130-200 parts, Cinnamomum cassia 90-145 parts, Paeonia lactiflora 110-160 parts, Forsythia suspensa 120-160 parts, Atractylodes macrocephala 140-200 parts, Platycodon grandiflorus 110-160 parts, Zingiber officinale 45-70 parts, Ziziphus jujuba 45-80 parts, Citrus reticulata 120-160 parts, Isatis indigotica 300-400 parts, and Glycyrrhiza uralensis 45-80 parts.

[0027] In a more specific embodiment, the active pharmaceutical ingredient used to prepare the traditional Chinese medicine composition of this application includes or is composed of the following pharmaceutical ingredients:

[0028] Astragalus membranaceus 450g, Lonicera japonica 365g, Saposhnikovia divaricata 150g, Cinnamomum cassia 100g, Paeonia lactiflora 120g, Forsythia suspensa 140g, Atractylodes macrocephala 160g, Platycodon grandiflorus 130g, Zingiber officinale 65g, Ziziphus jujuba 50g, Citrus reticulata 150g, Isatis indigotica 355g, Glycyrrhiza uralensis 50g; or

[0029] Astragalus membranaceus 365g, Lonicera japonica 335g, Saposhnikovia divaricata 180g, Cinnamomum cassia 100g, Paeonia lactiflora 150g, Forsythia suspensa 130g, Atractylodes macrocephala 180g, Platycodon grandiflorus 130g, Zingiber officinale 60g, Ziziphus jujuba 50g, Citrus reticulata 150g, Isatis indigotica 335g, Glycyrrhiza uralensis 50g; or

[0030] Astragalus membranaceus 320g, Lonicera japonica 380g, Saposhnikovia divaricata 180g, Cinnamomum cassia 120g, Paeonia lactiflora 130g, Forsythia suspensa 150g, Atractylodes macrocephala 200g, Platycodon grandiflorus 150g, Zingiber officinale 50g, Ziziphus jujuba 65g, Citrus reticulata 140g, Isatis indigotica 365g, Glycyrrhiza uralensis 60g; or

[0031] Astragalus membranaceus 335g, Lonicera japonica 400g, Saposhnikovia divaricata 160g, Cinnamomum cassia 130g, Paeonia lactiflora 150g, Forsythia suspensa 130g, Atractylodes macrocephala 155g, Platycodon grandiflorus 120g, Zingiber officinale 55g, Ziziphus jujuba 70g, Citrus reticulata 135g, Isatis indigotica 330g, Glycyrrhiza uralensis 70g; or

[0032] Astragalus membranaceus 345g, Lonicera japonica 345g, Saposhnikovia divaricata 173g, Cinnamomum cassia 115g, Paeonia lactiflora 138g, Forsythia suspensa 138g, Atractylodes macrocephala 173g, Platycodon grandiflorus 138g, Zingiber officinale 58g, Ziziphus jujuba 58g, Citrus reticulata 138g, Isatis indigotica 345g, Glycyrrhiza uralensis 58g.

[0033] The composition of this application may also include optional pharmaceutically acceptable excipients, examples of which include, but are not limited to, one or more selected from flavoring agents, preservatives, fillers, thickeners, binders, diluents, dispersants, colorants, etc.

[0034] In a specific embodiment, the composition of this application includes sucralose and maltodextrin as excipients.

[0035] The composition of this application can be used to treat colds or to prepare medicines for treating colds. In a specific embodiment, the cold is a qi deficiency cold, characterized by aversion to wind, chills, nasal congestion, runny nose, fever, sore throat, accompanied by fatigue, weakness, shortness of breath, reluctance to speak, spontaneous sweating, and pale complexion. Biden, etc.

[0036] The compositions of this application can be used for antiviral purposes or for the preparation of antiviral drugs. In specific embodiments, the virus is influenza A virus, influenza B virus, rhinovirus, respiratory syncytial virus, parainfluenza virus, enterovirus, coronavirus, or adenovirus. In more specific embodiments, the influenza A virus is preferably influenza A H1N1 virus or influenza A H3N2 virus, the rhinovirus is preferably human rhinovirus, and the respiratory syncytial virus is preferably human respiratory syncytial virus.

[0037] The compositions of this application may also be used for antipyretics, anti-inflammatory, antitussive, expectorant, analgesic, or immunomodulatory purposes, or for the preparation of antipyretics, anti-inflammatory, antitussive, expectorant, analgesic, or immunomodulatory drugs.

[0038] In a specific implementation, the immune regulation is preferably immunosuppression or nonspecific immune regulation.

[0039] The composition of this application can also be used to protect close contacts of H1N1 influenza, or to prepare a drug for protecting close contacts of H1N1 influenza.

[0040] The compositions of this application can be prepared into oral formulations by known methods, including but not limited to, liquid formulations, tablets, granules, powders, capsules, etc.

[0041] The composition of this application can be prepared by, for example, the following method, but is not limited thereto. The method includes: weighing the raw material drug according to the prescription amount, adding water and decocting three times. For the first decoction, add 8-12 times the mass of water and decoct for 1-2 hours. For the second and third decoctions, add 6-10 times the mass of water and decoct for 1-2 hours each time. Filter the solution and concentrate the filtrate under reduced pressure.

[0042] In a specific embodiment, the method includes: weighing the raw materials according to the prescription amount, adding water and decocting three times. The first time, about 10 times the mass of water is added and decocted for about 1.5 hours. The second and third times, about 8 times the mass of water is added and decocted for about 1 hour each time. The solution is filtered and the filtrate is concentrated under reduced pressure.

[0043] In a more specific embodiment, the method includes concentrating the filtrate under reduced pressure to an extract with a relative density of 1.10 to 1.20 (60°C).

[0044] In a more specific embodiment, the method includes drying the obtained extract to form a dry extract powder.

[0045] In a more specific embodiment, the method includes granulating the obtained dry powder with pharmaceutically acceptable excipients.

[0046] Example

[0047] Example 1 Preparation Example

[0048] Raw material prescription:

[0049] Astragalus membranaceus 450g, Lonicera japonica 365g, Saposhnikovia divaricata 150g, Cinnamomum cassia 100g, Paeonia lactiflora 120g, Forsythia suspensa 140g, Atractylodes macrocephala 160g, Platycodon grandiflorus 130g, Zingiber officinale 65g, Ziziphus jujuba 50g, Citrus reticulata 150g, Isatis indigotica 355g, Glycyrrhiza uralensis 50g; or

[0050] Astragalus membranaceus 365g, Lonicera japonica 335g, Saposhnikovia divaricata 180g, Cinnamomum cassia 100g, Paeonia lactiflora 150g, Forsythia suspensa 130g, Atractylodes macrocephala 180g, Platycodon grandiflorus 130g, Zingiber officinale 60g, Ziziphus jujuba 50g, Citrus reticulata 150g, Isatis indigotica 335g, Glycyrrhiza uralensis 50g; or

[0051] Astragalus membranaceus 320g, Lonicera japonica 380g, Saposhnikovia divaricata 180g, Cinnamomum cassia 120g, Paeonia lactiflora 130g, Forsythia suspensa 150g, Atractylodes macrocephala 200g, Platycodon grandiflorus 150g, Zingiber officinale 50g, Ziziphus jujuba 65g, Citrus reticulata 140g, Isatis indigotica 365g, Glycyrrhiza uralensis 60g; or

[0052] Astragalus membranaceus 335g, Lonicera japonica 400g, Saposhnikovia divaricata 160g, Cinnamomum cassia 130g, Paeonia lactiflora 150g, Forsythia suspensa 130g, Atractylodes macrocephala 155g, Platycodon grandiflorus 120g, Zingiber officinale 55g, Ziziphus jujuba 70g, Citrus reticulata 135g, Isatis indigotica 330g, Glycyrrhiza uralensis 70g; or

[0053] Astragalus membranaceus 345g, Lonicera japonica 345g, Saposhnikovia divaricata 173g, Cinnamomum cassia 115g, Paeonia lactiflora 138g, Forsythia suspensa 138g, Atractylodes macrocephala 173g, Platycodon grandiflorus 138g, Zingiber officinale 58g, Ziziphus jujuba 58g, Citrus reticulata 138g, Isatis indigotica 345g, Glycyrrhiza uralensis 58g.

[0054] Preparation of dry extract powder:

[0055] (1) Extraction and concentration

[0056] Weigh the above thirteen raw materials according to the prescription, add water and decoct three times. For the first decoction, add 10 times the weight of water and decoct for 1.5 hours. For the second and third decoctions, add 8 times the weight of water and decoct for 1 hour each. Filter, and concentrate the filtrate under reduced pressure to obtain an extract with a relative density of 1.10–1.20 (60℃). Filter again and set aside. The yield of extract from raw materials is 36.5 ± 5.5%.

[0057] (2) Drying

[0058] Take the above extract, dry it, collect the dried extract powder, and set it aside for later use.

[0059] Optionally, mix the dry extract powder, sucralose, and maltodextrin according to the following formula, granulate by dry method to obtain granules, and package them into bags of 4.5g each.

[0060] Formulation prescription:

[0061] Dry ointment powder 811±122g

[0062] 3g of sucralose

[0063] Maltodextrin 64-308g (adjust the amount of maltodextrin according to the amount of each batch of dry extract powder)

[0064] A total of 1000g was produced.

[0065] Example 2: In vitro antiviral pharmacodynamic study

[0066] 1. Research Objectives

[0067] This study used the CPE method, selecting oseltamiviric acid and ribavirin as positive control drugs, to investigate the in vitro antiviral activity of the test substance TCM-074 against influenza A subtypes H3N2 and H1N1, influenza B virus strains, human rhinovirus, human respiratory syncytial virus, parainfluenza virus, enterovirus, coronavirus, and adenovirus.

[0068] 2. Test materials

[0069] 2.1 Test substance

[0070] The dry extract powder of Example 1 has the following properties: brownish-yellow powder; slightly fragrant odor and slightly sweet taste; manufactured by Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0071] 2.2 Positive control drug

[0072] Oseltamivir, manufactured by Shanghai Yuanye Biotechnology Co., Ltd., is a positive control drug for influenza A subtypes H3N2 and H1N1 strains and influenza B virus strains.

[0073] Ribavirin, manufactured by Shanghai Yuanye Biotechnology Co., Ltd., is a positive control drug for rhinovirus, human respiratory syncytial virus, parainfluenza virus, enterovirus, coronavirus and adenovirus.

[0074] 2.3 Virus strains

[0075] 2.4 Cell lines

[0076] 2.5 Main Reagents

[0077] 3. Research Content

[0078] 3.1 In vitro antiviral

[0079] 3.1.1 Culture medium preparation

[0080] 3.1.1.1 Complete cell culture medium

[0081] Add 10% FBS to DMEM medium, mix well, and store at 2–8°C for later use.

[0082] 3.1.1.2 Virus maintenance solution

[0083] The virus maintenance medium is DMEM medium with 2% FBS. For influenza virus, an additional 2 μg / mL of TPCK-treated trypsin needs to be added. Mix well and store at 2–8°C for later use.

[0084] 3.1.2 Cell Culture

[0085] MDCK, Hep-2, LLC-MK2, and HeLa cells, all adherent cell lines, were cultured in DMEM medium containing 10% FBS. The culture method was as follows: A monolayer of adherent cells was collected, the medium was discarded, and the cells were washed twice with 1×PBS. Then, 1 mL of 0.25% Trypsin-EDTA was added for digestion. The cells were incubated for 2–5 minutes. Under a microscope, the cells were observed to become rounded and about to detach. 3 mL of complete culture medium was added to stop the digestion. The cells were gently pipetted to form a single-cell suspension, transferred to EP tubes, and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in an appropriate amount of fresh complete culture medium. The cells were then seeded into new culture flasks at a specific ratio and cultured at 37°C in a 5% CO2 incubator. The cells were passaged every 2–3 days depending on their growth.

[0086] 3.1.3 Cytotoxicity test of the test substance

[0087] (1) Experimental groups: Blank control group: serum-free DMEM medium only; Cell control group: cells only; Test drug group and positive control drug group: serially diluted with serum-free DMEM medium at 6 concentrations; 3 replicates were set for each concentration in each group. See Table 1 for specific concentrations.

[0088] Table 1. Concentration Settings for Cytotoxicity Assays

[0089] (2) Cell plating: Cells were prepared into plates with a density of 5 × 10⁶ cells / cm². 4 Single-cell suspensions of 100 μL / well were seeded into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0090] (3) Drug addition detection: Discard the culture medium and wash the cells twice with 1×PBS. Add 100 μL of culture medium containing different concentrations of drugs for each experimental group to each well. Incubate at 37℃ and 5% CO2 for 48 h. Add 10 μL of CCK-8 to each well and incubate for 1–4 h. Measure the OD on a microplate reader. 450nm The values ​​were calculated, and the cell inhibition rate and the half-maximal inhibitory concentration (IC50) of the test drug on the cells were determined. 50 ).

[0091] Cell inhibition rate (%) = (Cell control group OD) 450nm Value - Drug group OD 450nm (value) / (cell control group OD) 450nm (value) × 100%

[0092] 3.1.4 Viral amplification

[0093] Influenza virus culture: Select healthy 9-day-old chicken embryos and mark the boundary between the air cell and allantoic membrane. Place the marked embryos with the air cell facing upwards on a candler. Disinfect the embryos with 75% alcohol. Drill a hole in the air cell end using a sterile awl. Then, draw an appropriate amount of virus solution with a syringe, insert the needle into the drilled hole to puncture the shell membrane, and inject 0.1 mL of virus solution into the allantoic cavity. Incubate at 33°C for 3 days. Check the growth of the embryos daily and discard any dead embryos. Before harvesting, place the embryos in a 4°C refrigerator overnight. After disinfecting the eggshell of the air cell with a 75% alcohol swab, break the shell with sterile forceps, gently peel off the eggshell of the air cell area, remove the shell membrane and chorioallantoic membrane, and gently push the embryo to one side to hold it in place. Aspirate the virus solution and transfer it to a sterile EP tube. Centrifuge at 4000 rpm for 10 minutes, and collect the supernatant as the harvested virus. Store at -80°C for later use.

[0094] Other virus cultures: Host cells were inoculated into culture flasks. When the cell confluence reached 70%–80%, the culture medium was discarded. The cells were washed twice with 1×PBS. Then, 1 mL of the corresponding virus solution was added to the T25 culture flask and incubated in an incubator. The culture plate was gently shaken every 15–30 min to allow the virus to be evenly adsorbed onto the cell surface. After 2 h, the virus solution was discarded. The cells were washed twice with 1×PBS to remove free virus. Then, an appropriate amount of virus maintenance medium was added and the flask was placed in an incubator for continued culture. Specific culture conditions are shown in Table 2.

[0095] Table 2 Virus culture conditions and corresponding hosts

[0096] Observe cell morphology daily under an inverted microscope. When 80%–100% of cells swell and become round, intercellular spaces increase, cell nuclei condense or rupture, or in severe cases, cells partially or completely detach, stop culturing, use repeated freeze-thaw cycles to rupture the cells, centrifuge at 4000 rpm for 10 minutes, collect the supernatant and aliquot it into cryovials for further experiments or for cryopreservation and future use.

[0097] 3.1.5 Tissue culture infectious dose (TCID) 50 Determination of )

[0098] The virus fluid collected in 3.1.4 was subjected to TCID. 50 The specific method for determining [the value] is as follows:

[0099] (1) Cell plating: Cells were prepared into plates with a density of 5 × 10⁶ cells / cm². 4 Single-cell suspensions of 1 cell / mL were seeded into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0100] (2) Virus inoculation: The virus stock solution was serially diluted 10-fold with DMEM medium to obtain 6 different concentrations, each 10-fold. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Eight replicates were set up for each concentration. The original culture medium in the 96-well plate was discarded, and the plate was washed twice with 1×PBS. Then, 100 μL of the corresponding concentration of virus dilution was added to each well. At the same time, two replicates were set up for each dilution with uninoculated cell wells as negative controls.

[0101] (3) Culture and observation: After virus inoculation, 96-well cell culture plates were incubated at 35–37°C in a 5% CO2 incubator for 2 hours. Every 30 minutes, the plates were removed and gently shaken to promote virus adsorption. After adsorption, the medium was changed, with 100 μL of virus maintenance medium in each well, and culture continued. The cytopathic effect was then observed daily under an inverted microscope, and the number of wells with CPE was recorded. The endpoint was reached when no further cytopathic effect was observed at the highest dilution. TCID was calculated using the Reed-Muench formula. 50 :

[0102] TCID 50 = Highest logarithm of virus dilution above 50% CPE + Distance ratio × Logarithm of dilution factor

[0103] The distance ratio is calculated as follows: (Percentage of CPE above 50% - 50%) / (Percentage of CPE above 50% - Percentage of CPE below 50%).

[0104] 3.1.6 In vitro antiviral test of test subject

[0105] (1) Experimental grouping: The concentration was set according to the results of the cytotoxicity test and the literature reports on the antiviral effect of the positive control drug. The virus + drug group, where the drug group is the test substance group or the positive control drug group (diluted to 5 concentrations with virus maintenance solution), the virus control group (with virus but no drug), and the cell control group (with only virus maintenance solution), with 4 replicates for each concentration in each group.

[0106] (2) Cell plating: Cells were prepared into plates with a density of 5 × 10⁶ cells / cm². 4 Single-cell suspensions of 100 μL / well were seeded into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0107] (3) Observation and record of drug administration: Discard the culture medium, wash twice with 1×PBS, and then add 100 TCID. 50Add 50 μL of virus solution per well, allow it to adsorb for 2 h, discard the virus solution, wash twice with 1×PBS, and then add 100 μL of culture medium containing different concentrations of drugs according to the above group settings for further culture.

[0108] Cytopathic effect (CPE) was observed, and culture was terminated when the viral control group showed approximately 75% CPE. The degree of CPE was expressed as the proportion of cells exhibiting CPE, judged according to the following 5-level criteria: (-) Normal cell growth, no cytopathic effect; (+) Cytopathic effect less than 25% of the entire cell monolayer; (++) 25%–50% of cells exhibiting cytopathic effect; (+++) 50%–75% of cells exhibiting cytopathic effect; (++++) 75%–100% of cells exhibiting cytopathic effect. The inhibition rate was calculated using the Reed-Muench method based on the degree of CPE to determine the half-maximal effective concentration (EC50) of the drug. 50 ).

[0109] Table 3. Concentrations of drugs with in vitro antiviral effects

[0110] 3.1.7 Analytical Methods and Result Interpretation

[0111] All data in this experimental summary report were rounded to two decimal places. Original data were analyzed and inhibition rates were calculated using Microsoft Excel 2010. Then, based on the inhibition rates, curve fitting was performed using GraphPad Prism 8.0.1 software to obtain the IC50. 50 or EC 50 Therapeutic Index (TI) Calculation Method: TI = IC 50 / EC 50 .

[0112] 4. Experimental Results

[0113] 4.1 Cytotoxicity Results

[0114] Table 4 shows that the IC50 of the test substance on MDCK, Hep-2, LLC-MK2, and HeLa cells was measured. 50 The values ​​were 2705-3367, 2251-2750, 2264-2840, and 4901-6158 μg / mL, respectively.

[0115] Table 4 IC50 of test substance / positive control drug 50 Summary of results (n=3)

[0116] 4.2 Virus titer determination results

[0117] As shown in Table 5, the TCID values ​​for influenza A virus subtypes H3N2 and H1N1, influenza B virus strains, human rhinovirus, human respiratory syncytial virus, parainfluenza virus, enterovirus (EV71), coronavirus (HCoV-229E), and adenovirus are... 50 The test results are shown in the table below. 100 TCID samples were taken. 50 To conduct subsequent antiviral tests.

[0118] Table 5. Virus infection concentration in cells and tissues (TCID5) 50 Summary (n=8)

[0119] 4.3 In vitro antiviral results

[0120] Table 6. In vitro antiviral EC 50 Summary of results (n=4)

[0121] 5. Conclusion and Evaluation

[0122] The test substance was administered to host cells infected with H3N2, H1N1, IBV, RhV, RSV, PIV, EV71, HCoV-229E, and ADV viruses at different concentrations 2 hours after infection. After a period of culture, cytopathic effects were observed in the virus control group, with cell morphological changes including shrinkage, overlapping, or detachment. After treatment with the test substance, the number of cells was significantly increased compared to the normal cells in the virus control group. Furthermore, within the safe concentration range, the viral inhibition rate increased with increasing drug concentration, and its EC50... 50 The treatment index (TI) is shown in Table 6.

[0123] The results above indicate that the test substance has a certain inhibitory effect on cytopathic effects caused by H3N2, H1N1, IBV, RhV, RSV, PIV, EV71, HCoV-229E and ADV viruses, and has significant antiviral activity.

[0124] Example 3: In vivo antiviral pharmacodynamic study

[0125] Experiment 1: Effects of the test substance on a mouse model of rhinovirus infection

[0126] 1. Experimental Objective

[0127] This study established an in vivo viral infection model in BALB / c mice using nasal drops of ribavirin granules as a positive control. The efficacy of the test drug in the BALB / c mouse nasal virus infection model was evaluated by observing the lung index, immune cells in bronchoalveolar lavage fluid, five-part differential white blood cell count in bronchoalveolar lavage fluid, inflammatory factors in bronchoalveolar lavage fluid, and viral load in the lungs of each group of animals, as well as by examining the airway mechanical barrier function and histopathological examination.

[0128] 2. Experimental materials

[0129] 2.1 Test substances

[0130] The dry extract powder of Example 1, properties of the dry extract powder: brownish-yellow powder, slightly fragrant smell, slightly sweet taste, production unit: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0131] 2.2 Reference substances

[0132] Ribavirin granules, production unit: Kuihua Pharmaceutical Group (Hengshui) Defier Co., Ltd.

[0133] 2.3 Experimental animals

[0134] 130 SPF-level BALB / c mice, half male and half female, with body weight of​​​​​​​​​​​​​​​​​​​​​​​​​​​​(1) Cell plating: Cells were prepared into plates with a density of 5 × 10⁶ cells / cm². 4 Single-cell suspensions of 1 cell / mL were seeded into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0144] (2) Virus inoculation: The virus stock solution was serially diluted 10-fold with DMEM medium to obtain 6 different concentrations, each 10-fold. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Eight replicates were set up for each concentration. The original culture medium in the 96-well plate was discarded, and the plate was washed twice with 1×PBS. Then, 100 μL of the corresponding concentration of virus dilution was added to each well. At the same time, two replicates were set up for each dilution with uninoculated cell wells as negative controls.

[0145] (3) Culture and observation: After virus inoculation, 96-well cell culture plates were incubated at 35–37°C in a 5% CO2 incubator for 2 hours. Every 30 minutes, the plates were removed and gently shaken to promote virus adsorption. After adsorption, the medium was changed, with 100 μL of virus maintenance medium in each well, and culture continued. The cytopathic effect was then observed daily under an inverted microscope, and the number of wells showing cytopathic effect (CPE) was recorded. The endpoint was reached when no further cytopathic effect was observed at the highest dilution. TCID was calculated using the Reed-Muench formula. 50 :

[0146] TCID 50 = Highest dilution logarithm of virus with CPE above 50% + Distance ratio value × Logarithm of dilution factor, where Distance ratio value = (Percentage of CPE above 50% - 50%) / (Percentage of CPE above 50% - Percentage of CPE below 50%).

[0147] 3.2 Grouping, Modeling, and Drug Administration

[0148] One hundred and twenty qualified SPF-grade BALB / c mice, half male and half female, weighing 11.1–17.1 g, were randomly divided into six groups according to sex and weight: normal control group, model control group, ribavirin granule group (75 mg / kg), and low, medium, and high dose groups of the test drug (2.5, 5, and 10 g crude drug / kg, respectively), with 20 animals in each group. Before administration, the drug was prepared to the appropriate concentration using pure water. Each group of animals was administered the corresponding concentration of drug solution orally at a dose of 20 mL / kg once daily for 7 consecutive days. The normal control group and model control group were administered an equal volume of pure water orally. On the fourth day after administration, each group of mice was lightly anesthetized with ether and then administered 100 μL of rhinovirus stock solution nasally. The drug was administered 2 hours after infection. The normal control group was administered an equal volume of blank culture medium nasally.

[0149] 3.3 Indicator Testing

[0150] 3.3.1 General Physiological Observation

[0151] Animals in each group were weighed daily during the drug administration period to compare changes in body weight. The animals' physiological state was observed daily before and after drug administration, including symptoms such as runny nose, sneezing, and coughing.

[0152] 3.3.2 Lung Index

[0153] On the day following the last administration, animals in each group were euthanized by cervical dislocation, and their lungs were dissected, weighed, and the lung index was calculated as follows: lung index = lung wet weight / body weight.

[0154] 3.3.3 Detection of immune cells in bronchoalveolar lavage fluid

[0155] On the day following the last administration, patients in groups 1F01–1F05, 1M01–1M05…6F01–6F05, and 6M01–6M05 were euthanized with cervical dislocation to expose the lungs. After ligation of the left lung, the middle and lower lobes of the right lung were repeatedly lavaged with 0.9% sodium chloride injection. The lavage fluid was collected, and macrophages (M1, M2) and lymphocytes (CD4+) in the bronchoalveolar lavage fluid were detected by flow cytometry. + CD8 + )quantity.

[0156] 3.3.4 Detection of five-part differential white blood cell count and inflammatory factors in bronchoalveolar lavage fluid

[0157] The day after the last administration, 1F06–1F10, 1M06–1M10…6F06–6F10, 6M06–6M10 from each group were euthanized with cervical dislocation to expose the lungs. After ligating the right lung, the left lung was repeatedly lavaged with 0.9% sodium chloride injection. The lavage fluid was collected, and the white blood cell count in the bronchoalveolar lavage fluid was detected using a veterinary five-part differential hematology analyzer. The levels of TNF-α, IL-6, and IL-10 in the bronchoalveolar lavage fluid were detected using an ELISA kit.

[0158] 3.3.5 Viral load detection

[0159] On the day following the last administration, 1F01–1F05, 1M01–1M05…6F01–6F05, and 6M01–6M05 from each group were selected. The upper lobe of the right lung and the bronchus were dissected and placed in a homogenizer. RNA extraction solution was added at a ratio of 1:2 (bronchus-lung mass (mg): RNA extraction solution (μL)). The mixture was homogenized using a high-speed homogenizer to prepare a bronchus-lung suspension. The suspension was centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was collected. The extracted RNA was reverse transcribed into c-DNA, and the rhinovirus load in the bronchus-lung tissue was detected using real-time quantitative PCR.

[0160] 3.3.6 Histopathological examination

[0161] On the day following the last administration, animals 1F01–1F05, 1M01–1M05…6F01–6F05, and 6M01–6M05 from each group were selected. The nose, pharynx, trachea, and left lung were dissected, fixed in 10% neutral formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and examined under a light microscope for histopathological changes in the nasal, pharyngeal, tracheal, and lung tissues. Additionally, tissues from four animals in each group were randomly selected for immunohistochemical staining to detect the expression of occludin-1, E-cadherin, and TLR7 receptors in the lung tissue.

[0162] Immunohistochemical staining: Five fields of view were randomly selected for each animal, and the average optical density value of the positive staining (brownish) in each field of view was measured using Image Plus software. The average optical density value of the five fields of view was used as the receptor expression level of the animal.

[0163] 3.4 Grouping and Dosage Design

[0164] Table 7-1 Grouping and Dosage Design

[0165] 3.5 Data Processing and Statistical Analysis

[0166] The data in this experiment were rounded to the nearest whole number and statistical analysis was performed according to the standard operating procedure (SOP). SPSS 23.0 was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation. The results were presented, and tests for normality and homogeneity of variance were performed. If normality was satisfied (P>0.05), one-way ANOVA was used for statistical analysis, and either LSD+Dunnet (homogeneous variance) or Tamhane's T2 (non-homogeneous variance) was selected for comparison based on the homogeneity of variance. If normality was not satisfied (P≤0.05), the Kruskal-Wallis test was used, and pairwise comparisons were performed using the Mann-Whitney test. Statistical results were presented with α = 0.05 as the test limit, where P≤0.05 indicated statistical significance, and P≤0.01 indicated highly significant differences.

[0167] 4. Experimental Results

[0168] 4.1 Rhinovirus TCID 50 Measurement

[0169] Rhinovirus TCID 50 10 -5.42 / 0.1mL.

[0170] 4.2 General Physiological Observation

[0171] After modeling, all mice exhibited reduced spontaneous activity, piloerection, and rapid breathing. These symptoms persisted in the ribavirin granule group, as well as the low, medium, and high dose groups of the test substance after administration. However, the symptoms of each group were alleviated to varying degrees in the later stages of administration.

[0172] As shown in Table 7-2, the body weight of mice in the model control group was significantly lower than that of the normal control group from day 5 to the day after the last administration (P≤0.01). Compared with the model control group, the body weight of mice in the low-dose test substance group and the ribavirin granule group was significantly increased on day 5 (P≤0.05), the body weight of mice in the low, medium, and high-dose test substance groups and the ribavirin granule group was significantly increased on day 6 (P≤0.05), the body weight of mice in the medium and high-dose test substance groups was significantly increased on the day after the last administration (P≤0.05), and the body weight of mice in the high-dose test substance group was significantly increased on the day after the last administration (P≤0.05).

[0173] Table 7-2 Effects of the test substances on the body weight of rhinovirus mice ( n=20) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0174] Table 7-2 (Continued) Effects of the test substance on the body weight of rhinovirus mice n=20) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05.

[0175] 4.3 Effects on Lung Index

[0176] As shown in Table 7-3, compared with the normal control group, the lung index of mice in the model control group was significantly increased (P≤0.01); compared with the model control group, the lung index of mice in the medium and high dose groups of the test substance and the ribavirin granule group was significantly decreased (P≤0.05 or P≤0.01).

[0177] Table 7-3 Effects of the test substances on the lung index of rhinovirus in mice ( n=20) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0178] 4.4 Effects on immune cells in bronchoalveolar lavage fluid

[0179] As shown in Table 7-4, compared with the normal control group, the CD4 count in the bronchoalveolar lavage fluid of the model control group mice was higher. + CD4 + / CD8 + M2 was significantly reduced (P≤0.01), CD8 + M1 and M1 / M2 were significantly increased (P≤0.05 or P≤0.01); compared with the model control group, CD4 in the bronchoalveolar lavage fluid of mice in the low, medium and high dose groups of the test substance and the ribavirin granule group was significantly increased. + M2 was significantly increased (P≤0.05 or P≤0.01), and the M1 / M2 ratio was significantly decreased (P≤0.01). CD8+ levels in the bronchoalveolar lavage fluid of mice in the medium- and high-dose groups of the test substance and the ribavirin granule group were significantly increased. + Significantly reduced (P≤0.05), CD4 + / CD8 + The levels of M1 in the bronchoalveolar lavage fluid of mice were significantly increased (P≤0.01), while those in the low- and medium-dose groups of the test substance were significantly decreased (P≤0.01).

[0180] Table 7-4 Effects of the test substances on immune cells in bronchoalveolar lavage fluid from mice with rhinovirus ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0181] Table 7-4 (Continued) Effects of the test substance on immune cells in bronchoalveolar lavage fluid of rhinovirus mice n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0182] 4.5 Effect on the five-part differential count of white blood cells in bronchoalveolar lavage fluid

[0183] As shown in Table 7-5, compared with the normal control group, the levels of WBC and Neu in the bronchoalveolar lavage fluid of mice in the model control group were significantly increased (P≤0.05 or P≤0.01); compared with the model control group, the levels of WBC in the bronchoalveolar lavage fluid of mice in the high-dose test substance group and the ribavirin granule group were significantly decreased (P≤0.05).

[0184] Table 7-5 Effects of the test substances on the five-part differential count of white blood cells in mouse bronchoalveolar lavage fluid containing rhinovirus ( n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0185] Table 7-5 continues the discussion of the effects of the test substance on the five-part differential count of white blood cells in the bronchoalveolar lavage fluid of rhinovirus mice. n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0186] 4.6 Effects on inflammatory factors in bronchoalveolar lavage fluid

[0187] As shown in Table 7-6, compared with the normal control group, the IL-6 level in the bronchoalveolar lavage fluid of mice in the model control group was significantly increased (P≤0.01), and the IL-10 level was significantly decreased (P≤0.05). Compared with the model control group, the IL-6 level in the bronchoalveolar lavage fluid of mice in the low-, medium-, and high-dose groups of the test substance and the ribavirin granule group was significantly decreased (P≤0.05 or P≤0.01), while the IL-10 level in the bronchoalveolar lavage fluid of mice in the high-dose group of the test substance and the ribavirin granule group was significantly increased (P≤0.05).

[0188] Table 7-6 Effects of the test substances on inflammatory factors in bronchoalveolar lavage fluid from mice with rhinovirus ( n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0189] 4.7 Impact on viral load

[0190] As shown in Table 7-7, compared with the normal control group, the viral load in the bronchopulmonary tissue of mice in the model control group was significantly increased (P≤0.01); compared with the model control group, the viral load in the bronchopulmonary tissue of mice in the low, medium and high dose groups of the test substance and the ribavirin granule group was significantly decreased (P≤0.05 or P≤0.01).

[0191] Table 7-7 Effects of the test substances on viral load in mouse bronchopulmonary tissue of rhinovirus ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0192] 4.8 Histopathological examination

[0193] As shown in Figure 1, microscopic observation of the nose, pharynx, trachea of ​​each group of animals and the lung tissue of the normal control group on the day after the last administration showed no significant changes, and no inflammatory cell infiltration was observed. The lung tissue of mice in the model control group showed inflammatory and hemorrhagic exudation, alveolar epithelial destruction, alveolar wall thickening and inflammatory cell infiltration. The lung lesions of mice in the low, medium and high dose groups of the test substance and the ribavirin granule group were all reduced to varying degrees compared with the model control group.

[0194] As shown in Tables 7-8, compared with the normal control group, the lung tissue pathology score of the model control group was significantly increased on the day after the last administration (P≤0.01); compared with the model control group, the lung tissue pathology scores of mice in the high-dose test substance group and the ribavirin granule group were significantly reduced (P≤0.01).

[0195] Table 7-8 Effects of the test substances on the pathological scores of rhinovirus-induced mouse lung tissue ( n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0196] 4.9 Immunohistochemical staining

[0197] As shown in Tables 7-9 and Figure 2, compared with the normal control group, the mean optical density values ​​of E-cadherin and Occludin in the lung tissue of the model control group were significantly reduced (P≤0.05 or P≤0.01), while the mean optical density value of TLR7 was significantly increased (P≤0.01). Compared with the model control group, the mean optical density value of E-cadherin in the lung tissue of mice in the medium- and high-dose groups of the test substance and the ribavirin granule group was significantly increased (P≤0.05 or P≤0.01), while the mean optical density value of TLR7 in the lung tissue of mice in the high-dose group of the test substance was significantly reduced (P≤0.05).

[0198] Table 7-9 Effects of the test substances on the immune barrier of rhinovirus in mouse lung tissue ( n=4) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0199] 5. Experiment Summary

[0200] The test substance significantly increased body weight in rhinovirus-infected mice, reduced lung index and viral load in lung tissue, inhibited the production of white blood cells (WBCs), restored the levels of inflammatory factors in bronchoalveolar lavage fluid and the proportion of related immune cells such as lymphocytes and macrophages, upregulated the expression level of E-cadherin in lung tissue, downregulated the expression level of TLR7 in lung tissue, and significantly improved the degree of lung lesions in model mice, suggesting that the test substance has a significant therapeutic effect on rhinovirus-infected mice, with an effective dose of low dose (2.5 g crude drug / kg).

[0201] Effect of the Test Substance on a Mouse Model Infected with Influenza A (H1N1) Virus

[0202] 1. Experimental Purpose

[0203] In this study, a BALB / c mouse model of in - vivo influenza A (H1N1) virus infection was established by intranasal instillation of influenza A (H1N1) virus. Oseltamivir phosphate granules were selected as the positive control drug. By observing the lung index, immune cells in bronchoalveolar lavage fluid (BALF), five - category white blood cells in BALF, inflammatory factors in BALF, viral load in the lungs of animals in each group, and conducting airway mechanical barrier function and histopathological examinations, the efficacy of the test substance on the influenza A (H1N1) virus - infected BALB / c mouse model was evaluated.

[0204] 2. Experimental Materials

[0205] 2.1 Test Substance

[0206] The dry extract powder of Example 1, production unit: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0207] 2.2 Reference Substance

[0208] Oseltamivir phosphate granules, production unit: Yichang Dong阳光Yangtze River Pharmaceutical Co., Ltd.

[0209] 2.3 Experimental Animals

[0210] 130 SPF - level BALB / c mice, half male and half female, with body weight of 11.5 - 15.4 g, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (SCXK(Xiang)2019 - 0004).

[0211] 2.4 Virus Strain

[0212] Influenza A subtype H1N1 strain, provided by ATCC in the United States, number: VR - 1736. Cultured and amplified in the Pathogenic Microorganism Laboratory of Hunan Prima Pharmaceutical Research Center.

[0213] 3. Experimental Methods

[0214] 3.1 Culture, Amplification and Virulence Detection of Influenza A (H1N1) Virus

[0215] 3.1.1 Virus Culture and Amplification

[0216] Select healthy 9-day-old chicken embryos and mark the boundary between the air cell and allantoic cavity. Place the marked embryos with the air cell facing upwards on a candler. Disinfect the embryos with 75% alcohol and drill a hole in the air cell end using a sterile awl. Draw an appropriate amount of virus solution into a syringe, insert the needle into the drilled hole to puncture the shell membrane, and inject 0.2 mL of virus solution into the allantoic cavity. Incubate the embryos at 34°C for 2 days. (Check the embryo growth daily; discard any embryos that die within 24 hours as non-specific deaths.) Before harvesting, freeze the embryos overnight at 4°C to kill them and coagulate the red blood cells. Disinfect the air cell portion of the eggshell with 75% alcohol swabs after freezing. Using sterile tweezers, break the sterilized shell, gently peel off the eggshell from the air cell, remove the shell membrane and chorioallantoic membrane, gently push the chicken embryo to one side, press it down, aspirate the virus fluid, centrifuge at 4000 rpm for 10 minutes, collect the supernatant and aliquot it into cryovials, store at -80℃ for later use.

[0217] 3.1.2 Toxicity Detection

[0218] The titer of the virus amplified in 3.1.1 was determined using the following method:

[0219] Add 50 μL of 0.9% sodium chloride injection to each well of a 96-well disposable reaction plate, except for the first well. Add 100 μL of sample to well 1; then transfer 50 μL of liquid from well 1 to well 2 and gently pipette to mix. Continue this dilution up to well 11, then discard 50 μL of liquid from well 11. Add 50 μL of 0.9% sodium chloride injection to well 12 as a negative control. Add 50 μL of 1.0% chicken erythrocyte suspension to each well. Gently mix the reaction plate and incubate at room temperature for 20–45 min, observing the results. When interpreting the results, tilt the reaction plate slightly (not upright) and read from the back side of the plate. Read the results within approximately 5 seconds. Express the results using log2, where n log2 indicates that agglutination occurs when the virus is diluted to the power of n.

[0220] Results Interpretation: "—" indicates no agglutination, with red blood cells deposited at the bottom of the tube in a neatly shaped disc; "+" indicates trace agglutination, with red blood cells deposited at the bottom of the tube in a disc-shaped disc with indistinct edges; "++" indicates agglutination, with red blood cells deposited at the bottom of the tube in a ring shape with small agglutinated clumps around the edges; "+++" indicates substantial agglutination, with red blood cells forming granular agglutinations with irregular edges and a tendency to droop; "++++" indicates complete agglutination, with red blood cells evenly distributed at the bottom of the tube. The highest dilution that results in complete agglutination is taken as the blood coagulation titer.

[0221] 3.2 Grouping, Modeling, and Drug Administration

[0222] One hundred and twenty qualified SPF-grade BALB / c mice, half male and half female, weighing 11.5–15.4 g, were randomly divided into six groups according to sex and weight: normal control group, model control group, oseltamivir phosphate granule group (25 mg / kg), and low, medium, and high dose groups of the test substance (2.5, 5, and 10 g crude drug / kg, respectively), with 20 animals in each group. Before administration, the drug was prepared to the appropriate concentration using pure water. Each group of animals was administered the corresponding concentration of drug solution orally at a dose of 20 mL / kg once daily for seven consecutive days. The normal control group and model control group were administered an equal volume of pure water orally. On the fourth day after administration, each group of mice was lightly anesthetized with ether and then intranasally instilled with 100 μL / mouse of H1N1 influenza A virus stock solution. The drug was administered 2 hours after infection. The normal control group was intranasally instilled with an equal volume of blank culture medium.

[0223] 3.3 Indicator Testing

[0224] 3.3.1 General Physiological Observation

[0225] Animals in each group were weighed daily during the drug administration period to compare changes in body weight. The animals' physiological state was observed daily before and after drug administration, including symptoms such as runny nose, sneezing, and coughing.

[0226] 3.3.2 Lung Index

[0227] On the day following the last administration, animals in each group were euthanized by cervical dislocation, and their lungs were dissected, weighed, and the lung index was calculated as follows: lung index = lung wet weight / body weight.

[0228] 3.3.3 Detection of immune cells in bronchoalveolar lavage fluid

[0229] On the day following the last administration, patients in groups 1F01–1F05, 1M01–1M05…6F01–6F05, and 6M01–6M05 were euthanized with cervical dislocation to expose the lungs. After ligation of the left lung, the middle and lower lobes of the right lung were repeatedly lavaged with 0.9% sodium chloride injection. The lavage fluid was collected, and macrophages (M1, M2) and lymphocytes (CD4+) in the bronchoalveolar lavage fluid were detected by flow cytometry. + CD8 + )quantity.

[0230] 3.3.4 Detection of five-part differential white blood cell count and inflammatory factors in bronchoalveolar lavage fluid

[0231] The day after the last administration, 1F06–1F10, 1M06–1M10…6F06–6F10, 6M06–6M10 from each group were euthanized with cervical dislocation to expose the lungs. After ligating the right lung, the left lung was repeatedly lavaged with 0.9% sodium chloride injection. The lavage fluid was collected, and the white blood cell count in the bronchoalveolar lavage fluid was detected using a veterinary five-part differential hematology analyzer. The levels of TNF-α, IL-6, and IL-10 in the bronchoalveolar lavage fluid were detected using an ELISA kit.

[0232] 3.3.5 Viral load detection

[0233] On the day following the last administration, 1F01–1F05, 1M01–1M05…6F01–6F05, and 6M01–6M05 from each group were selected. The upper lobe of the right lung and the bronchus were dissected and placed in a homogenizer. RNA extraction solution was added at a ratio of 1:2 (bronchus-lung mass (mg): RNA extraction solution (μL)). The mixture was homogenized using a high-speed homogenizer to prepare a bronchus-lung suspension. The suspension was centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was collected. The extracted RNA was reverse transcribed into c-DNA, and the rhinovirus load in the bronchus-lung tissue was detected using real-time quantitative PCR.

[0234] 3.3.6 Blood coagulation titer detection

[0235] On the day following the last administration, 1F01–1F05, 1M01–1M05…6F01–6F05, and 6M01–6M05 from each group were selected. The upper lobe of the right lung and the bronchus were dissected and placed in a homogenizer. 0.9% sodium chloride injection was added at a ratio of bronchus-lung mass (g): 0.9% sodium chloride injection (mL) = 1:9. The mixture was manually ground and homogenized to prepare a mouse bronchus-lung suspension. The viral titer in the bronchus-lung tissue was detected using a hemagglutination inhibition test.

[0236] 3.3.7 Histopathological examination

[0237] On the day following the last administration, 1F01–1F05, 1M01–1M05…6F01–6F05, and 6M01–6M05 from each group were selected. Nasal, pharyngeal, tracheal, and left lung tissues were dissected, fixed in 10% neutral formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for pathological changes in the nasal, pharyngeal, tracheal, and lung tissues. Additionally, tissues from four animals in each group were randomly selected for immunohistochemical staining to detect the expression of occludin-1, E-cadherin, and TLR7 receptors in the lung tissue.

[0238] Immunohistochemical staining: Five fields of view were randomly selected for each animal, and the average optical density value of the positive staining (brownish) in each field of view was measured using Image Plus software. The average optical density value of the five fields of view was used as the receptor expression level of the animal.

[0239] 3.4 Dosage Design

[0240] For specific groupings and dosages, please refer to Table 8-1.

[0241] Table 8-1 Grouping and Dosage Design

[0242] 3.5 Data Processing and Statistical Analysis

[0243] The process is the same as "Data Processing and Statistical Analysis" in Experiment 1 of this embodiment.

[0244] 4. Experimental Results

[0245] 4.1 H1N1 Influenza Virus Titer

[0246] The hemagglutination titer of the H1N1 influenza virus was 7 log2.

[0247] 4.2 General Physiological Observation

[0248] After modeling, all mice exhibited reduced spontaneous activity, piloerection, and rapid breathing. The above symptoms persisted in the oseltamivir phosphate granule group, as well as the low, medium, and high dose groups of the test substance after administration. However, the symptoms of the animals in each group were alleviated to varying degrees in the later stages of administration.

[0249] As shown in Table 8-2, the body weight of mice in the model control group was significantly lower than that of the normal control group from D5 to the day after the last administration (P≤0.01); compared with the model control group, the body weight of the high-dose group of the test substance was significantly higher after the last administration (P≤0.05), and the body weight of the medium- and high-dose groups of the test substance and the oseltamivir phosphate granule group was significantly higher on the day after the last administration (P≤0.05 or P≤0.01).

[0250] Table 8-2 Effects of the test substances on the body weight of mice infected with H1N1 influenza A virus ( n=20) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0251] Continued from Table 8-2: Effects of the test substance on the body weight of mice infected with H1N1 influenza A virus. n=20) Note: Compared with the normal control group+ P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0252] 4.3 Effects on Lung Index

[0253] As shown in Table 8-3, compared with the normal control group, the lung index of mice in the model control group was significantly increased (P≤0.01); compared with the model control group, the lung index of mice in the medium and high dose groups of the test substance and the oseltamivir phosphate granule group was significantly decreased (P≤0.01).

[0254] Table 8-3 Effects of the test substances on the lung index of mice with H1N1 influenza A virus ( n=20) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0255] 4.4 Effects on immune cells in bronchoalveolar lavage fluid

[0256] As shown in Table 8-4, compared with the normal control group, the CD4 content in the bronchoalveolar lavage fluid of the model control group mice was significantly higher. + CD4 + / CD8 + M2 was significantly reduced (P≤0.01), CD8 + M1 and M1 / M2 were significantly increased (P≤0.01); compared with the model control group, CD4 in the bronchoalveolar lavage fluid of mice in the low, medium and high dose groups of the test substance and the oseltamivir phosphate granule group was significantly increased. + M2 was significantly increased (P≤0.05 or P≤0.01), while M1 and M1 / M2 were significantly decreased (P≤0.05 or P≤0.01). CD8+ levels in bronchoalveolar lavage fluid of mice in the high-dose test substance group and the oseltamivir phosphate granule group were significantly increased. + The CD4 count in bronchoalveolar lavage fluid of mice in the medium- and high-dose groups of the test substance and the oseltamivir phosphate granule group was significantly reduced (P≤0.01). + / CD8 + Significantly elevated (P≤0.05 or P≤0.01).

[0257] Table 8-4 Effects of the test substances on immune cells in mouse bronchoalveolar lavage fluid containing H1N1 influenza A virus ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group *P≤0.05, ** P≤0.01.

[0258] Table 8-4 (Continued) Effects of the test substance on immune cells in bronchoalveolar lavage fluid of mice infected with H1N1 influenza A virus ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0259] 4.5 Effect on the five-part differential count of white blood cells in bronchoalveolar lavage fluid

[0260] As shown in Table 8-5, compared with the normal control group, the WBC level in the bronchoalveolar lavage fluid of mice in the model control group was significantly increased (P≤0.01), and the Eos level was significantly decreased (P≤0.01). Compared with the model control group, the WBC level in the bronchoalveolar lavage fluid of mice in the medium- and high-dose groups of the test substance and the oseltamivir phosphate granule group was significantly decreased (P≤0.05 or P≤0.01), and the Eos level in the bronchoalveolar lavage fluid of mice in the medium-dose group of the test substance was significantly increased (P≤0.05).

[0261] Table 8-5 Effects of the test substances on the five-part differential count of white blood cells in bronchoalveolar lavage fluid of mice infected with H1N1 influenza A virus. n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0262] Table 8-5 continues the discussion of the effects of the test substance on the five-part differential count of white blood cells in the bronchoalveolar lavage fluid of mice infected with H1N1 influenza A virus. n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0263] 4.6 Effects on inflammatory factors in bronchoalveolar lavage fluid

[0264] As shown in Table 8-6, compared with the normal control group, the levels of IL-6 and TNF-α in the bronchoalveolar lavage fluid of mice in the model control group were significantly increased (P≤0.05 or P≤0.01), and the level of IL-10 was significantly decreased (P≤0.01). Compared with the model control group, the levels of IL-6 in the bronchoalveolar lavage fluid of mice in the low-, medium-, and high-dose groups of the test substance and the oseltamivir phosphate granule group were significantly decreased (P≤0.01), while the levels of IL-10 in the bronchoalveolar lavage fluid of mice in the high-dose group of the test substance and the oseltamivir phosphate granule group were significantly increased (P≤0.05 or P≤0.01), and the levels of TNF-α in the bronchoalveolar lavage fluid of mice in the high-dose group of the test substance were significantly decreased (P≤0.05).

[0265] Table 8-6 Effects of the test substances on inflammatory factors in bronchoalveolar lavage fluid of mice infected with H1N1 influenza A virus ( n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0266] 4.7 Impact on viral load

[0267] As shown in Table 8-7, compared with the normal control group, the viral load in the bronchopulmonary tissue of mice in the model control group was significantly increased (P≤0.01); compared with the model control group, the viral load in the bronchopulmonary tissue of mice in the low, medium and high dose groups of the test substance and the oseltamivir phosphate granule group was significantly decreased (P≤0.01).

[0268] Table 8-7 Effects of the test substances on viral load of H1N1 influenza virus in mouse bronchopulmonary tissue (H1N1 influenza A virus) n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0269] 4.8 Effect on blood coagulation titer

[0270] As shown in Table 8-8, compared with the normal control group, the viral titer in the bronchopulmonary tissue of mice in the model control group was significantly increased (P≤0.01); compared with the model control group, the viral titer in the bronchopulmonary tissue of mice in the low, medium and high dose groups of the test substance and the oseltamivir phosphate granule group was significantly decreased (P≤0.05 or P≤0.01).

[0271] Table 2-8 Effects of the test substances on hemagglutination titers of mouse bronchopulmonary tissues containing H1N1 influenza A virus ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0272] 4.9 Histopathological examination

[0273] As shown in Figure 3, microscopic observation of the nose, pharynx, trachea of ​​each group of animals and the lung tissue of the normal control group on the day after the last administration showed no significant changes, and no inflammatory cell infiltration was observed. The lung tissue of mice in the model control group showed inflammatory and hemorrhagic exudation, alveolar epithelial destruction, alveolar wall thickening and inflammatory cell infiltration. The lung lesions of mice in the low, medium and high dose groups of the test substance and the oseltamivir phosphate granule group were all reduced to varying degrees compared with the model control group.

[0274] As shown in Tables 8-9, compared with the normal control group, the lung tissue pathology score of the model control group was significantly increased on the day after the last administration (P≤0.01); compared with the model control group, the lung tissue pathology scores of mice in the high-dose test substance group and the oseltamivir phosphate granule group were significantly reduced (P≤0.05 or P≤0.01).

[0275] Table 8-9 Effects of the test substances on the pathological scores of mouse lung tissues infected with H1N1 influenza A virus ( n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0276] 4.10 Immunohistochemical staining

[0277] As shown in Tables 8-10 and Figure 4, compared with the normal control group, the mean optical density values ​​of E-cadherin and Occludin in the lung tissue of the model control group were significantly reduced (P≤0.05), while the mean optical density value of TLR7 was significantly increased (P≤0.01). Compared with the model control group, the mean optical density value of E-cadherin in the lung tissue of mice in the medium- and high-dose groups of the test substance and the oseltamivir phosphate granule group was significantly increased (P≤0.05 or P≤0.01), the mean optical density value of Occludin in the lung tissue of mice in the medium- and high-dose groups of the test substance was significantly increased (P≤0.05 or P≤0.01), and the mean optical density value of TLR7 in the lung tissue of mice in the medium- and high-dose groups of the test substance and the oseltamivir phosphate granule group was significantly reduced (P≤0.05).

[0278] Table 8-10 Effects of the test substances on the immune barrier of mouse lung tissue against H1N1 influenza A virus ( n=4) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0279] 5. Experiment Summary

[0280] The test substance significantly increased body weight and eosinophil levels in mice infected with H1N1 influenza A virus, reduced lung index, viral load and viral titer in lung tissue, inhibited the production of white blood cells (WBCs), restored the levels of inflammatory factors in bronchoalveolar lavage fluid and the proportion of related immune cells such as lymphocytes and macrophages, upregulated the expression levels of E-cadherin and Occludin in lung tissue, downregulated the expression level of TLR7 in lung tissue, and significantly improved the degree of lung lesions in model mice. This suggests that the test substance has a significant therapeutic effect on mice infected with H1N1 influenza A virus, with the effective dose being a low dose (2.5 g crude drug / kg).

[0281] Example 4: Pharmacological effect of the test substance on an aged mouse rhinovirus infection model.

[0282] 1. Experimental Objective

[0283] This study established an in vivo viral infection model by intranasal infection of 17-month-old C57BL / 6J mice with rhinovirus. Ribavirin granules were used as a positive control. The efficacy of the test drug on the rhinovirus infection model of aged C57BL / 6J mice was investigated by observing changes in body weight, lung index, bronchoalveolar lavage fluid inflammatory factors, humoral and cellular immune indicators, and viral load in the lungs of each group of animals.

[0284] 2. Experimental Materials

[0285] 2.1 Test substance

[0286] The dry ointment powder of Example 1 was manufactured by Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0287] 2.2 Reference Standard

[0288] Ribavirin granules, manufactured by Sunflower Pharmaceutical Group (Hengshui) Defeier Co., Ltd.

[0289] 2.3 Laboratory Animals

[0290] 120 SPF-grade C57BL / 6J mice, half male and half female, 17 months old, were purchased from Pizhou Oriental Breeding Co., Ltd. (SCXK(Su)2022-0005).

[0291] 2.4 Virus strains

[0292] The human rhinovirus strain, provided by the American Technical Commission (ATCC) and designated VR-1187, was cultured and amplified in the Pathogenic Microbiology Laboratory of the Hunan Prima Pharmaceutical Research Center.

[0293] 2.5 Main Reagents

[0294] 3. Experimental Methods

[0295] 3.1 Rhinovirus culture, amplification, and virulence detection

[0296] 3.1.1 Virus culture and amplification

[0297] HeLa host cells were seeded into culture flasks. When the cell confluence reached 70%–80%, the culture medium was discarded, and the cells were washed twice with 1×PBS. Then, 1 mL of the corresponding virus solution was added to the T25 culture flask and incubated. The culture flask was gently shaken every 15–30 minutes to ensure that the virus was evenly adsorbed onto the cell surface. After 2 hours, the virus solution was discarded, and the cells were washed twice with 1×PBS to remove free virus. An appropriate amount of virus maintenance medium was added, and the flask was placed in an incubator for continued culture. Cell morphology was observed daily under an inverted microscope. When 80%–100% of the cells swelled and became round, the intercellular spaces increased, and the nuclei condensed or ruptured, or in severe cases, the cells partially or completely detached, the culture was stopped. The cells were ruptured by repeated freeze-thaw cycles, centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected, aliquoted into cryovials, and stored at ultra-low temperature for later use.

[0298] 3.1.2 Toxicity Detection

[0299] TCID was performed on the virus amplified in section 3.1.1. 50 The specific method for measurement is as follows:

[0300] (1) Cell plating: Cells were prepared into plates with a density of 5 × 10⁶ cells / cm². 4 Single-cell suspensions of 1 cell / mL were seeded into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0301] (2) Virus inoculation: The virus stock solution was serially diluted 10-fold with DMEM medium to obtain 6 different concentrations, each 10-fold. -1 10 -2 10 -3 10 -4 10 -5 10-6 Eight replicates were set up for each concentration. The original culture medium in the 96-well plate was discarded, and the plate was washed twice with 1×PBS. Then, 100 μL of the corresponding concentration of virus dilution was added to each well. At the same time, two replicates were set up for each dilution with uninoculated cell wells as negative controls.

[0302] (3) Culture and observation: After virus inoculation, 96-well cell culture plates were incubated at 35–37°C in a 5% CO2 incubator for 2 hours. Every 30 minutes, the plates were removed and gently shaken to promote virus adsorption. After adsorption, the medium was changed, with 100 μL of virus maintenance medium in each well, and culture continued. The cytopathic effect was then observed daily under an inverted microscope, and the number of wells with CPE was recorded. The endpoint was reached when no further cytopathic effect was observed at the highest dilution. The median tissue culture infection dose (TCID) was calculated using the Reed-Muench formula. 50 ):

[0303] TCID 50 = Highest dilution logarithm of virus with CPE above 50% + Distance ratio value × Logarithm of dilution factor, where Distance ratio value = (Percentage of CPE above 50% - 50%) / (Percentage of CPE above 50% - Percentage of CPE below 50%).

[0304] 3.2 Grouping, Modeling, and Drug Administration

[0305] One hundred and twenty 17-month-old BALB / c mice (half male and half female, weighing 30.0–47.4 g) that passed quarantine inspection and were SPF grade were randomly divided into six groups according to sex and weight: normal control group, model control group, ribavirin granule group (75 mg / kg), and low, medium, and high dose groups of the test drug (2.5, 5, and 10 g crude drug / kg, respectively), with 20 animals in each group. Before administration, the drug was prepared to the appropriate concentration using pure water. Each group of animals was administered the corresponding concentration of drug solution orally at a dose of 20 mL / kg once daily for seven consecutive days. The normal control group and model control group were administered an equal volume of pure water orally. On the fourth day after administration, each group of mice was lightly anesthetized with ether and then instilled with 100 μL / mouse (50 μL / well) of rhinovirus stock solution via nasal drip. Administration was performed 2 hours after infection. The normal control group was instilled with an equal volume of DMEM culture medium via nasal drip.

[0306] 3.3 Indicator Testing

[0307] 3.3.1 General Physiological Observation

[0308] Animals in each group were weighed daily during the drug administration period to compare changes in body weight. The animals' physiological state before and after drug administration was observed, including symptoms such as runny nose, sneezing, and coughing.

[0309] 3.3.2 Lung Index

[0310] On the day following the last administration, animals in each group were euthanized by cervical dislocation, and their lungs were dissected, weighed, and the lung index was calculated as follows: lung index = lung wet weight / body weight.

[0311] 3.3.3 Detection of viral titer in lung tissue

[0312] The day after the last administration, animals in each group (F01-F05, M01-M05) were selected, and right lung tissue was dissected and placed in a homogenizer. 0.9% sodium chloride injection was added at a ratio of right lung mass (mg): 0.9% sodium chloride injection (mL) = 1:9. The mixture was centrifuged at 4000 rpm for 7 minutes, and the supernatant was collected. 1*10 4 HeLa cells were seeded at 10⁶ / mL in 24-well plates and incubated at 37°C with 5% CO₂ for 24 h. The plates were then divided into blank wells, virus control wells, and sample wells. DMEM was used to isolate the sample and control wells at a ratio of 10⁶ / mL. -1 10 -2 10 -3 10 -4 10 -5 Dilute the virus solution by adding 0.1 mL of diluted virus solution to each well of a cell culture plate, with two parallel wells for each dilution. Incubate the cell culture plate at 37°C with 5% CO2 for 1 hour to allow virus adsorption, tilting and shaking the plate every 30 minutes. After 1 hour of adsorption, discard the supernatant. Wash the cells once with DMEM. Incubate the agar in a 56°C water bath with DMEM at 37°C. Mix agar and DMEM at a 1:1 ratio, and add 1 mL of the mixture to each well of a 24-well plate. Place the cell culture plate in a biosafety cabinet until the agar solidifies, then incubate upside down at 37°C with 5% CO2 for 2–5 days. Once white spots appear, stain with crystal violet, adding 1 mL to each well and incubating at 37°C for 4 hours. After removing the stain, count the empty spots.

[0313] 3.3.4 Detection of immune indicators

[0314] On the day following the last administration, blood was collected from the jugular vein of each group of animals (F01–F05, M01–M05) and placed in EDTA-K2 anticoagulant tubes. Flow cytometry was used to detect the number of CD4+ lymphocytes in the whole blood. + CD8 +Number of animals: Select animals from each group (F06-F10, M06-M10), collect blood from the jugular vein and place it in tubes without anticoagulant. Use a mouse ELISA kit to detect the serum levels of IgA, IgG, and IgM. Euthanize the mice in each group (F01-F05, M01-M05) by cervical dislocation, dissect and take the left lung, and use flow cytometry to detect macrophages (M1, M2) in the lung tissue. Also dissect and take the spleen and thymus, and use flow cytometry to detect the percentage of helper T cells (Th17), regulatory T cells (Treg), dendritic cells (DC), and NK cells in the spleen and thymus tissues, respectively.

[0315] 3.3.5 Detection of inflammatory factors in bronchoalveolar lavage fluid

[0316] The day after the last administration, animals from each group (F06-F10, M06-M10) were euthanized by cervical dislocation to expose the lungs. After ligating the left lung, the right lung was repeatedly irrigated with 0.9% sodium chloride injection. The irrigating fluid was collected, and the levels of TNF-α and IL-1β in the bronchoalveolar lavage fluid were detected using an ELISA kit.

[0317] 3.3.6 Histopathological examination

[0318] On the day following the last administration, each group (F06-F10, M06-M10) was selected, and the left lung was dissected, fixed in 10% neutral formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for pathological changes in lung tissue.

[0319] 3.4 Dosage Design

[0320] For specific groupings and dosages, please refer to Table 9-1.

[0321] Table 9-1 Grouping and Dosage Design

[0322] 3.5 Data Processing and Statistical Analysis

[0323] The process is the same as "Data Processing and Statistical Analysis" in Experiment 1 of Example 3.

[0324] 4. Experimental Results

[0325] 4.1 Rhinovirus TCID 50 Measurement

[0326] The rhinovirus TCID used in this experiment was tested and found to be positive. 50 10 -4.55 / 0.1mL.

[0327] 4.2 General Physiological Observation

[0328] After modeling, all mice exhibited reduced spontaneous activity and piloerection. These symptoms persisted in the ribavirin granule group, as well as the low, medium, and high dose groups of the test substance after administration. However, the symptoms in each group showed varying degrees of relief in the later stages of administration. As shown in Table 9-2, from 24 hours after modeling to D4, the body weight of mice in the model control group was significantly lower than that of the normal control group (P≤0.01). Compared with the model control group, there were no significant differences in body weight among the low, medium, and high dose groups of the test substance and the ribavirin granule group.

[0329] Table 9-2 Effects of the test substances on the body weight of rhinovirus-infected mice ( n=20) Note: Compared with the normal control group ++ P≤0.01.

[0330] 4.3 Effects on Lung Index

[0331] As shown in Table 9-3, compared with the normal control group, the lung index of mice in the model control group was significantly increased (P≤0.01); compared with the model control group, the lung index of mice in the medium and high dose groups of the test substance and the ribavirin granule group was significantly decreased (P≤0.05).

[0332] Table 9-3 Effects of the test substances on the lung index of rhinovirus-infected mice ( n=20) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group * P≤0.05.

[0333] 4.4 Effect on viral titer in lung tissue

[0334] As shown in Table 9-4, compared with the normal control group, the viral titer in the lung tissue of mice in the model control group was significantly increased (P≤0.01); compared with the model control group, the viral titer in the lung tissue of mice in the low, medium and high dose groups of the test substance and the ribavirin granule group was significantly decreased (P≤0.01).

[0335] Table 9-4 Effects of test substances on viral titers in lung tissue of rhinovirus-infected mice ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group ** P≤0.01.

[0336] 4.5 Effects on cellular immunity

[0337] 4.5.1 Effects on lymphocytes

[0338] As shown in Table 9-5, compared with the normal control group, the CD8+ level in the serum of mice in the model control group was significantly lower.+ The percentage increased significantly (P≤0.05), CD4 + / CD8 + The levels of CD8 in the blood of mice in the low-, medium-, and high-dose groups of the test substance and the ribavirin granule group were significantly reduced (P≤0.01); compared with the model control group, the levels of CD8 in the blood of mice in the low-, medium-, and high-dose groups of the test substance and the ribavirin granule group were significantly reduced. + The percentage was significantly reduced (P≤0.05 or P≤0.01) in the serum of mice in the low, medium, and high dose groups of the test substance, with CD4 levels significantly decreased. + / CD8 + All were significantly elevated (P≤0.05 or P≤0.01).

[0339] Table 9-5 Effects of the test substances on rhinovirus-infected mouse lymphocytes ( n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0340] 4.5.2 Effects on lung tissue macrophages

[0341] As shown in Table 9-6, compared with the normal control group, the M2% of lung tissue in the model control group mice was significantly reduced, and the M1 / M2 ratio was significantly increased (P≤0.01). Compared with the model control group, the M1 / M2 ratio of lung tissue in mice in the low-dose group, high-dose group of the test substance and ribavirin granule group was significantly reduced (P≤0.01). The M1 / M2 ratio in the medium-dose group of the test substance showed a decreasing trend, but there was no statistical difference (P≥0.05).

[0342] Table 9-6 Effects of the test substances on macrophages in the lung tissue of mice infected with rhinovirus ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group ** P≤0.01.

[0343] 4.5.3 Effects on NK, DC, Th17, and Treg cells in the spleen

[0344] As shown in Table 9-7, compared with the normal control group, the number of NK cells, DCs, and Treg / Th17 ratio in the spleen tissue of mice in the model control group were significantly decreased (P≤0.01); compared with the model control group, the number of NK cells in the spleen tissue of mice in the low, medium, and high dose groups of the test substance and the ribavirin granule group were significantly increased (P≤0.05 or P≤0.01), the number of DC cells in the spleen tissue of mice in the medium and high dose groups was significantly increased (P≤0.01), and the Treg / Th17 ratio in the spleen tissue of mice in the high dose group of the test substance and the ribavirin granule group was significantly increased (P≤0.05 or P≤0.01).

[0345] Table 9-7 Effects of the test substances on NK, DC, Th17, and Treg cells in the spleen of mice infected with rhinovirus ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0346] 4.5.4 Effects on thymic NK, DC, Th17, and Treg cells

[0347] As shown in Table 9-8, compared with the normal control group, there were no significant differences in NK cells, DCs, and Treg / Th17 ratio in the thymus tissue of mice in the model control group; compared with the model control group, the percentage of DC cells in the thymus tissue of mice in the low, medium, and high dose groups of the test substance and the ribavirin granule group was significantly increased (P≤0.05 or P≤0.01).

[0348] Table 9-8 Effects of the test substances on thymic NK, DC, Th17, and Treg cells in mice infected with rhinovirus ( n=10) Note: Compared with the model control group * P≤0.05, ** P≤0.01.

[0349] 4.6 Effects on humoral immunity (immunoglobulins)

[0350] As shown in Table 9-9, compared with the normal control group, the levels of IgA, IgG, and IgM in the blood of mice in the model control group were significantly decreased (P≤0.01); compared with the model control group, the levels of IgA, IgG, and IgM in the blood of mice in the medium- and high-dose groups of the test substance and the ribavirin granule group were significantly increased (P≤0.01).

[0351] Table 9-9 Effects of the test substances on immunoglobulins in rhinovirus-infected mice ( n=10) Note: Compared with the normal control group++ P≤0.01; compared with the model control group ** P≤0.01.

[0352] 4.7 Effects on cytokines in bronchoalveolar lavage fluid

[0353] As shown in Tables 9-10, compared with the normal control group, the levels of IL-1β and TNF-α in the bronchoalveolar lavage fluid of mice in the model control group were significantly increased (P≤0.05 or P≤0.01); compared with the model control group, the levels of TNF-α in the bronchoalveolar lavage fluid of mice in the low-, medium-, and high-dose groups of the test substance and the ribavirin granule group were significantly decreased (P≤0.05 or P≤0.01), and the levels of IL-1β in the bronchoalveolar lavage fluid of mice in the medium- and high-dose groups were significantly decreased (P≤0.05 or P≤0.01).

[0354] Table 9-10 Effects of the test substances on cytokines in bronchoalveolar lavage fluid of rhinovirus-infected mice ( n=10) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the model control group * P≤0.05, ** P≤0.01.

[0355] 4.8 Histopathological examination

[0356] As shown in Figure 7, the lung tissue of the normal control group animals showed no obvious changes and no inflammatory cell infiltration was observed; the lung tissue of the model control group mice showed inflammatory and hemorrhagic exudation, alveolar epithelial destruction, alveolar wall thickening and inflammatory cell infiltration; the lung tissue lesions of mice in the low, medium and high dose groups of the test substance and the ribavirin granule group were all reduced to varying degrees compared with the model control group.

[0357] As shown in Tables 9-11, compared with the normal control group, the pathological scores of hemorrhagic exudation, inflammatory cell infiltration, and total score of lung tissue in the model control group were significantly increased (P≤0.01); compared with the model control group, the pathological scores of inflammatory exudation and hemorrhagic exudation in the low-dose and high-dose groups of the test substance were significantly reduced (P≤0.05), the total pathological score of lung tissue in the medium-dose and high-dose groups of the test substance was significantly reduced (P≤0.05), and the total pathological score of lung tissue in mice in the ribavirin granule group was significantly reduced (P≤0.05).

[0358] Table 9-11 Effects of test substances on lung tissue pathological scores in rhinovirus-infected mice ( n=10) Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group * P≤0.05.

[0359] 5. Conclusion and Evaluation

[0360] The purpose of this study was to investigate the therapeutic effect of the test substance on upper respiratory tract infection in aged animals. An upper respiratory tract infection model was established by nasally infecting 17-month-old C57BL / 6J mice with rhinovirus. The age of the animals used was approximately equivalent to 55-56 years in humans. The efficacy of the test substance against respiratory viral infection in aged animals was evaluated by observing changes in body weight, lung index, bronchoalveolar lavage fluid inflammatory factors, humoral and cellular immune indicators, and viral load in the lungs of each group of animals.

[0361] (1) Antiviral efficacy of the test substance: Viral titer is the gold standard for virus identification and antigen analysis. Viral titer reflects viral load and predicts the severity of infection. Our results showed that the viral titer in the lung tissue of aged mice infected with rhinovirus was increased, indicating the successful establishment of a rhinovirus intranasal infection model. After administration of different concentrations of the test substance to the model mice, the test substance significantly inhibited the increase in lung index, significantly reduced viral titer in lung tissue, and significantly alleviated the degree of lung lesions in the model mice, suggesting that the test substance has significant in vivo antiviral efficacy and can improve clinical symptoms (lung index and histopathological changes) after respiratory viral infection.

[0362] (2) Effects of the test substance on cellular immunity: Cellular immune response, humoral immune response, and inflammatory response play key roles in the pathogenesis of viral infectious diseases, and immune dysfunction is a key factor contributing to disease development. The immune system is an important defense against viral invasion. Among them, macrophage M1 mainly secretes pro-inflammatory factors and plays an important role in the early stage of inflammation, while M2 expresses inhibitory inflammatory factors, playing a role in inhibiting inflammatory response and tissue repair. CD4+ in T lymphocytes... + Primarily expressed on helper T cells, CD4 cells are activated and release signaling molecules when a virus invades the body. These molecules regulate the immune response by recognizing and binding antigens, thereby exerting an antiviral effect. CD8... + Macrophages and T lymphocytes are a subset of T lymphocytes that specifically recognize and bind to virus-infected cells. Therefore, macrophages and T lymphocytes play a crucial role in assessing the body's immune function. Our results show that after rhinovirus infection of aged mice, the serum CD8+ level... + The M1 / M2 ratio in lung tissue was significantly increased, and the blood CD4+ level was also elevated. + / CD8 +The significantly reduced proportions suggest that the immune system balance is disrupted after viral infection in mice, indicating that the model mice are in the early stage of infection. After administration of different concentrations of the test substance, the proportions of lymphocytes in the blood and macrophages in the lung tissue of the model mice significantly recovered, suggesting that the test substance can improve the body's immune level and inhibit the release of inflammatory factors. Furthermore, the levels of inflammatory factors in the immune system are closely related to the degree of infection. Among them, TNF-α and IL-1β are cytokines produced by immune cells and can reflect changes in inflammation. Our results show that after rhinovirus infection of aged mice, the levels of TNF-α and IL-1β in the bronchoalveolar lavage fluid of the model control mice were significantly increased, suggesting that an inflammatory response occurred in the lungs of aged mice after viral infection. After administration of different concentrations of the test substance to the model mice, the test substance significantly inhibited the levels of pro-inflammatory factors TNF-α and IL-1β, suggesting that the test substance can improve the body's inflammatory response.

[0363] The results of this study showed that after rhinovirus infection of aged mice, the number of NK cells, DC cells, and Treg / Th17 ratio in the spleen tissue of the model control mice were significantly reduced, suggesting that the virus attacks and interferes with the immune system of aged mice, leading to an imbalance in the animal's immune response. After the model mice were given different concentrations of the test substance, the test substance could upregulate the number of NK cells, DC cells, and Treg / Th17 ratio in the spleen tissue of aged mice to varying degrees, suggesting that the test substance can regulate the imbalance of cellular immunity.

[0364] (3) Effects of the test substance on humoral immunity: The results of this study showed that after rhinovirus infection of aged mice, the levels of IgG, IgA and IgM in the blood of mice in the model control group were significantly reduced, suggesting that the immunity of aged mice decreased after viral infection. After the model mice were given different concentrations of the test substance, the test substance could significantly increase the levels of IgG, IgA and IgM in the blood of aged mice, suggesting that the test substance could enhance the immune function of aged mice.

[0365] In summary, the test substance significantly reduced the lung index and viral titer in lung tissue, significantly reduced the level of inflammatory factors in bronchoalveolar lavage fluid, and significantly improved the degree of lung lesions in model mice, suggesting that the test substance has a significant pharmacological effect on rhinovirus infection in aged mice, with an effective dose of low dose (2.5 g crude drug / kg).

[0366] Example 5: Study on the preventive effect of the test substance on the respiratory tract of guinea pigs infected with influenza A virus.

[0367] Experiment 1: The preventive effect of the test substance on the respiratory tract of guinea pigs infected with influenza A virus (interrupting transmission).

[0368] 1. Experimental Objective

[0369] In this study, Hartley guinea pigs were instilled with influenza A (H1N1) virus (A / Virgina / ATCC1 / 2009). The close contact transmission was induced by co-housing virus-infected animals and non-infected animals in a 1:1 ratio to construct an influenza A virus transmission and infection model. Drugs were administered to the virus-infected animals (to cut off the transmission). By observing the virus titers in the close contact animals by hemagglutination inhibition assay / plaque assay (nasal tissue, trachea, lung tissue) and histopathological changes (lung tissue, trachea and bronchi), the preventive effect of the test substance on guinea pig respiratory tract infection caused by influenza A virus was evaluated.

[0370] 2. Experimental materials

[0371] 2.1 Test substance

[0372] The dry extract powder of Example 1, production unit: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0373] 2.2 Reference substance

[0374] Oseltamivir phosphate granules, production unit: Yichang Dongyangguang Yangtze River Pharmaceutical Co., Ltd.

[0375] 2.3 Experimental animals

[0376] 54 SPF-grade BALB / c mice, half male and half female, with body weight of 10.0 - 11.8 g, animal quality certificate: 430727241100855914, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (SCXK(Xiang)2019 - 0004); 20 SPF-grade Hartley guinea pigs, half male and half female, with body weight of 257.6 - 293.7 g, animal quality certificate: 44829700029919, purchased from Guangdong Vital River Experimental Animal Technology Co., Ltd. (SCXK(Yue)2022 - 0063). The above animals were used for the exploration of the influenza A virus transmission and infection model;

[0377] 110 SPF-grade Hartley guinea pigs, half male and half female, with body weight of 260.8 - 326.4 g, animal quality certificate: 370823241100139815, purchased from Qingdao Kangda Aibo Biotechnology Co., Ltd. (SCXK(Lu)2021 - 0003), were used for the preventive effect of the test substance on guinea pig respiratory tract infection caused by influenza A virus (cutting off the transmission).

[0378] 2.4 Virus strain

[0379] Influenza A subtype H1N1 strain, serial number: VR-1736, A / Virgina / ATCC1 / 2009. Influenza A subtype H3N2 strain, serial number: VR-1679, A / HONG KONG / 8 / 68 / 2011. Both virus strains were provided by the American Testing Center (ATCC) and cultured and amplified in the Pathogenic Microbiology Laboratory of the Hunan Prima Pharmaceutical Research Center.

[0380] 3. Experimental Methods

[0381] 3.1 Virus culture and amplification

[0382] Select healthy 9-day-old chicken embryos and mark the boundary between the air cell and allantoic cavity. Place the marked embryos with the air cell facing upwards on a candler. Disinfect the embryos with 75% alcohol and drill a hole in the air cell end using a sterile awl. Draw an appropriate amount of virus solution into a syringe, insert the needle into the drilled hole to puncture the shell membrane, and inject 0.1 mL of virus solution into the allantoic cavity. Incubate the embryos at 34°C for 2 days (check the embryo growth daily; discard any embryos that die within 24 hours as non-specific death). Before harvesting, freeze the embryos overnight at 4°C to kill them and coagulate the red blood cells. Disinfect the air cell shell of the frozen embryos with 75% alcohol swabs. Using sterile tweezers, break the sterilized shell, gently peel off the eggshell from the air cell, remove the shell membrane and chorioallantoic membrane, gently push the chicken embryo to one side, press it down, aspirate the virus fluid, centrifuge at 4000 rpm for 5 minutes, collect the supernatant and aliquot it into cryovials, store at -80℃ for later use.

[0383] 3.2 Toxicity Detection

[0384] The titer of the virus amplified in 3.1.1 was determined using the following method:

[0385] Add 50 μL of 0.9% sodium chloride injection to each well of a 96-well disposable reaction plate, except for the first well. Add 100 μL of sample to well 1; then transfer 50 μL of liquid from well 1 to well 2 and gently pipette to mix. Continue this dilution up to well 11, then discard 50 μL of liquid from well 11. Add 50 μL of 0.9% sodium chloride injection to well 12 as a negative control. Add 50 μL of 1.0% chicken erythrocyte suspension to each well. Gently mix the reaction plate and incubate at room temperature for 20–45 min, observing the results. When interpreting the results, tilt the reaction plate slightly (not upright) and read from the back side of the plate. Read the results within approximately 5 seconds. Express the results using log2, where n log2 indicates that agglutination occurs when the virus is diluted to the power of n.

[0386] Results Interpretation: "—" indicates no agglutination, with red blood cells deposited at the bottom of the tube in a neatly shaped disc; "+" indicates slight agglutination, with red blood cells deposited at the bottom of the tube in a disc-shaped disc with indistinct edges; "++" indicates agglutination, with red blood cells deposited at the bottom of the tube in a ring shape with small agglutinated clumps around the edges; "+++" indicates mostly agglutination, with red blood cells forming granular agglutinations with irregular edges and a tendency to droop; "++++" indicates complete agglutination, with red blood cells evenly distributed at the bottom of the tube. The highest dilution that results in complete agglutination is taken as the blood coagulation titer.

[0387] 3.3 Establishment of a Transmission and Infection Model for Influenza A Virus

[0388] 3.3.1 Exploration of a mouse model of transmission and infection

[0389] Fifty-four qualified SPF-grade BALB / c mice (half male and half female) were randomly divided into five groups according to sex and weight: a normal control group (n=6), a virus infection group 1 (n=12), a virus infection group 2 (n=12), a close contact transmission group 1 (n=12), and a close contact transmission group 2 (n=12). Mice in the virus infection group 1 were lightly anesthetized with ether and then instilled with 100 μL / mice (50 μL / well) of influenza A H1N1 virus stock solution via nasal drip. Mice in the virus infection group 2 were lightly anesthetized with ether and then instilled with 100 μL / mice (50 μL / well) of influenza A H3N2 virus stock solution via nasal drip. The normal control group and mice in close contact transmission groups 1 and 2 were instilled with an equal volume of 0.9% sodium chloride injection via nasal drip. Animals in the virus infection group 1 were housed in a 1:1 ratio with animals in the close contact transmission group 1 the day after infection, and animals in the virus infection group 2 were housed in a 1:1 ratio with animals in the close contact transmission group 2 the day after infection. Observations were conducted for 7 consecutive days.

[0390] 3.3.2 Exploration of a Guinea Pig Transmission and Infection Model

[0391] Twenty qualified SPF-grade Hartley guinea pigs (half male and half female) were randomly divided into five groups according to sex and weight: a normal control group (n=4), a virus infection group 1 (n=4), a virus infection group 2 (n=4), a close contact transmission group 1 (n=4), and a close contact transmission group 2 (n=4). Guinea pigs in the virus infection group 1 were lightly anesthetized with ether and then instilled with 500 μL / animal (250 μL / well) of H1N1 influenza A virus stock solution via nasal drip. Guinea pigs in the virus infection group 2 were lightly anesthetized with ether and then instilled with 500 μL / animal (250 μL / well) of H3N2 influenza A virus stock solution via nasal drip. The normal control group and close contact transmission groups 1 and 2 received an equal volume of 0.9% sodium chloride injection via nasal drip. Animals in the virus infection group 1 were housed in a 1:1 ratio with animals in the close contact transmission group 1 the day after infection, and animals in the virus infection group 2 were housed in a 1:1 ratio with animals in the close contact transmission group 2 the day after infection. Observations were conducted for 7 consecutive days.

[0392] 3.3.3 Detection Indicators

[0393] On day 7 of co-cultivation, nasal lavage fluid, nasal tissue, trachea, lung tissue, and bronchoalveolar lavage fluid were collected from normal control group, virus-infected groups 1 and 2, and close contact transmission groups 1 and 2 (mice and guinea pigs) for virus titer detection. Nasal tissue, trachea, and lung tissue from normal control group and close contact transmission groups 1 and 2 were fixed in 10% neutral formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for pathological changes in nasal tissue, trachea, and lung tissue.

[0394] 3.4 The preventive effect of the test substance on the respiratory tract of guinea pigs infected with influenza A virus (interrupting transmission)

[0395] 3.4.1 Grouping, Modeling, and Drug Administration

[0396] One hundred and one hundred and fifty SPF-grade Hartley guinea pigs, weighing 260.8–326.4 g, were selected and divided into a normal control group (n=10), a virus infection group (n=50), and a close contact transmission group (n=50). The virus infection group animals were randomly divided into five subgroups according to sex and weight: virus infection-model control group, virus infection-oseltamivir phosphate granule group (13 mg / kg), and virus infection-low, medium, and high dose groups of test substance (1.5, 3.0, and 6.0 g crude drug / kg), with ten animals in each subgroup. The close contact transmission group animals were also randomly divided into five subgroups according to sex and weight: close contact transmission-model control group, close contact transmission-oseltamivir phosphate granule group, and close contact transmission-low, medium, and high dose groups of test substance, with ten animals in each subgroup. Before administration, the drug was prepared to the appropriate concentration using pure water. Animals in each virus infection group were administered the corresponding concentration of drug solution orally at a dose of 10 mL / kg, once daily for 10 consecutive days. Animals in the normal control group and each close contact transmission group were administered an equal volume of pure water orally. On the 4th day after administration, animals in each virus infection group were lightly anesthetized with ether and then instilled with 500 μL / animal (250 μL / well) of H1N1 influenza A virus stock solution via nasal drip. Drug administration was administered 2 hours post-infection. Animals in the normal control group and each close contact transmission group were instilled with an equal volume of 0.9% sodium chloride injection via nasal drip. The day after infection, animals in the virus infection group were housed in a 1:1 ratio with animals in the close contact transmission group (2 animals / cage) and observed for 7 consecutive days.

[0397] 3.4.2 Indicator Testing

[0398] 3.4.2.1 General Physiological Observations

[0399] During the administration period, the physiological state of each group of animals was observed before and after administration, including symptoms such as coughing, runny nose, and piloerection.

[0400] 3.4.2.2 Virus titer detection

[0401] (1) Hemagglutination inhibition method: On the 7th day after the animals were put into the cage, the right upper lobe of the lung, trachea and nasal tissue of each group of animals (F01~F05, M01~M05) were collected. 0.9% sodium chloride injection was added at a mass (g): 0.9% sodium chloride injection (mL) = 1:9. The mixture was homogenized to make a suspension. The viral titer of the nasal tissue, trachea and lung tissue was detected by the hemagglutination inhibition test. The specific detection method of viral titer is the same as in 3.2.

[0402] (2) Plaque assay: On the 7th day after the animals were put into the same cage, tissues of the upper lobe of the right lung, trachea and nasal tissues were collected from the normal control group and the animals in each close contact transmission group (F01~F05, M01~M05). 0.9% sodium chloride injection was added at a mass ratio of 1:9 and homogenized to prepare a suspension. The viral titer of the nasal tissue, trachea and lung tissue was detected by plaque assay.

[0403] The specific detection procedure for viral titer is as follows:

[0404] (1) Cell plating: After digesting the MDCK cells that were growing in sheets in the cell flask, the cells were plated in a 24-well cell plate and incubated at 37°C with 5% CO2 for 24 hours.

[0405] (2) Virus inoculation: Dilute the virus 10-fold with DMEM, add 0.1 mL of diluted virus solution to each well of the cell culture plate, and set up 2 parallel wells for each dilution; incubate the cell plate at 37℃ with 5% CO2 to allow the virus to adsorb for 1 h, and tilt and shake the cell plate once every 30 minutes; after adsorption for 1 h, discard the supernatant and wash the cells once with DMEM; microwave melt the agar and place it in a 56℃ water bath, incubate DMEM at 37℃, mix agar and DMEM 1:1, and then add 1 mL of the mixture to each well of a 24-well plate; place the cell plate in a safety cabinet until the agar solidifies, invert it and incubate at 37℃ with 5% CO2 for 2-5 days. When white spots appear, they can be stained with crystal violet, add 1 mL of crystal violet to each well and incubate at 37℃ for 4 h, remove the staining solution and count the empty spots.

[0406] Virus titer (PFU / mL) = (average number of empty plaques per well × reciprocal of virus dilution) / virus inoculation amount per well (mL).

[0407] 3.4.2.3 Histopathological examination

[0408] On the 7th day after being put into cages, the left lung tissue, trachea and bronchus of the normal control group and each close contact transmission group (F01~F05, M01~M05) were collected, fixed in 10% neutral formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for pathological changes in the left lung tissue, trachea and bronchus.

[0409] 3.5 Dosage Design

[0410] For specific groupings and dosages, please refer to Table 10-1.

[0411] Table 10-1 Grouping and Dosage Design

[0412] 3.6 Data Processing and Statistical Analysis

[0413] The process is the same as "Data Processing and Statistical Analysis" in Experiment 1 of Example 3.

[0414] 4. Experimental Results

[0415] 4.1 Exploration of Influenza A Virus Transmission and Infection Models

[0416] 4.1.1 Detection of viral titers of pathogenic microorganisms

[0417] The hemagglutination titer of the H1N1 influenza virus used in the exploratory experiment of influenza A virus transmission and infection in mice was 7 log2; the hemagglutination titer of the H3N2 influenza virus used in the exploratory experiment of influenza A virus transmission and infection in guinea pigs was 6 log2.

[0418] 4.1.2 Impact on viral titer

[0419] As shown in Table 10-2, in the mouse infection experiment of influenza A virus, compared with the normal control group, the viral titer in the nasal tissue of the virus-infected group 2 and the close contact transmission groups 1 and 2 was significantly increased (P≤0.01), the viral titer in the tracheal tissue of the virus-infected group 2 and the close contact transmission group 1 was significantly increased (P≤0.01 or P≤0.05), and the viral titer in the lung tissue of the virus-infected group 1 and 2 and the close contact transmission groups 1 and 2 was significantly increased (P≤0.01).

[0420] As shown in Table 10-3, in the guinea pig infection experiment of influenza A virus transmission, compared with the normal control group, the viral titers in the nasal tissue and trachea of ​​the virus-infected group 2 and the close contact transmission groups 1 and 2 were significantly increased (P≤0.05), and the viral titers in the lung tissue of the virus-infected group 1 were significantly increased (P≤0.05).

[0421] Table 10-2 Effects of influenza A virus on viral titers in different tissues of BALB / c mice n=6) Note: Compared with the normal control group + P≤0.05, ++ P≤0.01.

[0422] Table 10-3 Effects of influenza A virus on viral titers in different tissues of Hartley guinea pigs ( n=4) Note: Compared with the normal control group + P≤0.05.

[0423] 4.1.3 Histopathological examination

[0424] As shown in Table 10-4 and Figure 6-1, in the mouse infection experiment of influenza A virus, compared with the normal control group, the nasal tissue of the two close-contact transmission groups and the tracheal tissue of the two close-contact transmission groups showed lesions, but there was no statistical difference (P≥0.05).

[0425] As shown in Table 10-5 and Figure 6-2, in the experiment of influenza A virus transmission and infection of guinea pigs, compared with the normal control group, the pathological scores of nasal tissue in the close contact transmission group 1 were significantly increased (P≤0.05), and the pathological scores of tracheal tissue in close contact transmission groups 1 and 2 were significantly increased (P≤0.05).

[0426] Table 10-4 Effects of influenza A virus on different histopathological scores in BALB / c mice ( n=6)

[0427] Table 10-5 Effects of Influenza A Virus on Different Histopathological Scores in Hartley Guinea Pigs n=4) Note: Compared with the normal control group + P≤0.05.

[0428] 4.1.4 Model Exploration Conclusions

[0429] Based on the results of the above exploration of mouse and guinea pig transmission and infection models, it can be seen that both H1N1 and H3N2 influenza A virus strains can be used to construct transmission and infection models in guinea pigs, and both can infect close contacts. However, only guinea pigs infected with the virus can induce significant histopathological damage (nasal cavity and bronchi). Therefore, guinea pigs will be used in subsequent formal experiments to investigate the efficacy of the test drug against influenza A rhinovirus transmission and infection.

[0430] 4.2 The preventive effect of the test substance on the respiratory tract of guinea pigs infected with influenza A virus (interrupting transmission)

[0431] 4.2.1 Detection of viral titers of pathogenic microorganisms

[0432] The hemagglutination titer of the H1N1 influenza virus used in this experiment was 7log2.

[0433] 4.2.2 General Physiological Observations

[0434] After modeling, all guinea pigs exhibited reduced spontaneous activity, piloerection, and rapid breathing; after being put into cages together, animals in the close contact transmission groups also showed piloerection.

[0435] 4.2.3 Effect on viral titer (hemagglutination inhibition method)

[0436] As shown in Table 10-6, compared with the normal control group, the viral titers in the nasal tissue, trachea, and lung tissue of guinea pigs in the virus infection-model control group were significantly increased (P≤0.01); compared with the virus infection-model control group, the viral titers in the nasal tissue and trachea of ​​guinea pigs in the virus infection-high dose group and the virus infection-oseltamivir phosphate granule group were significantly decreased (P≤0.01 or P≤0.05), and the viral titers in the lung tissue of guinea pigs in the virus infection-medium and high dose groups and the virus infection-oseltamivir phosphate granule group were significantly decreased (P≤0.01 or P≤0.05).

[0437] As shown in Table 10-7, compared with the normal control group, the viral titers in the nasal tissue, trachea, and lung tissue of guinea pigs in the close contact transmission-model control group were significantly increased (P≤0.01); compared with the close contact transmission-model control group, the viral titers in the nasal tissue of guinea pigs in the close contact transmission-high dose group and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.01 or P≤0.05), the viral titers in the trachea of ​​guinea pigs in the close contact transmission-low, medium, and high dose groups and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.01 or P≤0.05), and the viral titers in the lung tissue of guinea pigs in the close contact transmission-high dose group were significantly decreased (P≤0.05).

[0438] Table 10-6 Effects of test substances on viral titers in guinea pig respiratory model of influenza A virus infection (interrupted transmission) - hemagglutination inhibition method n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the virus infection-model control group * P≤0.05, ** P≤0.01.

[0439] Table 10-7 Effects of test substances on viral titers in a close-contact (interrupted transmission) influenza A virus-infected guinea pig respiratory model - hemagglutination inhibition method ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the close contact transmission-model control group * P≤0.05, ** P≤0.01.

[0440] 4.2.4 Effect on viral titer (plaque method)

[0441] As shown in Table 10-8, compared with the normal control group, the viral titers in the nasal tissue, trachea, and lung tissue of guinea pigs in the close contact transmission-model control group were significantly increased (P≤0.01); compared with the close contact transmission-model control group, the viral titers in the nasal tissue, trachea, and lung tissue of guinea pigs in the close contact transmission-low, medium, and high dose groups of the test substance and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.01 or P≤0.05).

[0442] Table 10-8 Effects of test substances on viral titers in a close-contact (interrupted transmission) influenza A virus-infected guinea pig respiratory model - plaque assay n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the close contact transmission-model control group * P≤0.05, ** P≤0.01.

[0443] 4.2.5 Histopathological examination

[0444] As shown in Figure 6-3, microscopic observation on the day after the last administration revealed no significant changes in the trachea and bronchi of the normal control group, and no inflammatory cell infiltration was observed. In the close contact transmission-model control group, the trachea and bronchi showed mucosal congestion and edema, ciliated cell damage and shedding, and inflammatory cell infiltration. The tracheal and bronchial lesions of the guinea pigs in the close contact transmission-low, medium, and high dose groups of the test substance, as well as the close contact transmission-oseltamivir phosphate granule group, were all reduced to varying degrees compared with the close contact transmission-model control group.

[0445] As shown in Figure 6-4, microscopic observation of the lung tissue of guinea pigs in the normal control group on the day after the last administration showed no significant changes and no inflammatory cell infiltration. In the close contact transmission-model control group, alveolar epithelial destruction, alveolar septal thickening, inflammatory / hemorrhagic exudation, and inflammatory cell infiltration were observed in the lung tissue. The lung lesions of guinea pigs in the close contact transmission-low, medium, and high dose groups of the test substance and the close contact transmission-oseltamivir phosphate granule group were all reduced to varying degrees compared with the close contact transmission-model control group.

[0446] As shown in Table 10-9, compared with the normal control group, the lung tissue, trachea and bronchus pathological scores of guinea pigs in the close contact transmission-model control group were significantly increased (P≤0.01); compared with the close contact transmission-model control group, the trachea and bronchus pathological scores of guinea pigs in the close contact transmission-high dose group were significantly decreased (P≤0.05), and the lung tissue pathological scores of guinea pigs in the close contact transmission-medium and high dose groups and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.05).

[0447] Table 10-9 Effects of the test substances on the pathological scores of close-contact transmission (interrupted transmission) animals in a guinea pig respiratory model of influenza A virus infection. n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the close contact transmission-model control group * P≤0.05.

[0448] 5. Experiment Summary

[0449] Both influenza A subtypes H1N1 and H3N2 can replicate and spread in BABL / c mice and Hartley guinea pigs. Based on viral titer and pathological scores, H1N1 was more effective at inducing infection in Hartley guinea pigs, with the virus primarily distributed in nasal, tracheal, and lung tissues. Therefore, subsequent studies used H1N1 to induce infection in Hartley guinea pigs to establish a viral transmission and infection model. The test substance significantly reduced viral titers in the trachea, nasal, and lung tissues of infected animals and close contacts, and decreased pathological scores in lung tissue, trachea, and bronchi, suggesting a significant effect in interrupting influenza A transmission. The effective dose was a low dose (1.5 g crude drug / kg).

[0450] Experiment 2: The preventive effect of the test substance on the respiratory tract of guinea pigs infected with influenza A virus (protection of close contacts).

[0451] 1. Experimental Objective

[0452] This study used Hartley guinea pig nasal drops of H1N1 influenza A virus (A / Virgina / ATCC1 / 2009) to induce close contact transmission by co-occulting infected and uninfected animals in a 1:1 ratio, thus establishing an influenza A virus transmission and infection model. The test substance was administered to the infected animals (while protecting the close contacts). The preventive effect of the test substance on respiratory tract infection in guinea pigs caused by influenza A virus was evaluated by observing the virus titer-hemagglutination inhibition / plaque assay (nasal tissue, trachea, and lung tissue) and histopathological changes (lung tissue, trachea, and bronchi) in the infected animals.

[0453] 2. Experimental Materials

[0454] 2.1 Test substance

[0455] The dry ointment powder of Example 1 was produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0456] 2.2 Reference Standard

[0457] Oseltamivir phosphate granules, manufactured by Yichang Dongyangguang Changjiang Pharmaceutical Co., Ltd.

[0458] 2.3 Laboratory Animals

[0459] 110 SPF - level Hartley guinea pigs, with an equal number of males and females, weighing 228.9 - 350.5 g. Animal quality certificate: 370823241100139953, purchased from Qingdao Kangda Aibo Biotechnology Co., Ltd. (SCXK(Lu)2021 - 0003).

[0460] 2.4 Virus strain

[0461] Influenza A subtype H1N1 strain, provided by ATCC in the United States, numbered: VR - 1736, A / Virgina / ATCC1 / 2009. Cultured and amplified in the Pathogenic Microorganism Laboratory of Hunan Prima Pharmaceutical Research Center.

[0462] 3. Experimental methods

[0463] 3.1 Virus culture and amplification

[0464] Select 9 - day - old chicken embryos in good condition, and mark the boundary between the air chamber and the allantois of the chicken embryo. Place the marked chicken embryo with the air chamber facing up on the egg candler. Disinfect the chicken embryo with 75% alcohol, and drill a hole at the air chamber end with a sterile awl. Use a syringe to draw an appropriate amount of virus solution, insert the injection needle into the hole to pierce the shell membrane of the small hole, and inject 0.1 mL of virus solution into the allantoic cavity. Culture the chicken embryos in an incubator at 34°C for 2 days (note that the growth of chicken embryos should be checked every day, and chicken embryos that die within 24 hours are considered non - specifically dead and should be discarded). Before harvesting, place the chicken embryos in a 4°C refrigerator overnight to freeze the chicken embryos and coagulate the red blood cells. Disinfect the eggshell of the air chamber part of the frozen chicken embryo with a 75% alcohol cotton ball. Use sterile forceps to break the disinfected shell, gently remove the eggshell at the air chamber part, tear off the shell membrane and the chorioallantoic membrane, gently push the chicken embryo to one side, hold it down, suck out the virus solution, centrifuge at 4000 rpm for 5 minutes, take the supernatant and aliquot it into cryotubes, and store it at - 80°C for standby.

[0465] 3.2 Virulence detection

[0466] Perform titer determination on the virus amplified in 3.1.1. The specific method is as follows:

[0467] Add 50 μL of 0.9% sodium chloride injection to each well of a 96-well disposable reaction plate, except for the first well. Add 100 μL of sample to well 1; then transfer 50 μL of liquid from well 1 to well 2 and gently pipette to mix. Continue this dilution up to well 11, then discard 50 μL of liquid from well 11. Add 50 μL of 0.9% sodium chloride injection to well 12 as a negative control. Add 50 μL of 1.0% chicken erythrocyte suspension to each well. Gently mix the reaction plate and incubate at room temperature for 20–45 min, observing the results. When interpreting the results, tilt the reaction plate slightly (not upright) and read from the back side of the plate. Read the results within approximately 5 seconds. Express the results using log2, where n log2 indicates that agglutination occurs when the virus is diluted to the power of n.

[0468] Results Interpretation: "—" indicates no agglutination, with red blood cells deposited at the bottom of the tube in a neatly shaped disc; "+" indicates slight agglutination, with red blood cells deposited at the bottom of the tube in a disc-shaped disc with indistinct edges; "++" indicates agglutination, with red blood cells deposited at the bottom of the tube in a ring shape with small agglutinated clumps around the edges; "+++" indicates mostly agglutination, with red blood cells forming granular agglutinations with irregular edges and a tendency to droop; "++++" indicates complete agglutination, with red blood cells evenly distributed at the bottom of the tube. The highest dilution that results in complete agglutination is taken as the blood coagulation titer.

[0469] 3.3 Grouping, Modeling, and Drug Administration

[0470] One hundred and ten SPF-grade Hartley guinea pigs (half male and half female, weighing 228.9–350.5 g) were selected and divided into a normal control group (n=10), a virus infection group (n=50), and a close contact transmission group (n=50). Animals in the virus infection group were randomly divided into five subgroups according to sex and weight: virus infection-model control group, virus infection-oseltamivir phosphate granule group, and virus infection-low, medium, and high dose test substance group, with ten animals in each subgroup. Animals in the close contact transmission group were also randomly divided into five subgroups according to sex and weight: close contact transmission-model control group, close contact transmission-oseltamivir phosphate granule group (13 mg / kg), and close contact transmission-low, medium, and high dose test substance group (1.5, 3.0, and 6.0 g crude drug / kg), with ten animals in each subgroup. Before administration, the drug was prepared to the appropriate concentration using pure water. Animals in each close contact transmission group were administered the corresponding concentration of drug solution orally at a dose of 10 mL / kg once a day for 10 consecutive days. Animals in the normal control group and each virus infection group were administered an equal volume of pure water orally. On the 4th day after administration, animals in each virus infection group were lightly anesthetized with ether and then instilled with 500 μL / animal (250 μL / well) of H1N1 influenza A virus stock solution via nasal drip. The drug was administered 2 hours after infection. Animals in the normal control group and each close contact transmission group were instilled with an equal volume of 0.9% sodium chloride injection via nasal drip. The day after infection, animals in the virus infection group were housed in a 1:1 ratio with animals in the close contact transmission group (2 animals / cage) and observed for 7 consecutive days.

[0471] 3.4 Indicator Testing

[0472] 3.4.1 General Physiological Observation

[0473] During the administration period, the physiological state of each group of animals was observed before and after administration, including symptoms such as coughing, runny nose, and piloerection.

[0474] 3.4.2 Virus titer detection

[0475] 3.4.2.1 Blood coagulation inhibition method

[0476] On the 7th day after co-cultivation, tissues from the upper lobe of the right lung, trachea, and nose of each group of animals (F01~F05, M01~M05) were collected. 0.9% sodium chloride injection was added at a ratio of 1:9 (g:mL) and homogenized to form a suspension. The viral titer in the nasal tissue, trachea, and lung tissue was detected using a hemagglutination inhibition test. The specific detection procedure for viral titer was the same as in 3.2.

[0477] 3.4.2.2 Spatter Method

[0478] On the 7th day after co-cultivation, tissues from the upper lobe of the right lung, trachea, and nose of animals in the normal control group and each close contact transmission group (F01-F05, M01-M05) were collected. 0.9% sodium chloride injection was added at a mass ratio of 1:9 and homogenized to prepare a suspension. The viral titer of the nasal tissue, trachea, and lung tissue was detected by the plaque method.

[0479] The specific detection procedure for viral titer is as follows:

[0480] (1) Cell plating: After digesting the MDCK cells that were growing in sheets in the cell flask, the cells were plated in a 24-well cell plate and incubated at 37°C with 5% CO2 for 24 hours.

[0481] (2) Virus inoculation: Dilute the virus 10-fold with DMEM, add 0.1 mL of diluted virus solution to each well of the cell culture plate, and set up 2 parallel wells for each dilution; incubate the cell plate at 37℃ with 5% CO2 to allow the virus to adsorb for 1 h, and tilt and shake the cell plate once every 30 minutes; after adsorption for 1 h, discard the supernatant and wash the cells once with DMEM; microwave melt the agar and place it in a 56℃ water bath, incubate DMEM at 37℃, mix agar and DMEM 1:1, and then add 1 mL of the mixture to each well of a 24-well plate; place the cell plate in a safety cabinet until the agar solidifies, invert it and incubate at 37℃ with 5% CO2 for 2-5 days. When white spots appear, they can be stained with crystal violet, add 1 mL of crystal violet to each well and incubate at 37℃ for 4 h, remove the staining solution and count the empty spots.

[0482] Virus titer (PFU / mL) = (average number of empty plaques per well × reciprocal of virus dilution) / virus inoculation amount per well (mL).

[0483] 3.4.3 Histopathological examination

[0484] On the 7th day after being put into cages, the left lung tissue, trachea and bronchus of the normal control group and each close contact transmission group (F01~F05, M01~M05) were collected, fixed in 10% neutral formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for pathological changes in the left lung tissue, trachea and bronchus.

[0485] 3.5 Dosage Design

[0486] For specific groupings and dosages, please refer to Table 11-1.

[0487] Table 11-1 Grouping and Dosage Design

[0488] 3.6 Data Processing and Statistical Analysis

[0489] The process is the same as "Data Processing and Statistical Analysis" in Experiment 1 of Example 3.

[0490] 4. Experimental Results

[0491] 4.1 H1N1 Influenza Virus Titer

[0492] The hemagglutination titer of the H1N1 influenza virus used in this experiment was 7log2.

[0493] 4.2 General Physiological Observation

[0494] After modeling, all guinea pigs exhibited reduced spontaneous activity, piloerection, and rapid breathing; after being put into cages together, animals in the close contact transmission groups also showed piloerection.

[0495] 4.3 Effect on viral titer (hemagglutination inhibition method)

[0496] As shown in Table 11-2, compared with the normal control group, the viral titers in the nasal tissue, trachea, and lung tissue of the virus-infected model control group were significantly increased (P≤0.01).

[0497] As shown in Table 11-3, compared with the normal control group, the viral titers in the nasal tissue, trachea, and lung tissue of guinea pigs in the close contact transmission-model control group were significantly increased (P≤0.01); compared with the close contact transmission-model control group, the viral titers in the nasal tissue of guinea pigs in the close contact transmission-low, medium, and high dose groups of the test substance and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.01 or P≤0.05), the viral titers in the trachea of ​​guinea pigs in the close contact transmission-high dose group of the test substance and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.05), and the viral titers in the lung tissue of guinea pigs in the close contact transmission-high dose group of the test substance were significantly decreased (P≤0.01).

[0498] Table 11-2 Effects of test substances on viral titers in guinea pig respiratory model of influenza A virus infection (protected close contacts) - hemagglutination inhibition method ( n=10) Note: Compared with the normal control group ++ P≤0.01.

[0499] Table 11-3 Effects of test substances on viral titers in a guinea pig respiratory model of influenza A virus infection (protected close contacts) - hemagglutination inhibition method ( n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the close contact transmission-model control group * P≤0.05, ** P≤0.01.

[0500] 4.4 Effect on viral titer (plaque assay)

[0501] As shown in Table 11-4, compared with the normal control group, the viral titers in the nasal tissue, trachea, and lung tissue of guinea pigs in the close contact transmission-model control group were significantly increased (P≤0.01); compared with the close contact transmission-model control group, the viral titers in the trachea, lung tissue, and nasal tissue of guinea pigs in the close contact transmission-low, medium, and high dose groups of the test substance and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.01).

[0502] Table 11-4 Effects of test substances on viral titers in a guinea pig respiratory model of influenza A virus infection (protected close contacts) - plaque assay n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the close contact transmission-model control group ** P≤0.01.

[0503] 4.5 Histopathological examination

[0504] As shown in Figure 7-1, microscopic observation on the day after the last administration revealed no significant changes in the trachea and bronchi of the normal control group, and no inflammatory cell infiltration was observed. In the close contact transmission-model control group, the trachea and bronchi showed mucosal congestion and edema, ciliated cell damage and shedding, and inflammatory cell infiltration. The tracheal and bronchial lesions of the guinea pigs in the close contact transmission-low, medium, and high dose groups of the test substance, as well as the close contact transmission-oseltamivir phosphate granule group, were all reduced to varying degrees compared with the close contact transmission-model control group.

[0505] As shown in Figure 7-2, microscopic observation of the lung tissue of guinea pigs in the normal control group on the day after the last administration showed no significant changes and no inflammatory cell infiltration. In the close contact transmission-model control group, alveolar epithelial destruction, alveolar septal thickening, inflammatory / hemorrhagic exudation, and inflammatory cell infiltration were observed in the lung tissue. The lung lesions of guinea pigs in the close contact transmission-low, medium, and high dose groups of the test substance and the close contact transmission-oseltamivir phosphate granule group were all reduced to varying degrees compared with the close contact transmission-model control group.

[0506] As shown in Table 11-5, compared with the normal control group, the pathological scores of lung tissue, trachea and bronchus of guinea pigs in the close contact transmission-model control group were significantly increased (P≤0.01); compared with the close contact transmission-model control group, the pathological scores of trachea and bronchus of guinea pigs in the close contact transmission-high dose group and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.05), and the pathological scores of lung tissue of guinea pigs in the close contact transmission-low, medium and high dose groups and the close contact transmission-oseltamivir phosphate granule group were significantly decreased (P≤0.01).

[0507] Table 11-5 Effects of the test substances on pathological scores in close-contact transmission models of influenza A virus in guinea pigs (protected close contacts). n=10) Note: Compared with the normal control group ++ P≤0.01; compared with the close contact transmission-model control group * P≤0.05, ** P≤0.01.

[0508] 5. Experiment Summary

[0509] The test substance significantly reduced the viral titer in the nasal, tracheal, and lung tissues of close contacts, and reduced the pathological scores of the lung tissue, trachea, and bronchi, suggesting that it has a significant protective effect against influenza A in close contacts. The effective dose is a low dose (1.5 g crude drug / kg).

[0510] Example 6: Study on the depyrogenic effect of the test substance

[0511] Experimental Objective

[0512] The antipyretic effect of the test substance was observed by inducing fever in rats through intraperitoneal injection of lipopolysaccharide (LPS).

[0513] 1. Experimental Materials

[0514] 1.1 Test sample

[0515] 1.1.1 Name: Dry ointment powder of Example 1.

[0516] 1.1.2 Physical characteristics: Brownish-yellow powder; slightly fragrant odor and slightly sweet taste.

[0517] 1.1.3 Source: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0518] 1.2 Positive reagents and main reagents

[0519] 1.2.1 Positive drug: Ibuprofen granules, manufacturer: CSPC Ouyi Pharmaceutical Co., Ltd.

[0520] 1.2.2 Main Reagents

[0521] LPS: SIGMA.

[0522] 1.3 Experimental System

[0523] 1.3.1 Animal strain: SD rat.

[0524] 1.3.2 Animal grade: SPF grade.

[0525] 1.3.3 Animal sex and number: 60 males, 50 of which were selected for the formal experiment.

[0526] 1.3.4 Animal age: Animals were approximately 5 weeks old at the time of receipt and approximately 6 weeks old at the time of administration.

[0527] 1.3.5 Animal weight: The proposed animal weight range is 160-180g, the actual weight of the animals received is 157.4-181.9g, and the weight at the time of administration is 196.9-231.2g.

[0528] 1.3.6 Animal source: Purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0529] 1.3.7 Husbandry conditions: Rats were caged, with 12 hours of light per day, a temperature of 20-26℃, and a relative humidity of 40-70%.

[0530] 1.3.8 Quarantine process: Animals were kept in an adaptive environment and weighed once a day for 5 consecutive days. During this period, animal No. 40 suffered an accidental fracture of its right forelimb and was removed after the quarantine period ended. The remaining animals had normal drinking and feeding, were in good health, and showed no signs of disease or death.

[0531] 2. Test Methods

[0532] 2.1 Basis for Experimental Design

[0533] 2.1.1 Standards adopted: "Classification of Traditional Chinese Medicine Registration and Requirements for Application Materials", "Good Laboratory Practice for Non-Clinical Studies of Drugs" and related literature.

[0534] 2.1.2 Experimental System Selection Instructions: LPS is the active ingredient of Gram-negative bacterial endotoxin (ET). Its fever-inducing effect is mainly due to the activation of inflammatory cells (mostly mononuclear macrophages) to produce pyrogens (EP). The LPS-induced fever model in rats is widely used for the evaluation of antipyretic traditional Chinese medicine. Since pharmacological doses of estrogen have anti-inflammatory and immunosuppressive effects, male animals are generally selected.

[0535] 2.2 Dosage and Grouping

[0536] See Table 12 for details.

[0537] Table 12 Effects of test substances on LPS-induced fever in rats: test groups and dosage settings

[0538] 2.3 Administration method

[0539] Administer the medication via gavage.

[0540] 2.4 Preparation and storage of test samples

[0541] Weigh 2.4002g (low), 4.2001g (medium), and 8.4003g (high) of the test substance, grind them with sterile water for injection, and make up to 60mL; take one packet of ibuprofen granules, grind them with sterile water for injection, and make up to 75mL. All test drugs should be prepared fresh before use.

[0542] 2.5 Administration of the test sample

[0543] The drug was administered using a disposable syringe at a volume of 20 mL / kg. The model group animals were given the same volume of solvent once.

[0544] 2.6 Steps and Observation Indicators

[0545] Newly received experimental animals were numbered and acclimatized for 5 days. Afterward, rectal temperature measurement and gavage were performed for 3 consecutive days to reduce temperature fluctuations caused by operational stress during the formal measurement. The insertion depth of the electronic thermometer was controlled to approximately 4 cm. Before the formal experiment, a basal body temperature was measured once and recorded as T0. Fifty animals with moderate basal body temperatures were selected and grouped for drug administration. Animals were fasted for at least 15 hours before administration, but allowed free access to water. Immediately after administration, 20 μg / kg LPS (prepared with 0.9% physiological saline) was injected intraperitoneally at a volume of 5 mL / kg. Body temperature was measured approximately every hour after injection and recorded as Tx for 5 consecutive hours. ΔT was calculated as ΔT = Tx - T0.

[0546] 2.7 Statistical Methods

[0547] The experimental data were analyzed using SPSS 18.0 statistical software, with significance levels of 0.05 and 0.01. Results are expressed as mean ± standard deviation. The method involves first performing a normality test. For data that conform to a normal distribution, one-way ANOVA is used to compare the means. If the variances are homogeneous, the least significant difference (LSD) test is used for pairwise comparisons. If the variances are not homogeneous, Dunnett's T3 test is used for pairwise comparisons. If the data does not conform to a normal distribution, nonparametric tests are used.

[0548] 3. Results

[0549] As shown in Tables 13 and 14, compared with the model group, the body temperature and body temperature difference ΔT of rats in the positive drug ibuprofen group were significantly reduced at 3h, 4h and 5h after administration (P<0.01). The body temperature of rats in the high-dose test substance group was significantly reduced at 3h after administration (P<0.05), and a decreasing trend was also observed at other time points. The low- and medium-dose test substance groups also showed a decreasing trend at different time points, but no significant difference was observed (P>0.05).

[0550] Table 13 Results of the experiment on the effect of the test substance on LPS-induced fever in rats Note: Compared with the model group: **P<0.01, *P<0.05.

[0551] Table 14 Results of the experiment on the effect of the test substance on LPS-induced fever in rats Note: Compared with the model group: **P<0.01.

[0552] 4. Conclusion

[0553] In the high-dose group, rats showed a significant decrease in body temperature 3 hours after administration, with a similar decreasing trend observed at other time points. The low- and medium-dose groups also showed a decreasing trend at different time points. This indicates that the test substance has an inhibitory effect on fever induced by intraperitoneal injection of LPS in rats.

[0554] Example 7: Study on the anti-inflammatory effect of the test substance

[0555] Experimental Objective

[0556] A mouse ear swelling model was established by applying xylene, a chemical inflammatory substance, to one ear. The effect of the test substance on xylene-induced inflammation was observed by measuring the degree of swelling, i.e., the weight difference between the two ears.

[0557] 1. Experimental Materials

[0558] 1.1 Test sample

[0559] 1.1.1 Name: Dry ointment powder of Example 1.

[0560] 1.1.2 Physical characteristics: Brownish-yellow powder; slightly fragrant odor and slightly sweet taste.

[0561] 1.1.3 Source: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0562] 1.2 Positive Drug

[0563] Positive drug: Aspirin enteric-coated tablets, specification: 100mg / tablet, manufacturer: Bayer SPA.

[0564] 1.3 Experimental System

[0565] 1.3.1 Animal strain: KM mouse.

[0566] 1.3.2 Animal grade: SPF grade.

[0567] 1.3.3 Animal sex and number: Half male and half female, a total of 60 animals, from which 50 animals of suitable weight (25 male and 25 female) were selected for the formal experiment.

[0568] 1.3.4 Animal age: Animals were approximately 4 weeks old at the time of receipt and approximately 5 weeks old at the time of drug administration.

[0569] 1.3.5 Animal weight: The proposed animal weight range is 18-20g, the actual weight of the animals received is 16.4-19.3g, and the weight at the time of the first administration is 23.7-28.0g.

[0570] 1.3.6 Animal source: Purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0571] 1.3.7 Husbandry conditions: Mice are cage-raised with 12 hours of light per day, a temperature of 20-26℃, and a relative humidity of 40-70%.

[0572] 1.3.8 Quarantine process: Animals were acclimatized and weighed once a day for 5 consecutive days. During this period, the animals drank water and ate normally, were in good health, and showed no signs of disease or death.

[0573] 2. Test Methods

[0574] 2.1 Basis for Experimental Design

[0575] 2.1.1 Standards adopted: "Classification of Traditional Chinese Medicine Registration and Requirements for Application Materials", "Good Laboratory Practice for Non-Clinical Studies of Drugs" and related literature.

[0576] 2.1.2 Experimental System Selection Instructions: Xylene, croton oil, etc. are often used as inflammatory agents in acute inflammation models. Xylene is a chemical reagent that, when applied or injected locally in animals (such as ears, toes, etc.), can cause local capillary dilation and increased permeability, leading to inflammatory infiltration. It is often used as one of the routine methods for evaluating anti-inflammatory drugs.

[0577] 2.2 Dosage and Grouping

[0578] See Table 15 for details.

[0579] Table 15 Effects of the test substance on xylene-induced ear swelling in mice: test groups and dosage settings

[0580] 2.3 Administration method

[0581] Administer the medication via gavage.

[0582] 2.4 Preparation and storage of test samples

[0583] Weigh approximately 0.4g (low), 0.9g (medium), and 1.8g (high) of the test substance, grind them separately with sterile water for injection, and bring the volume to 10mL; take 2 aspirin enteric-coated tablets, grind them with sterile water for injection, and bring the volume to 8mL. All test drugs should be prepared fresh before use.

[0584] 2.5 Administration of the test sample

[0585] The drug was administered using a disposable syringe at a volume of 20 mL / kg. The model group animals were given the corresponding volume of solvent. The drug was administered once a day for 3 consecutive days.

[0586] 2.6 Steps and Observation Indicators

[0587] Newly received experimental animals were numbered, acclimatized for 5 days, and grouped as described previously. Immediately after the last administration, the animals were immobilized, the right ear was exposed, and xylene was applied to the middle of the mouse's right ear using a pipette, 15 μl on each side, spreading evenly. The left ear was left untreated. Approximately 40 minutes later, the animals were euthanized by cervical dislocation. Ear flaps were punched out from symmetrical locations on both ears using a 7 mm diameter punch, weighed using a precision analytical balance, and the weight was recorded. The degree of swelling and the swelling inhibition rate were calculated.

[0588] Swelling degree = Weight of right earpiece - Weight of left earpiece

[0589] Swelling inhibition rate = (average swelling degree of model group - average swelling degree of drug treatment group) / average swelling degree of model group × 100%.

[0590] 2.7 Statistical Methods

[0591] The statistical method is the same as that used in Example 6.

[0592] 3. Results

[0593] As shown in Table 16, compared with the model group, the ear swelling of mice in the positive drug aspirin group was significantly reduced (P<0.01), with a swelling inhibition rate of 31.14%. The ear swelling of mice in the medium and high dose groups of the test substance was significantly reduced (P<0.05), with swelling inhibition rates of 16.17% and 16.77%, respectively. The ear swelling of mice in the low dose group of the test substance showed a decreasing trend, with a swelling inhibition rate of 4.79%.

[0594] Table 16 Results of the effect of the test substance on xylene-induced ear swelling in mice Note: Compared with the model group: **P<0.01, *P<0.05.

[0595] 4. Conclusion

[0596] The ear swelling of mice in the medium and high dose groups of the test substance was significantly reduced, while the ear swelling of mice in the low dose group showed a decreasing trend, indicating that the test substance has an anti-xylene-induced local inflammation effect.

[0597] Example 8: Study on the antitussive effect of the test substance

[0598] Experimental Objective

[0599] This experiment used ammonia spray to induce coughing in mice to evaluate the antitussive effect of the test substance.

[0600] 1. Experimental Materials

[0601] 1.1 Test sample

[0602] 1.1.1 Name: Dry ointment powder of Example 1.

[0603] 1.1.2 Physical characteristics: Brownish-yellow powder; slightly fragrant odor and slightly sweet taste.

[0604] 1.1.3 Source: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0605] 1.2 Positive Drug

[0606] Dextromethorphan hydrobromide tablets, specification: 15mg / tablet, manufacturer: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0607] 1.3 Experimental System

[0608] 1.3.1 Animal strain: KM mouse.

[0609] 1.3.2 Animal grade: SPF grade.

[0610] 1.3.3 Animal sex and number: Half male and half female, a total of 60 animals, from which 50 animals of suitable weight (25 male and 25 female) were selected for the formal experiment.

[0611] 1.3.4 Animal age: Animals were approximately 4 weeks old at the time of receipt and approximately 5 weeks old at the time of drug administration.

[0612] 1.3.5 Animal weight: The proposed animal weight range is 18-20g, the actual weight of the animals received is 16.4-19.5g, and the weight at the time of the first administration is 23.4-29.1g.

[0613] 1.3.6 Animal source: Purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0614] 1.3.7 Husbandry conditions: Mice are cage-raised with 12 hours of light per day, a temperature of 20-26℃, and a relative humidity of 40-70%.

[0615] 1.3.8 Quarantine process: Animals were acclimatized and weighed once a day for 5 consecutive days. During this period, the animals drank water and ate normally, were in good health, and showed no signs of disease or death.

[0616] 2. Test Methods

[0617] 2.1 Basis for Experimental Design

[0618] 2.1.1 Standards adopted: "Classification of Traditional Chinese Medicine Registration and Requirements for Application Materials", "Good Laboratory Practice for Non-Clinical Studies of Drugs" and related literature.

[0619] 2.1.2 Experimental System Selection Instructions: Irritating gases such as sulfur dioxide, ammonia, sulfuric acid, citric acid, etc., are inhaled into the respiratory tract of mice, stimulating the respiratory epithelial receptors and causing coughing. They are often used as models for screening antitussive test substances.

[0620] 2.2 Dosage and Grouping

[0621] See Appendix 17 for details.

[0622] Table 17 Effects of test substances on ammonia-induced cough in mice; experimental groups and dosage settings.

[0623] 2.3 Administration method

[0624] Administer the medication via gavage.

[0625] 2.4 Preparation and storage of test samples

[0626] Weigh approximately 0.4g (low), 0.9g (medium), and 1.8g (high) of the test substance, grind them separately with sterile water for injection, and bring the volume to 10mL; take one dextromethorphan hydrobromide tablet, grind it with sterile water for injection, and bring the volume to 15mL. All test drugs should be prepared fresh before use.

[0627] 2.5 Administration of the test sample

[0628] The drug was administered using a disposable syringe at a volume of 20 mL / kg. The model group animals were given the corresponding volume of solvent. The drug was administered once a day for 3 consecutive days.

[0629] 2.6 Steps and Observation Indicators

[0630] Newly received experimental animals were numbered and acclimatized for 5 days, grouped as described previously. Approximately 0.5 hours after the last administration, the animals were placed in a 10cm*10cm*10cm acrylic box to induce coughing. An ultrasonic nebulizer was set to level 2, and a 13% ammonia solution (prepared with 0.9% physiological saline) was sprayed at a constant rate for 6 seconds. The cough latency and the number of coughs within 3 minutes were recorded. The cough latency refers to the time required from the start of ammonia spraying to the first cough. Coughing in mice is characterized by abdominal muscle contraction (chest retraction) accompanied by either a wide or narrow mouth opening, sometimes accompanied by a coughing sound.

[0631] 2.7 Statistical Methods

[0632] The statistical method is the same as that used in Example 6.

[0633] 3. Results

[0634] As shown in Table 18, compared with the model group, the number of coughs in the positive drug dextromethorphan hydrobromide group was significantly reduced (P<0.05), while the cough latency period in the high-dose test drug group was significantly prolonged (P<0.05) and the number of coughs was significantly reduced (P<0.05).

[0635] Table 18 Effects of the test substances on ammonia-induced cough in mice. Cough results. Note: Compared with the model group: *P<0.05.

[0636] 4. Conclusion

[0637] Inhalation of ammonia via nebulization induced a significant cough response in mice. The high-dose group of mice showed a significantly prolonged cough latency and a significantly reduced frequency of coughs. This indicates that the test substance has an antitussive effect.

[0638] Example 9: Study on the expectorant effect of the test substance

[0639] Experimental Objective

[0640] The expectorant effect of the test substance is evaluated by measuring the amount of phenol red excreted from the trachea.

[0641] 1. Experimental Materials

[0642] 1.1 Test sample

[0643] 1.1.1 Name: Dry ointment powder of Example 1.

[0644] 1.1.2 Physical characteristics: Brownish-yellow powder; slightly fragrant odor and slightly sweet taste.

[0645] 1.1.3 Source: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0646] 1.2 Positive Drug

[0647] Ambroxol Hydrochloride Tablets, Specification: 30mg / tablet, Manufacturer: Chengdu Hengrui Pharmaceutical Co., Ltd.

[0648] 1.3 Experimental System

[0649] 1.3.1 Animal strain: KM mouse.

[0650] 1.3.2 Animal grade: SPF grade.

[0651] 1.3.3 Animal sex and number: Half male and half female, a total of 60 animals, from which 50 animals of suitable weight (25 male and 25 female) were selected for the formal experiment.

[0652] 1.3.4 Animal age: Animals were approximately 4 weeks old at the time of receipt and approximately 5 weeks old at the time of drug administration.

[0653] 1.3.5 Animal weight: The proposed animal weight range is 18-20g, the actual weight of the animals received is 16.9-20.9g, and the weight at the time of the first administration is 22.3-27.3g.

[0654] 1.3.6 Animal source: Purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0655] 1.3.7 Husbandry conditions: Mice are cage-raised with 12 hours of light per day, a temperature of 20-26℃, and a relative humidity of 40-70%.

[0656] 1.3.8 Quarantine process: Animals were acclimatized and weighed once a day for 5 consecutive days. During this period, the animals drank water and ate normally, were in good health, and showed no signs of disease or death.

[0657] 2. Test Methods

[0658] 2.1 Basis for Experimental Design

[0659] 2.1.1 Standards adopted: "Classification of Traditional Chinese Medicine Registration and Requirements for Application Materials", "Good Laboratory Practice for Non-Clinical Studies of Drugs" and related literature.

[0660] 2.1.2 Experimental System Selection Instructions: Most expectorants work by increasing respiratory secretions, thinning the sputum attached to the respiratory mucosa, making it easier to detach from the airway wall and cough up. The most commonly used evaluation methods are respiratory fluid volume measurement and goblet cell and alveolar type II cell secretion function measurement. Among them, the respiratory fluid volume measurement method often utilizes the characteristic that phenol red is partially excreted from the airway after intraperitoneal injection in mice to measure the amount of phenol red excreted from the mouse airway in order to determine the effect of the test substance on the amount of airway secretions.

[0661] 2.2 Dosage and Grouping

[0662] See Table 19 for details.

[0663] Table 19 Effects of test substances on phenol red excretion in mouse trachea; test groups and dosage settings.

[0664] 2.3 Administration method

[0665] Administer the medication via gavage.

[0666] 2.4 Preparation and storage of test samples

[0667] Weigh approximately 0.4g (low), 0.9g (medium), and 1.8g (high) of the test substance, grind them separately with sterile water for injection, and bring the volume to 10mL; take 2 tablets of ambroxol hydrochloride, grind them with sterile water for injection, and bring the volume to 10mL. All test drugs should be prepared fresh before use.

[0668] 2.5 Administration of the test sample

[0669] The drug was administered using a disposable syringe at a volume of 20 mL / kg. The model group animals were given the corresponding volume of solvent. The drug was administered once a day for 3 consecutive days.

[0670] 2.6 Steps and Observation Indicators

[0671] Newly received experimental animals were numbered, acclimatized for 5 days, and grouped as described above. Mice were fasted for at least 15 hours before the last administration, but allowed free access to water. Immediately after the last administration, mice were intraperitoneally injected with 2.5% phenol red (prepared with 0.9% physiological saline) at a volume of 20 mL / kg. Approximately 1 hour later, the animals were euthanized by cervical dislocation, exposing the trachea. The tracheal tube was connected to a syringe, and 0.8 mL of 5% NaHCO3 aqueous solution was slowly injected into the trachea, then gently aspirated. This process was repeated once. The mixture was centrifuged at 2500 rpm for 10 min to obtain a clear supernatant. The absorbance was measured at 545 nm, and the amount of phenol red excreted was calculated based on the phenol red standard curve.

[0672] Preparation of phenol red standard curve: Weigh 0.00103g of phenol red, dissolve it in 5% NaHCO3 aqueous solution, and make up to 10mL to prepare a phenol red solution of about 100μg / mL (stock solution). Dilute it successively to 32, 16, 8, 4, 2, 1 and 0.5μg / mL phenol red solutions, measure the absorbance values, and construct a standard curve with phenol red concentration as the x-axis and absorbance value as the y-axis.

[0673] 2.7 Statistical Methods

[0674] The statistical method is the same as that used in Example 6.

[0675] 3. Results

[0676] As shown in Table 20, compared with the model group, the amount of phenol red excreted in the trachea of ​​mice in the ambroxol group was significantly increased (P<0.05), and the amount of phenol red excreted in the medium and high dose groups of the test substance was significantly increased (P<0.05). The amount of phenol red excreted in the low dose group was increased, but no significant difference was observed (P>0.05).

[0677] Table 20 Results of the experiment on the effect of the test substance on phenol red excretion in mouse trachea. Note: Compared with the model group: *P<0.05.

[0678] 4. Conclusion

[0679] Under the conditions of this experiment, the test substance significantly increased the amount of phenol red excreted in the trachea of ​​mice, indicating that it can increase the amount of respiratory secretions, dilute sputum, and has an expectorant effect.

[0680] Example 10: Study on the analgesic effect of the test substance

[0681] Experimental Objective

[0682] In this experiment, mice were induced to writhe by intraperitoneal injection of 0.7% acetic acid to observe the effect of the test substance on pain.

[0683] 1. Experimental Materials

[0684] 1.1 Test sample

[0685] 1.1.1 Name: Dry ointment powder of Example 1.

[0686] 1.1.2 Physical characteristics: Brownish-yellow powder; slightly fragrant odor and slightly sweet taste.

[0687] 1.1.3 Source: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0688] 1.2 Positive Drug

[0689] Indomethacin capsules, specification: 25mg / capsule, manufacturer: Hebei Yongfeng Pharmaceutical Co., Ltd.

[0690] 1.3 Experimental System

[0691] 1.3.1 Animal strain: KM mouse.

[0692] 1.3.2 Animal grade: SPF grade.

[0693] 1.3.3 Animal sex and number: Half male and half female, a total of 60 animals, from which 50 animals of suitable weight (25 male and 25 female) were selected for the formal experiment.

[0694] 1.3.4 Animal age: Animals were approximately 4 weeks old at the time of receipt and approximately 5 weeks old at the time of drug administration.

[0695] 1.3.5 Animal weight: The proposed animal weight range is 18-20g, the actual weight of the animals received is 17.9-20.8g, and the weight at the time of the first administration is 24.5-27.9g.

[0696] 1.3.6 Animal source: Purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0697] 1.3.7 Husbandry conditions: Mice are cage-raised with 12 hours of light per day, a temperature of 20-26℃, and a relative humidity of 40-70%.

[0698] 1.3.8 Quarantine process: Animals were acclimatized and weighed once a day for 5 consecutive days. During this period, the animals drank water and ate normally, were in good health, and showed no signs of disease or death.

[0699] 2. Test Methods

[0700] 2.1 Basis for Experimental Design

[0701] 2.1.1 Standards adopted: "Classification of Traditional Chinese Medicine Registration and Requirements for Application Materials", "Good Laboratory Practice for Non-Clinical Studies of Drugs" and related literature.

[0702] 2.1.2 Experimental System Selection Instructions: The basic principle of this experiment is to inject a certain volume and concentration of chemical irritant (acetic acid) into the peritoneal cavity of mice, stimulating the visceral and parietal peritoneum, causing a deep, large-area, and prolonged pain response. This results in behavioral responses in the mice such as abdominal retraction, trunk and hind limb extension, and raised buttocks, known as the writhing response. This response occurs most frequently within 15 minutes after injection; therefore, the latency and frequency of the writhing response within 15 minutes after injection are often used as quantitative indicators of pain.

[0703] 2.2 Dosage and Grouping

[0704] See Appendix 21 for details.

[0705] Table 21 Effects of test substances on acetic acid-induced writhing in mice; test groups and dosage settings.

[0706] 2.3 Administration method

[0707] Administer the medication via gavage.

[0708] 2.4 Preparation and storage of test samples

[0709] Weigh approximately 0.4g (low), 0.9g (medium), and 1.8g (high) of the test substance, grind them separately with sterile water for injection, and bring the volume to 10mL; take one indomethacin capsule, grind it with sterile water for injection, and bring the volume to 100mL. All test drugs should be prepared fresh before use.

[0710] 2.5 Administration of the test sample

[0711] The drug was administered using a disposable syringe at a volume of 20 mL / kg. The model group animals were given the corresponding volume of solvent. The drug was administered once a day for 3 consecutive days.

[0712] 2.6 Steps and Observation Indicators

[0713] Newly received experimental animals were numbered and acclimatized for 5 days, grouped as described previously. Approximately 1 hour after the last administration, 10 mL / kg of 0.7% acetic acid (prepared with 0.9% physiological saline) was injected into the peritoneal cavity of the mice to stimulate the visceral and parietal peritoneum, causing a deep, large-area, and prolonged pain response. This resulted in writhing behaviors in the mice, such as abdominal retraction, trunk and hind limb extension, and raised buttocks. The latency and frequency of writhing responses within 15 minutes after injection were recorded as quantitative indicators of pain. If no writhing response was observed within 15 minutes, the latency was recorded as 900 seconds.

[0714] 2.7 Statistical Methods

[0715] The statistical method is the same as that used in Example 6.

[0716] 3. Results

[0717] As shown in Table 22, compared with the model group, the number of writhing movements in the indomethacin group was significantly reduced (P<0.05), the number of writhing movements in the high-dose group was significantly reduced (P<0.05), and the number of writhing movements in the low- and medium-dose groups was reduced compared with the model group, but the difference was not significant (P>0.05). There was no significant difference in the writhing latency between the drug administration groups and the model group (P>0.05).

[0718] Table 22 Results of the effects of the test substances on acetic acid-induced writhing in mice Note: Compared with the model group: *P<0.05.

[0719] 4. Conclusion

[0720] The test substance significantly reduced the number of writhing movements caused by acetic acid and inhibited the pain response induced by acetic acid stimulation of the peritoneum in mice.

[0721] Example 11: Study on the immunomodulatory effects of the test substance

[0722] Experimental Objective

[0723] A mouse model of immunosuppression was established by intraperitoneal injection of cyclophosphamide, and the immunomodulatory effects of the test substance were evaluated using the immune organ weight method.

[0724] 1. Experimental Materials

[0725] 1.1 Test sample

[0726] 1.1.1 Name: Dry ointment powder of Example 1.

[0727] 1.1.2 Physical characteristics: Brownish-yellow powder; slightly fragrant odor and slightly sweet taste.

[0728] 1.1.3 Source: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0729] 1.2 Positive Drug

[0730] Levamisole Hydrochloride Tablets, Specification: 25mg / tablet, Manufacturer: Renhetang Pharmaceutical Co., Ltd.

[0731] 1.3 Experimental System

[0732] 1.3.1 Animal strain: BALB / c mouse.

[0733] 1.3.2 Animal grade: SPF grade.

[0734] 1.3.3 Animal sex and number: half male and half female, a total of 70 animals, from which 60 animals of suitable weight (30 male and 30 female) were selected for the formal experiment.

[0735] 1.3.4 Animal age: Animals were approximately 6 weeks old at the time of receipt and approximately 7 weeks old at the time of first administration.

[0736] 1.3.5 Animal weight: The proposed animal weight range is 18-20g, the actual weight of the animals received is 17.7-20.8g, and the weight at the time of the first administration is 18.5-21.8g.

[0737] 1.3.6 Animal source: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0738] 1.3.7 Husbandry conditions: Mice are cage-raised with 12 hours of light per day, a temperature of 20-26℃, and a relative humidity of 40-70%.

[0739] 1.3.8 Quarantine process: Animals were acclimatized and weighed once a day for 5 consecutive days. The animals drank water and ate normally, were in good health, and showed no signs of disease or death.

[0740] 2. Test Methods

[0741] 2.1 Basis for Experimental Design

[0742] 2.1.1 Standards adopted: "Classification of Traditional Chinese Medicine Registration and Requirements for Application Materials", "Good Laboratory Practice for Non-Clinical Studies of Drugs" and related literature.

[0743] 2.1.2 Experimental System Selection Instructions: Cyclophosphamide is a cytotoxic chemotherapeutic drug and also belongs to the alkylating agent class of immunosuppressants. It can damage DNA structure, block replication, and thus lead to cell death. Due to its strong immunosuppressive effect, cyclophosphamide is a commonly used drug in immunotoxicology for preparing immunosuppressive models. The main immune organs of the body are the thymus and spleen. The thymus is the site of T cell differentiation and maturation, and the spleen is one of the sites where mature T cells and B cells reside. It is also the main site for immune responses to these cells after stimulation by antigens and other foreign substances, playing an important role in cellular and humoral immunity. The effect of drugs on the weight of the thymus and spleen in experimental animals is often used as a preliminary indicator to observe the effect of drugs on immune function.

[0744] 2.2 Dosage and Grouping

[0745] See Table 23 for details.

[0746] Table 23 Effects of test substances on cyclophosphamide-induced immunosuppression in mice; test groups and dosage settings.

[0747] 2.3 Administration method

[0748] The administration was performed via gavage, consistent with the intended route of administration in clinical practice.

[0749] 2.4 Preparation and storage of test samples

[0750] Weigh approximately 0.4g (low), 0.9g (medium), and 1.8g (high) of the test substance, grind them separately with sterile water for injection, and bring the volume to 10mL; take one levamisole hydrochloride tablet, grind it with sterile water for injection, and bring the volume to 10mL. All test drugs should be prepared fresh before use.

[0751] 2.5 Administration of the test sample

[0752] The drug was administered using a disposable syringe at a volume of 20 mL / kg. Animals in the normal and model groups were given the same volume of solvent. The drug was administered once a day for 5 consecutive days.

[0753] 2.6 Steps and Observation Indicators

[0754] Newly received experimental animals were numbered and acclimatized for 5 days, then grouped as described previously. Except for the normal control group, all other groups received an intraperitoneal injection of 100 mg / kg cyclophosphamide (prepared with 0.9% physiological saline, 10 mL / kg) on ​​day 3 of drug administration to establish a mouse immunosuppression model. The normal control group received the corresponding volume of 0.9% physiological saline. Approximately 5 hours after the last drug administration, the animals were euthanized by cervical dislocation. The thymus and spleen were dissected and weighed, and the thymus and spleen indices were calculated: Thymus index = thymus weight / body weight, Spleen index = spleen weight / body weight.

[0755] 2.7 Statistical Methods

[0756] The statistical method is the same as that used in Example 6.

[0757] 3. Results

[0758] As shown in Table 24, compared with the normal group, the thymus and spleen indices of mice in the model group were significantly reduced (P<0.01); compared with the model group, the spleen index of mice in the high-dose group and the thymus index of mice in the low-dose group were significantly increased (P<0.05), while the thymus index of mice in the high-dose group, the spleen index of mice in the low-dose group, and the thymus and spleen indices of mice in the medium-dose group and the levamisole group were all increased, but no significant differences were observed (P>0.05).

[0759] Table 24 Results of the experiment on the effect of the test substance on cyclophosphamide-induced immunosuppression in mice Note: Comparison between the model group and the normal group. ## P<0.01; Comparison between the drug-treated group and the model group: *P<0.05.

[0760] 4. Conclusion

[0761] Intraperitoneal injection of cyclophosphamide in mice significantly reduced the thymus and spleen indices, while the test substance increased the thymus and spleen indices in model mice, indicating an immunomodulatory effect.

[0762] Example 12 Study on the nonspecific immunomodulatory effects of the test substance

[0763] Experimental Objective

[0764] After a certain concentration of carbon particles is injected into the tail vein of mice, they are rapidly phagocytosed by the mononuclear macrophage system (RES) and cleared from the blood; this is known as the carbon particle clearance method. This method was used to investigate the effect of the test substance on RES and evaluate its role in non-specific immunity.

[0765] 1. Experimental Materials

[0766] 1.1 Test sample

[0767] 1.1.1 Name: Dry ointment powder of Example 1.

[0768] 1.1.2 Physical characteristics: Brownish-yellow powder; slightly fragrant odor and slightly sweet taste.

[0769] 1.1.3 Source: Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0770] 1.2 Positive reagents and main reagents

[0771] 1.2.1 Positive drug: Levamisole hydrochloride tablets, specification: 25mg / tablet, manufacturer: Renhetang Pharmaceutical Co., Ltd.

[0772] 1.2.2 Main Reagents

[0773] Indian ink: Solarbio.

[0774] 1.3 Experimental System

[0775] 1.3.1 Animal strain: BALB / c mouse.

[0776] 1.3.2 Animal grade: SPF grade.

[0777] 1.3.3 Animal sex and number: 70 males in total, of which 60 animals of suitable weight were selected for the formal experiment.

[0778] 1.3.4 Animal age: Animals were approximately 7 weeks old at the time of receipt and approximately 8 weeks old at the time of first administration.

[0779] 1.3.5 Animal weight: The proposed animal weight range is 22-24g, the actual weight of the animals received is 20.4-25.0g, and the weight at the time of the first administration is 23.0-25.3g.

[0780] 1.3.6 Animal source: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0781] 1.3.7 Husbandry conditions: Mice are cage-raised with 12 hours of light per day, a temperature of 20-26℃, and a relative humidity of 40-70%.

[0782] 1.3.8 Quarantine process: Animals were acclimatized and weighed once a day for 5 consecutive days. The animals drank water and ate normally, were in good health, and showed no signs of disease or death.

[0783] 2. Test Methods

[0784] 2.1 Basis for Experimental Design

[0785] 2.1.1 Standards adopted: "Classification of Traditional Chinese Medicine Registration and Requirements for Application Materials", "Good Laboratory Practice for Non-Clinical Studies of Drugs" and related literature.

[0786] 2.1.2 Experimental System Selection Instructions: The carbon particle clearance method is a commonly used method in immunology for non-specific immune function assays. When particulate foreign bodies (such as carbon particles) enter the bloodstream, they can be rapidly phagocytosed and cleared by mononuclear macrophages. Therefore, the phagocytic function of the mononuclear macrophage system (RES) can be reflected by measuring the rate of carbon particle disappearance in the blood, often characterized by the phagocytic index K. The phagocytic index K reflects the phagocytic clearance capacity and rate of the RES for injected carbon particles. Estrogen has a certain influence on the phagocytic activity of the RES; therefore, male animals should generally be used.

[0787] 2.2 Dosage and Grouping

[0788] See Table 25 for details.

[0789] Table 25 Effects of test substances on carbon particle clearance ability in mice; test groups and dosage settings.

[0790] 2.3 Administration method

[0791] Administer the medication via gavage.

[0792] 2.4 Preparation and storage of test samples

[0793] Weigh approximately 0.4g (low), 0.9g (medium), and 1.8g (high) of the test substance, grind them separately with sterile water for injection, and bring the volume to 10mL; take one levamisole hydrochloride tablet, grind it with sterile water for injection, and bring the volume to 10mL. Prepare the test drug immediately before use.

[0794] 2.5 Administration of the test sample

[0795] The drug was administered using a disposable syringe at a volume of 20 mL / kg. Animals in the model group were given the same volume of solvent. The drug was administered once a day for 5 consecutive days.

[0796] 2.6 Steps and Observation Indicators

[0797] Newly received experimental animals were numbered and acclimatized for 5 days, grouped as described previously. Approximately 1 hour after the last administration, all animals were injected intravenously with 20% India ink (diluted with 0.9% physiological saline, injection volume 10 mL / kg). Blood samples were collected from the orbital sinus immediately after injection (T1, which can be recorded as zero) and 10 minutes after injection (T2). 20 μL of each sample was added to 2 mL of 0.1% Na2CO3 solution, and the absorbance values ​​OD1 (absorbance measured immediately after injection) and OD2 (absorbance measured 10 minutes after injection) were measured at 675 nm. Using 0.1% Na2CO3 solution as a blank, the phagocytic index K was calculated: Phagocytic index K = (lgOD1 - lgOD2) / (T2 - T1)

[0798] 2.7 Statistical Methods

[0799] The statistical method is the same as that used in Example 6.

[0800] 3. Results

[0801] As shown in Table 26, compared with the model group, the carbon particle phagocytosis index K of mice in the levamisole group and the medium dose group of the test substance was significantly increased (P<0.05), and it was also increased in the low and high dose groups of the test substance, but no significant difference was observed (P>0.05).

[0802] Table 26 Results of the test results on the effect of the test substances on the carbon particle clearance ability of mice. Note: Compared with the model group: *P<0.05. Tail vein injection failed in some animals, hence the different number of animals in each group.

[0803] 4. Conclusion

[0804] The test substance significantly increased the carbon particle phagocytic index K in mice, and had a regulatory effect on non-specific immune function.

Claims

1. A traditional Chinese medicine composition, the composition being made from the following raw materials and optional pharmaceutical excipients, said raw materials including or composed of the following drugs: Astragalus membranaceus, Lonicera japonica, Saposhnikovia divaricata, Cinnamomum cassia, Paeonia lactiflora, Forsythia suspensa, Atractylodes macrocephala, Platycodon grandiflorus, Zingiber officinale, Ziziphus jujuba, Citrus reticulata, Isatis indigotica and Glycyrrhiza uralensis.

2. The traditional Chinese medicine composition according to claim 1, wherein, By weight, the active pharmaceutical ingredient comprises or consists of the following drugs Composition: Astragalus membranaceus 250-500 parts, Lonicera japonica 250-450 parts, Saposhnikovia divaricata 120-220 parts, Cinnamomum cassia 80-150 parts, Paeonia lactiflora 100-180 parts, Forsythia suspensa 100-180 parts, Atractylodes macrocephala 120-220 parts, Platycodon grandiflorus 100-180 parts, Zingiber officinale 40-80 parts, Ziziphus jujuba 40-80 parts, Citrus reticulata 100-180 parts, Isatis indigotica 250-450 parts, Glycyrrhiza uralensis 40-80 parts.

3. The traditional Chinese medicine composition according to claim 1 or 2, wherein, By weight, the active pharmaceutical ingredient comprises or consists of the following drugs Composition: Astragalus membranaceus 300-480 parts, Lonicera japonica 300-440 parts, Saposhnikovia divaricata 130-200 parts, Cinnamomum cassia 90-145 parts, Paeonia lactiflora 110-160 parts, Forsythia suspensa 120-160 parts, Atractylodes macrocephala 140-200 parts, Platycodon grandiflorus 110-160 parts, Zingiber officinale 45-70 parts, Ziziphus jujuba 45-80 parts, Citrus reticulata 120-160 parts, Isatis indigotica 300-400 parts, Glycyrrhiza uralensis 45-80 parts.

4. The traditional Chinese medicine composition according to any one of claims 1 to 3, wherein, Based on parts by weight, the active pharmaceutical ingredient comprises or consists of the following pharmaceutical ingredients: Astragalus membranaceus 450g, Lonicera japonica 365g, Saposhnikovia divaricata 150g, Cinnamomum cassia 100g, Paeonia lactiflora 120g, Forsythia suspensa 140g, Atractylodes macrocephala 160g, Platycodon grandiflorus 130g, Zingiber officinale 65g, Ziziphus jujuba 50g, Citrus reticulata 150g, Isatis indigotica 355g, Glycyrrhiza uralensis 50g; or Astragalus membranaceus 365g, Lonicera japonica 335g, Saposhnikovia divaricata 180g, Cinnamomum cassia 100g, Paeonia lactiflora 150g, Forsythia suspensa 130g, Atractylodes macrocephala 180g, Platycodon grandiflorus 130g, Zingiber officinale 60g, Ziziphus jujuba 50g, Citrus reticulata 150g, Isatis indigotica 335g, Glycyrrhiza uralensis 50g; or Astragalus membranaceus 320g, Lonicera japonica 380g, Saposhnikovia divaricata 180g, Cinnamomum cassia 120g, Paeonia lactiflora 130g, Forsythia suspensa 150g, Atractylodes macrocephala 200g, Platycodon grandiflorus 150g, Zingiber officinale 50g, Ziziphus jujuba 65g, Citrus reticulata 140g, Isatis indigotica 365g, Glycyrrhiza uralensis 60g; or Astragalus membranaceus 335g, Lonicera japonica 400g, Saposhnikovia divaricata 160g, Cinnamomum cassia 130g, Paeonia lactiflora 150g, Forsythia suspensa 130g, Atractylodes macrocephala 155g, Platycodon grandiflorus 120g, Zingiber officinale 55g, Ziziphus jujuba 70g, Citrus reticulata 135g, Isatis indigotica 330g, Glycyrrhiza uralensis 70g; or Astragalus membranaceus 345g, Lonicera japonica 345g, Saposhnikovia divaricata 173g, Cinnamomum cassia 115g, Paeonia lactiflora 138g, Forsythia suspensa 138g, Atractylodes macrocephala 173g, Platycodon grandiflorus 138g, Zingiber officinale 58g, Ziziphus jujuba 58g, Citrus reticulata 138g, Isatis indigotica 345g, Glycyrrhiza uralensis 58g.

5. The traditional Chinese medicine composition according to any one of claims 1 to 4, wherein it is an oral preparation, preferably a liquid preparation, tablet, granule, powder or capsule.

6. The traditional Chinese medicine composition according to any one of claims 1 to 5, wherein the pharmaceutically acceptable excipients include sucralose and maltodextrin.

7. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of a medicament for treating colds, wherein the cold is preferably a cold due to qi deficiency.

8. The use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of antiviral drugs, wherein the virus is preferably influenza A virus, influenza B virus, rhinovirus, respiratory syncytial virus, parainfluenza virus, enterovirus, coronavirus, and adenovirus.

9. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of antipyretic drugs.

10. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of anti-inflammatory drugs.

11. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of antitussive drugs.

12. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of expectorant drugs.

13. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of analgesic drugs.

14. The use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of an immunomodulatory drug, wherein the immunomodulation is preferably immunosuppression or nonspecific immunomodulation.

15. The use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of a drug for preventing viral infection, wherein the virus is preferably an influenza A virus, and the influenza A virus is preferably an H1N1 virus or an H3N2 virus.

16. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of a drug for protecting close contacts of H1N1 influenza.

17. A method for preparing a traditional Chinese medicine composition according to any one of claims 1 to 6, the method comprising: Weigh the raw materials according to the prescription amount, add water and decoct three times. For the first decoction, add 8-12 times the weight of water, preferably about 10 times the weight of water, and decoct for 1-2 hours, preferably about 1.5 hours. For the second and third decoctions, add 6-10 times the weight of water, preferably about 8 times the weight of water, and decoct for 1-2 hours, preferably about 1 hour. Filter and concentrate the filtrate under reduced pressure.

18. The method for preparing the traditional Chinese medicine composition according to claim 17, wherein the method further comprises concentrating the filtrate under reduced pressure to an extract with a relative density of 1.10 to 1.20, preferably further comprising drying the extract to prepare a dry extract powder, more preferably further comprising granulating the dry extract powder with pharmaceutically acceptable excipients.

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