Application of traditional Chinese medicine composition in preventing influenza A virus replication at cellular level

Abstract

The invention provides an application of a traditional Chinese medicine composition in preventing influenza A virus replication at a cellular level. The traditional Chinese medicine composition is prepared from the following raw materials: cortex magnoliae officinalis, scorched betel nut, roasted amomum tsao-ko, herba ephedrae, semen armeniacae amarae, notopterygium root, ginger, pogostemon cablin, herba eupatorii, rhizoma atractylodis, bighead atractylodes rhizome, poria cocos, scorched hawthorn fruit, scorched medicated leaven, scorched malt, rhizoma dryopteris crassirhizomae, radix cynanchi paniculati, earthworm, gypsum and semen lepidii. It is confirmed for the first time that the traditional Chinese medicine composition can inhibit replication of influenza A viruses (such as influenza A H1N1 viruses) on the cellular level, has an obvious inhibiting effect on influenza A viruses, has the advantages of being clear in effective component, obvious in drug effect, small in taking dosage and the like, is good in application prospect, is suitable for industrial large-scale production, and has wide application prospects. The traditional Chinese medicine composition can be used for preparing medicines for preventing or treating influenza A virus infection, and has good economic benefits and social benefits.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to the application of a traditional Chinese medicine composition in preventing the replication of influenza A virus at the cellular level. Background Technology

[0002] Influenza is an acute respiratory infectious disease caused by the influenza virus. It is highly contagious and spreads rapidly. Influenza viruses are mainly classified into four types: A, B, C, and D. Among them, influenza A virus is the main culprit causing seasonal influenza and triggering global pandemics. Due to its antigenic variability, it leads to widespread susceptibility in the population, posing a long-term and severe challenge to global public health security.

[0003] Currently, the main methods for preventing and treating influenza A mainly include chemical antiviral drugs and traditional Chinese medicine (TCM) preparations. The most common chemical antiviral drugs are oseltamivir and ribavirin. Oseltamivir inhibits neuraminidase activity to prevent viral release and spread, while ribavirin interferes with viral replication. However, chemical antiviral drugs often have side effects such as nausea and vomiting, and long-term use may lead to viral mutations and reduced drug sensitivity. TCM preparations, due to their fewer side effects, have been widely used for the prevention or treatment of influenza A virus infection. These preparations mainly contain ingredients such as Isatis root, Schizonepeta, Saposhnikovia root, and licorice. However, existing TCM preparations primarily focus on symptom relief and have shortcomings such as unclear mechanisms of action, poor efficacy, and long treatment courses.

[0004] Therefore, finding and identifying traditional Chinese medicine compositions that can effectively prevent or treat influenza A virus infection is of great practical significance and clinical value for enriching clinical drug options and responding to influenza pandemics. Summary of the Invention

[0005] This invention provides an application of a traditional Chinese medicine composition in preventing the replication of influenza A virus at the cellular level. This traditional Chinese medicine composition can effectively inhibit the replication of influenza A virus, has high safety, no toxic side effects, and high application value.

[0006] This invention provides an application of a traditional Chinese medicine composition in preventing the replication of influenza A virus at the cellular level. The raw materials of the traditional Chinese medicine composition include Magnolia officinalis, Areca catechu (roasted), Amomum villosum (roasted), Ephedra sinica, Prunus armeniaca (bitter), Notopterygium incisum, Zingiber officinale, Pogostemon cablin, Eupatorium fortunei, Atractylodes lancea, Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida (roasted), Massa fermentata (roasted), Hordeum vulgare (roasted), Dryopteris crassirhizoma, Cynanchum paniculatum, Pheretima aspergillum, Gypsum fibrosum, and Lepidium apetalum.

[0007] The traditional Chinese medicine composition provided by this invention has high safety and no toxic side effects. It has a significant inhibitory effect on influenza A virus, especially on influenza A H1N1 virus.

[0008] Further, the traditional Chinese medicine composition comprises the following raw material components in parts by weight: Magnolia officinalis 200-300 parts, Areca catechu (roasted) 100-200 parts, Amomum villosum (roasted) 100-200 parts, Ephedra sinica 50-150 parts, Prunus armeniaca 100-200 parts, Notopterygium incisum 200-300 parts, Zingiber officinale 200-300 parts, Pogostemon cablin 200-300 parts, Eupatorium fortunei 100-200 parts, and Atractylodes lancea 100-200 parts. 200-300 parts of Atractylodes macrocephala, 400-600 parts of Atractylodes macrocephala, 700-800 parts of Poria cocos, 100-200 parts of charred hawthorn, 100-200 parts of charred Shenqu (medicated leaven), 100-200 parts of charred malt, 100-200 parts of Dryopteris crassirhizoma, 200-300 parts of Cynanchum paniculatum, 200-300 parts of earthworm, 200-300 parts of gypsum, and 200-300 parts of Lepidium apetalum.

[0009] Preferably, the traditional Chinese medicine composition comprises the following raw material components in parts by weight: Magnolia officinalis 250±10 parts, Areca catechu 150±10 parts, Amomum villosum 150±10 parts, Ephedra sinica 100±10 parts, Prunus armeniaca 150±10 parts, Notopterygium incisum 250±10 parts, Zingiber officinale 250±10 parts, Pogostemon cablin 250±10 parts, Eupatorium fortunei 150±10 parts, Atractylodes lancea 250±10 parts, Atractylodes macrocephala 500±10 parts, Poria cocos 750±10 parts, Crataegus pinnatifida 150±10 parts, Massa fermentata 150±10 parts, Hordeum vulgare 150±10 parts, Dryopteris crassirhizoma 150±10 parts, Cynanchum paniculatum 250±10 parts, Pheretima aspergillum 250±10 parts, Gypsum fibrosum 250±10 parts, and Lepidium apetalum 250±10 parts.

[0010] Furthermore, the effective active ingredients in the traditional Chinese medicine composition include gallic acid, norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, atractylodes ketone, amygdalin, epimedium glycoside, imperatorin, magnolol, imperatorin, leucogenin, apigenin, kaempferol, epicatechin, magnolol, coumarin, etc.

[0011] Furthermore, the dosage form of the traditional Chinese medicine composition is a powder.

[0012] Furthermore, the traditional Chinese medicine composition is a raw material for the cold-dispelling and dampness-resolving granules. This invention does not specifically limit the source of the raw material for the cold-dispelling and dampness-resolving granules; any commercially available raw material conforming to the above-mentioned traditional Chinese medicine formula is acceptable. In the embodiments of this invention, the raw material for the cold-dispelling and dampness-resolving granules is sourced from Jiangsu Kangyuan Pharmaceutical Co., Ltd., and its formula is as follows: 250g Magnolia officinalis, 150g Areca catechu (roasted), 150g Amomum villosum (roasted), 100g Ephedra sinica, 150g Prunus armeniaca (bitter), 250g Notopterygium incisum, 250g Zingiber officinale, 250g Pogostemon cablin, 150g Eupatorium fortunei, 250g Atractylodes lancea, 500g Atractylodes macrocephala, 750g Poria cocos, 150g Crataegus pinnatifida (roasted), 150g Massa fermentata (roasted), 150g Hordeum vulgare (roasted), 150g Dryopteris crassirhizoma, 250g Cynanchum paniculatum, 250g Pheretima aspergillum, 250g Gypsum fibrosum, and 250g Lepidium apetalum.

[0013] Furthermore, the influenza A virus is the influenza A H1N1 virus.

[0014] Furthermore, the concentration of the traditional Chinese medicine composition that effectively inhibits the H1N1 influenza virus is ≥0.690 mg / mL. Controlling the concentration of the traditional Chinese medicine composition within this range ensures both its superior antiviral effect and avoids potential cytotoxicity.

[0015] Preferably, the concentration at which the traditional Chinese medicine composition effectively inhibits the H1N1 influenza virus is 0.790±0.100 mg / mL.

[0016] Furthermore, the traditional Chinese medicine composition is used to inhibit the replication of the influenza A virus at the cellular level.

[0017] Furthermore, the prevention or treatment is used to prevent or treat fever, cough, sore throat, muscle aches, or lung damage caused by influenza A virus infection.

[0018] Furthermore, the application of the traditional Chinese medicine composition in medicines for the prevention or treatment of H1N1 influenza virus infection.

[0019] The present invention provides the beneficial effects of a traditional Chinese medicine composition on the prevention of influenza A virus replication at the cellular level: The traditional Chinese medicine composition of the present invention is prepared from raw materials including Magnolia officinalis, Areca catechu (roasted), Amomum villosum (roasted), Ephedra sinica, Prunus armeniaca (bitter), Notopterygium incisum, Zingiber officinale, Pogostemon cablin, Eupatorium fortunei, Atractylodes lancea, Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida (roasted), Massa fermentata (roasted), Hordeum vulgare (roasted), Dryopteris crassirhizoma, Cynanchum paniculatum, Pheretima aspergillum, Gypsum fibrosum, and Lepidium apetalum. This traditional Chinese medicine composition has high safety and few toxic side effects. The present invention is the first to demonstrate that this traditional Chinese medicine composition can inhibit the replication of influenza A virus (such as influenza A H1N1 virus) at the cellular level, and has a significant inhibitory effect on influenza A virus. It has the advantages of clear effective ingredients, significant efficacy, and small dosage, and has good application prospects. It is suitable for industrial-scale production. This traditional Chinese medicine composition can be used to prepare drugs for the prevention or treatment of influenza A virus infection, and has good economic and social benefits. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the experimental scheme in the embodiment.

[0022] Figure 2 It is the inhibitory effect of the raw material of the cold-dispelling and dampness-resolving granules on the H1N1 influenza virus. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0024] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0025] Example The traditional Chinese medicine composition in this embodiment is the raw material of Sanhan Huashi Granules, which can effectively inhibit the replication of H1N1 influenza A virus at the cellular level. This embodiment uses Sanhan Huashi Granules as experimental material to verify its direct inhibitory effect on the replication of H1N1 influenza A virus through in vitro cell experiments, thus confirming its inhibitory effect on H1N1 influenza A virus.

[0026] In this embodiment, the raw material for the cold-dispelling and dampness-resolving granules is sourced from Jiangsu Kangyuan Pharmaceutical Co., Ltd., and its formula is as follows: Magnolia officinalis 250g, Areca catechu 150g, Amomum villosum 150g, Ephedra sinica 100g, Prunus armeniaca 150g, Notopterygium incisum 250g, Zingiber officinale 250g, Pogostemon cablin 250g, Eupatorium fortunei 150g, Atractylodes lancea 250g, Atractylodes macrocephala 500g, Poria cocos 750g, Crataegus pinnatifida 150g, Massa fermentata 150g, Hordeum vulgare 150g, Dryopteris crassirhizoma 150g, Cynanchum paniculatum 250g, Pheretima aspergillum 250g, Gypsum fibrosum 250g, Lepidium apetalum 250g.

[0027] The experimental scheme and verification results of this embodiment are as follows: Experimental Protocol: To evaluate the inhibitory effect of the raw material of Sanhan Huashi Granules on H1N1 influenza virus replication at the cellular level. MDCK cells were used as the sensitive cells for H1N1 influenza virus. The selected H1N1 influenza A virus titer was 100 TCID. 50 / 100μL, cell density selected was 3×10 5 / mL.

[0028] 1. Formula: (1) Raw material formula of the medicine for dispelling cold and dampness: 250g of Magnolia officinalis, 150g of charred Areca catechu, 150g of roasted Amomum villosum, 100g of Ephedra sinica, 150g of bitter apricot kernel, 250g of Notopterygium incisum, 250g of fresh ginger, 250g of Pogostemon cablin, 150g of Eupatorium fortunei, 250g of Atractylodes lancea, 500g of Atractylodes macrocephala, 750g of Poria cocos, 150g of charred Crataegus pinnatifida, 150g of charred Shenqu (medicated leaven), 150g of charred malt, 150g of Dryopteris crassirhizoma, 250g of Cynanchum paniculatum, 250g of Pheretima aspergillum, 250g of gypsum, 250g of Lepidium apetalum, without starch and sucrose.

[0029] (2) Diluent for raw materials of the cold-dispelling and dampness-removing granules (DMEM culture medium), cell maintenance medium (DMEM culture medium containing 2.0% FBS), and cell culture medium (DMEM culture medium containing 10.0% FBS).

[0030] 2. Inhibitory effect of the raw material of the cold-dispelling and dampness-resolving granules on the replication of H1N1 influenza virus - based on the cellular level. See the experimental protocol. Figure 1 .

[0031] Experimental steps: (1) Cell preparation: Digest, resuspend, and count MDCK cells in good growth condition. Use 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μL of complete culture medium into 96-well plates. The plates were then incubated at 37°C with 5% CO2 for 18 hours to allow the cells to adhere and grow to approximately 80%-90% monolayer.

[0032] (2) Preparation of raw material solution of Sanhan Huashi Granules: The raw material of Sanhan Huashi Granules was prepared into a 2mg / ml stock solution with pure DMEM in a 50ml centrifuge tube, and diluted with Sanhan Huashi Granules raw material diluent at a volume ratio of 1:1 to prepare Sanhan Huashi Granules raw material with different dosage concentration gradients.

[0033] (3) Virus infection and administration: The pre-determined H1N1 influenza A virus was diluted to 100 TCID with cell maintenance medium. 50 100 μL of virus solution was added to each well (virus control well and experimental well). Cell control wells (100 μL of maintenance medium only, without virus or the raw material of the Sanhan Huashi granules) were also prepared. Virus control wells (100 μL of virus only, without the raw material of the Sanhan Huashi granules) were also prepared. Drug control wells (100 μL of the highest concentration of the Sanhan Huashi granules raw material was added only, without virus) and blank control wells (100 μL of maintenance medium only, resulting in no cells in the corresponding wells of the 96-well plate, without virus or the raw material of the Sanhan Huashi granules). The plates were incubated at 37°C in a 5% CO2 incubator.

[0034] (4) Effect detection: After culturing for 48 hours, observe the cytopathic effect (CPE, judgment criteria: cell rounding, shedding, breakage, etc.) of the virus control group under a microscope. It should reach 75%-90%; carefully aspirate the liquid in the well, add 100μL of CCK8 reagent to each well, and incubate for 1-2 hours; use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well at a wavelength of 640nm.

[0035] (5) The inhibition rate and EC were calculated using GraphPad Prism 9.5.1. 50 .

[0036] The inhibitory effects of the raw materials of the cold-dispelling and dampness-resolving granules on the H1N1 influenza virus are shown in Table 1. Figure 2 .

[0037] Table 1. Inhibitory effect of different concentrations of the raw material of the cold-dispelling and dampness-resolving granules on H1N1 influenza virus - CCK8 experiment The above results verify that the raw material of Sanhan Huashi Granules has a significant inhibitory effect on the H1N1 influenza virus (EC). 50 (0.6896 mg / ml) also showed good dose-dependent inhibition, and no obvious cell damage was observed at high concentrations, suggesting that it has a high safety profile.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a traditional Chinese medicine composition for preventing the replication of an influenza A virus at a cellular level, characterized in that, The raw materials of the traditional Chinese medicine composition include Magnolia officinalis, Ficus hirta, Evodia lepta, Ephedra sinica, bitter apricot kernel, Notopterygium incisum, Zingiber officinale, Pogostemonis herba, Pterocephalus hookeri, Atractylodes lancea, Atractylodes macrocephala, Poria cocos, Fructus Crataegi, Fructus Jujubae, Fructus Jujubae, Fructus Jujubae, Radix Pteroxygoni, Radix Rubiae, Lumbricus, Gypsum fibrosum and Semen Platycladi.

2. The use of the traditional Chinese medicine composition according to claim 1 for preventing the replication of influenza A virus at a cellular level, characterized in that, The traditional Chinese medicine composition comprises the following raw material components in mass parts: Magnolia officinalis 200-300 parts, Ficus hirta 100-200 parts, Evodia lepta 100-200 parts, Ephedra sinica 50-150 parts, bitter apricot kernel 100-200 parts, Notopterygium incisum 200-300 parts, Zingiber officinale 200-300 parts, Pogostemonis herba 200-300 parts, Pterocephalus hookeri 100-200 parts, Atractylodes lancea 200-300 parts, Atractylodes macrocephala 400-600 parts, Poria cocos 700-800 parts, Fructus Crataegi 100-200 parts, Fructus Jujubae 100-200 parts, Fructus Jujubae 100-200 parts, Fructus Jujubae 100-200 parts, Radix Pteroxygoni 100-200 parts, Radix Rubiae 200-300 parts, Lumbricus 200-300 parts, Gypsum fibrosum 200-300 parts and Semen Platycladi 200-300 parts.

3. The use of the traditional Chinese medicine composition according to claim 1 for preventing the replication of influenza A virus at a cellular level, characterized in that, The traditional Chinese medicine composition comprises the following raw material components in mass parts: Magnolia officinalis 200-300 parts, Ficus hirta 100-200 parts, Evodia lepta 100-200 parts, Ephedra sinica 50-150 parts, bitter apricot kernel 100-200 parts, Notopterygium incisum 200-300 parts, Zingiber officinale 200-300 parts, Pogostemonis herba 200-300 parts, Pterocephalus hookeri 100-200 parts, Atractylodes lancea 200-300 parts, Atractylodes macrocephala 400-600 parts, Poria cocos 700-800 parts, Fructus Crataegi 100-200 parts, Fructus Jujubae 100-200 parts, Fructus Jujubae 100-200 parts, Fructus Jujubae 100-200 parts, Radix Pteroxygoni 100-200 parts, Radix Rubiae 200-300 parts, Lumbricus 200-300 parts, Gypsum fibrosum 200-300 parts and Semen Platycladi 200-300 parts.

4. The use of the traditional Chinese medicine composition according to any one of claims 1-3 for preventing the replication of influenza A virus at a cellular level, characterized in that, The effective active ingredients in the traditional Chinese medicine composition include magnolol, ephedrine, pseudoephedrine, amygdalin, 6-gingerol and pogostone.

5. The use of the traditional Chinese medicine composition according to any one of claims 1-3 for preventing the replication of influenza A virus at a cellular level, characterized in that, The dosage form of the traditional Chinese medicine composition is powder.

6. The use of the traditional Chinese medicine composition according to claim 5 for preventing the replication of influenza A virus at a cellular level, characterized in that, The traditional Chinese medicine composition is a cold-damp dispelling granule raw material medicine.

7. The use of the traditional Chinese medicine composition according to any one of claims 1-6 for preventing the replication of influenza A virus at a cellular level, characterized in that, The influenza A virus is influenza A H1N1 virus. 8.The application of the traditional Chinese medicine composition in preventing the replication of influenza A virus at the cell level according to claim 7, characterized in that, The concentration of the traditional Chinese medicine composition for effectively inhibiting the influenza A H1N1 virus is ≥0.690 mg / mL.

9. The use of the traditional Chinese medicine composition according to any one of claims 1-8 for preventing the replication of influenza A virus at a cellular level, characterized in that, The traditional Chinese medicine composition is used for inhibiting the replication of the influenza A virus at a cellular level.

10. The use of the traditional Chinese medicine composition according to any one of claims 1-8 for preventing the replication of influenza A virus at a cellular level, characterized in that, The traditional Chinese medicine composition is used for preventing or treating fever, cough, sore throat, muscle soreness or lung injury caused by influenza A virus infection.

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