Compound traditional Chinese medicine powder and preparation method thereof

Abstract

The invention discloses compound traditional Chinese medicine powder which is prepared from the following raw materials: snake slough, toad skin and cockscomb. Researches show that the traditional Chinese medicine composition has an antiviral effect on various viruses. The traditional Chinese medicine is prepared from natural traditional Chinese medicinal materials, is good in safety and small in side effect and can be taken for a long time. The raw materials have a synergistic effect, mutually enhance the antiviral efficacy and improve the antiviral effect, have broad-spectrum antiviral activity, and can effectively inhibit the replication of various viruses and cope with various virus infections. The raw materials provided by the invention have anti-inflammatory and immunoregulation effects, and are helpful for alleviating inflammation caused by virus infection and enhancing the function of an immune system. The powder is convenient to take, can be taken anytime and anywhere, and is relatively low in price and high in cost performance. In conclusion, the traditional Chinese medicine composition has broad-spectrum antiviral activity and has potential in the aspect of preventing and treating virus infectious diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a compound traditional Chinese medicine powder and a preparation method thereof. Background Art

[0002] Viral infections are a major threat to public health worldwide and can cause a variety of diseases, including influenza, AIDS, hepatitis, measles, etc. The World Health Organization estimates that about 14 million people die from viral infections each year. Currently, antiviral drugs used to treat viral infections have many limitations: First, over time, viruses can easily develop resistance to existing antiviral drugs, thereby reducing the efficacy of the drugs. Second, antiviral drugs often have potential side effects, including liver damage, kidney damage, neurological side effects, etc. Finally, many antiviral drugs are only effective against specific types of viruses and cannot target multiple viral infections.

[0003] Compared with Western medicine, Chinese medicine has many potential advantages in antiviral treatment: first, Chinese medicine has a long history and rich experience in antiviral treatment, and many Chinese medicines have been shown to have antiviral activity; second, Chinese medicines usually have multi-target effects and can inhibit viral replication and transmission through multiple mechanisms; finally, Chinese medicines usually have lower side effects and are relatively safe. The problem of viral resistance is becoming increasingly serious, and there is an urgent need to develop new antiviral drugs to address the threat of viral resistance. At the same time, with the increase in globalization and population mobility, new viral infections continue to emerge, and new antiviral drugs need to be developed to address these new threats. The limitations of existing antiviral drugs, including resistance, side effects, and narrow scope of application, also require the development of new antiviral drugs to make up for these deficiencies. Therefore, the development of Chinese medicine antiviral drugs is of great significance and can provide new treatment options for dealing with viral infections. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a compound Chinese medicine powder which has broad-spectrum antiviral activity and is easy to use, highly safe and has little side effects, and a preparation method thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] Compound Chinese medicine powder, made from the following ingredients: snake slough, toad skin and cockscomb.

[0007] The compound Chinese medicine powder is prepared from the following raw materials in parts by weight: 0.5-4.5 parts of snake skin, 0.5-4.5 parts of toad skin and 3-7 parts of cockscomb.

[0008] The above-mentioned compound Chinese medicine powder is prepared from the following raw materials in parts by weight: 2.5 parts of snake skin, 2.5 parts of toad skin and 5 parts of cockscomb.

[0009] Powders are for oral administration.

[0010] The preparation method of the above-mentioned compound Chinese medicine powder comprises the following steps: washing and drying snake sloughs and toad skins respectively, mixing and crushing them into fine powder; soaking cockscombs and then boiling them, filtering them after boiling, and concentrating the filtrate into an extract; adding the fine powder into the extract and mixing them evenly, and then drying the extract to prepare a dry extract, which is then crushed to obtain the powder.

[0011] The above preparation method is carried out according to the following steps:

[0012] <1> Select dried snake skin and toad skin, soak them in clean water, rinse them with clean water, drain them and set aside;

[0013] <2> Dry the cleaned snake slough and toad skin, crush them and sieve them to obtain fine powder for later use;

[0014] <3> Put cockscomb into a container and add appropriate amount of water to soak;

[0015] <4> Adding Cockscomb to clear water and boiling, after boiling is completed, filter out the medicinal liquid to obtain the first medicinal juice for later use; adding clear water to the medicinal residue after the first boiling, continue boiling, after boiling is completed, filter out the medicinal liquid to obtain the second medicinal juice for later use;

[0016] <5> The decoctions after the two decoctions are mixed evenly; the mixed decoction is allowed to stand for clarification; the clarified decoction is filtered, and the clear decoction after filtration is concentrated to form an extract for later use;

[0017] <6> Step <1> Slowly add the fine powder <5> The extract is mixed evenly, and then the extract is dried to prepare a dry extract;

[0018] <7> The mixed dry extract is dried and crushed into a fine powder.

[0019] step <1> Medium: Soak for 1 hour.

[0020] step <2> Medium: Dry at 60℃ and sieve through 100 mesh.

[0021] step <3> Soak for 30 minutes; Steps <4> Each time, add 8 times the amount of water to the raw material, and boil for 2 hours each time; Step <5> The relative density of the extract is 1.36 at 80°C.

[0022] The above-mentioned compound Chinese medicine powder is used in the preparation of antiviral drugs, and the viruses are P2V virus, RV virus, CV-A9 virus, CV-B3 virus, RSV virus, VACV TT virus, EV71 virus, AdV virus, SARS virus, PEDV virus, ZIKA virus, and PV virus.

[0023] In view of the problems existing in current antiviral drugs, the inventor has developed a compound Chinese medicine powder made from the following raw materials: snake slough, toad skin and cockscomb. Studies have shown that the Chinese medicine composition has antiviral effects against a variety of viruses. Compared with the prior art, the present invention has the following outstanding advantages:

[0024] The invention is made of natural Chinese medicinal materials, has good safety, small side effects, and can be taken for a long time.

[0025] The raw materials of the present invention act synergistically, mutually enhance the antiviral efficacy, improve the antiviral effect, have broad-spectrum antiviral activity, can effectively inhibit the replication of multiple viruses, and cope with multiple viral infections.

[0026] The raw material of the present invention has anti-inflammatory and immunomodulatory effects, helps to alleviate inflammation caused by viral infection, and enhances the function of the immune system.

[0027] The powder of the present invention is convenient to take, can be taken anytime and anywhere, is relatively cheap, and has high cost performance.

[0028] In summary, the Chinese medicine composition of the present invention has a broad-spectrum antiviral activity and has potential in preventing and treating viral infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The present invention is a flow chart of the preparation method of the compound Chinese medicine powder.

[0030] Figure 2 The figure is a result diagram of the antiviral pharmacodynamics experiment (half effective concentration test and half cytotoxic concentration test) of the compound Chinese medicine powder of the present invention, in which: A P2V virus; B RV virus; C CV-A9 virus; D CV-B3 virus; E RSV virus; F VACV_TT virus; G EV71 virus; H AdV virus; I SARS virus; J PEDV virus; K ZIKA virus; L PV virus.

[0031] Figure 3 This is a graph showing the relative concentrations of the main components of flavonoids in the compound Chinese medicine powder of the present invention. DETAILED DESCRIPTION

[0032] 1. Preparation of compound Chinese medicine powder

[0033] Formula: 2.5 parts of snake skin, 2.5 parts of toad skin and 5 parts of cockscomb. (All raw medicinal materials meet the standards of Part I of the 2020 edition of the Pharmacopoeia of the People's Republic of China)

[0034] Preparation method (such as Figure 1 shown):

[0035] <1> Choose dried snake skin and toad skin, soak them in clean water for 1 hour to soften them, gently scrub them with your hands to remove dirt and impurities, rinse them with clean water until the water becomes clear, drain them and set aside;

[0036] <2> The cleaned snake slough and toad skin are placed in a drying oven at 60° C. for drying to remove moisture therein, and after drying, the snake slough and toad skin are placed in a grinder for grinding to make them into fine powder; the crushed snake slough and toad skin are sieved through 100 meshes to remove coarse particles, and fine powder with uniform fineness is obtained for standby use;

[0037] <3> Put cockscomb into a sterilized container, add appropriate amount of water to allow the Chinese medicine to absorb water and swell, and soak for 30 minutes;

[0038] <4> Add 8 times of water to the cockscomb and boil for 2 hours, stirring several times during the process to allow the cockscomb raw material to fully release its effective ingredients; after the boiling is completed, filter out the medicinal liquid to obtain the first medicinal juice for later use; add 8 times of water to the medicinal residue after the first boiling, continue boiling for 2 hours, after the boiling is completed, filter out the medicinal liquid to obtain the second medicinal juice for later use;

[0039] <5> Pour the decoctions from the two decoctions into a clean container respectively, ensure that the temperature of the decoctions is appropriate, mix the decoctions in the two containers evenly, and stir gently to fully blend the decoctions; let the mixed decoctions stand for a period of time, usually 10-15 minutes, to allow the residues to settle to the bottom of the container; after the residues have settled, carefully tilt the container to allow the clarified decoction to flow out slowly, and avoid stirring the residues; pour the clarified decoction into a new clean container, and filter with filter paper or gauze to remove any residual residues, and retain the clear decoction after filtration; further concentrate the decoction to an appropriate amount to form an extract (relative density is 1.36 at 80°C), and set aside;

[0040] <6> Step <1> Slowly add the fine powder <5> Add the extract and stir with a pestle until the mixture reaches a uniform paste, making sure there are no lumps or unmixed ingredients, then dry the extract to make a dry extract;

[0041] <7> The mixed dry extract is dried and crushed into fine powder, the fine powder is put into a sealed container and stored in a cool and dry place.

[0042] Mechanism of action (speculation): Snake skin is cool in nature, sweet and salty in taste, and enters the lung and liver meridians. It has the effects of dispelling wind and relieving itching, cooling blood and detoxifying, and improving eyesight; toad skin is cool in nature, sweet in taste, slightly toxic, and enters the lung and heart meridians. It has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and reducing swelling and relieving pain; cockscomb is cool in nature, sweet and astringent in taste, and enters the liver, lung, and large intestine meridians. It has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, stopping diarrhea and relieving pain. Modern pharmacological studies have shown that snake skin contains a variety of active ingredients such as snake skin spirit and snake skin protein, which have the effect of inhibiting virus replication and clearing virus particles; toad skin contains a variety of active ingredients such as toad toxin and toad skin skin, which have the effect of inhibiting virus adsorption and blocking virus entry into cells; cockscomb contains a variety of active ingredients such as crestinatin and crestina glycoside, which have the effect of inhibiting viral nucleic acid synthesis and inhibiting virus release. The combination of snake skin, toad skin and cockscomb has a synergistic antiviral effect, which can enhance the antiviral effect and reduce the side effects of drugs.

[0043] 2. Antiviral Pharmacodynamics Experiment

[0044] The following experiments used the compound Chinese medicine powder prepared in the above step 1 as the experimental medicine.

[0045] Test Example 1 Anti-GX P2V virus (ie P2V) pharmacodynamics experiment

[0046] 1. Half effective concentration (EC50) test: Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with GX_P2V virus with a final MOI of 0.01, and different concentrations of drugs were added at the same time. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After 2 hours of infection, PBS was washed 3 times and the medium containing the same concentration of drugs was added, and cultured for 48 hours. Observe under a microscope, collect cell nucleic acids, and use qRT-PCR to quantify viral nucleic acids. GAPDH was used for homogenization, and the half effective concentration (EC50) was analyzed using GraphPad Prism 8.0.

[0047] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to Vero E6 cells was measured by CellTiter-Blue assay. Specifically, Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 48 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0048] Test Example 2 Anti-VACV_TT Virus Pharmacodynamics Experiment

[0049] 1. EC50 test: Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with VACV TT virus at a final MOI of 0.01, and different concentrations of drugs were added at the same time. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and cultured for 36 hours. Observe under a microscope, collect cell nucleic acids, and quantify viral nucleic acids using qRT-PCR. GAPDH was used for homogenization, and the EC50 was analyzed using GraphPad Prism8.0.

[0050] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to Vero E6 cells was measured by CellTiter-Blue assay. Specifically, Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 36 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0051] Test Example 3 Anti-CV-A9 Virus Pharmacodynamics Experiment

[0052] 1. EC50 test: RD cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. Cells were infected with CV-A9 virus at a final MOI of 0.001, and different concentrations of drugs were added at the same time. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After 2 hours of infection, PBS was washed 3 times and the medium containing the same concentration of drugs was added, and cultured for 48 hours. Observe under a microscope, collect cell nucleic acids, and use qRT-PCR to quantify viral nucleic acids. GAPDH was used for homogenization, and the EC50 was analyzed using GraphPad Prism 8.0.

[0053] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to RD cells was measured by Cel 1Titer-Blue assay. Specifically, RD cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 48 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0054] Test Example 4 Anti-ADV-5 virus (ie, AdV) pharmacodynamics experiment

[0055] 1. EC50 test: Hela cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with ADV-5 virus with a final MOI of 0.1, and different concentrations of drugs were added at the same time. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After 2 hours of infection, PBS was washed 3 times and the medium containing the same concentration of drugs was added, and cultured for 48 hours. Observe under a microscope, collect cell nucleic acids, and use qRT-PCR to quantify viral nucleic acids. GAPDH was used for homogenization, and the EC50 was analyzed using GraphPad Prism 8.0.

[0056] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to HeLa cells was measured by Cel 1Titer-Blue assay. Specifically, HeLa cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 48 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0057] Test Example 5 Anti-CV-B3 Virus Pharmacodynamics Experiment

[0058] 1. EC50 test: Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with CVB3 virus at a final MOI of 0.1, and different concentrations of drugs were added at the same time. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and cultured for 24 hours. Observed under a microscope, cell nucleic acids were collected, viral nucleic acids were quantified using qRT-PCR, and GAPDH was used for homogenization. The EC50 was analyzed using GraphPad Prism 8.0.

[0059] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to Vero E6 cells was measured by CellTiter-Blue assay. Specifically, Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 24 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0060] Test Example 6 Anti-PEDV Virus Pharmacodynamics Experiment

[0061] 1. EC50 test: Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with PEDV virus at a final MOI of 0.01, and different concentrations of drugs were added at the same time. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and cultured for 36 hours. Observe under a microscope, collect cell nucleic acids, and quantify viral nucleic acids using qRT-PCR. GAPDH was used for homogenization, and the EC50 was analyzed using GraphPad Prism 8.0.

[0062] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to Vero E6 cells was measured by CellTiter-Blue assay. Specifically, Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 36 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0063] Test Example 7 Anti-EV-A71 virus (i.e. EV71) pharmacodynamics experiment

[0064] 1. Half effective concentration (EC50) test: RD cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with EV-A71 virus with a final MoI of 0.1, and different concentrations of drugs were added at the same time. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. The cells were cultured for 24 hours. Observed under a microscope, cell nucleic acids were collected, and viral nucleic acids were quantified by qRT-PCR. GAPDH was used for homogenization, and the half effective concentration (EC50) was analyzed using GraphPad Prism 8.0.

[0065] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to RD cells was measured by Cel 1Titer-Blue assay. Specifically, RD cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 24 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0066] Test Example 8 Anti-RSV Virus Pharmacodynamics Experiment

[0067] 1. EC50 test: Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with RSV virus at a final MOI of 0.01, and different concentrations of drugs were added at the same time, ranging from 10 mg / mL to 0.02 mg / mL, and cultured for 48 hours. Observe under a microscope, collect cell nucleic acids, and quantify viral nucleic acids using qRT-PCR. GAPDH was used for homogenization, and the EC50 was analyzed using qraphPad Prism 8.0.

[0068] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to Vero E6 cells was measured by CellTiter-Blue assay. Specifically, Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL~0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 48 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then the fluorescence signal was detected with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0

[0069] Test Example 9 Anti-rotavirus (RV) pharmacodynamics experiment

[0070] 1. Half effective concentration (EC50) test: MA-104 cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. Rotavirus was activated at 37°C for 30 min with trypsin (EDTA-free) (15050-065, Gibco) at a final concentration of 20ug / ml. Cells were infected with porcine rotavirus OSU strain with a final MOI=0.1. The infection maintenance solution contained 0.2% BSA (H1130, Solarbio) and 2ug / mv of trypsin (EDTA-free). Different concentrations of drugs were added at the same time. The drug concentration range was 10mg / mL to 0.02mg / mL, and cultured for 36 hours. Observe under a microscope, collect cell nucleic acids, and use qRT-PCR to quantify viral nucleic acids. GAPDH was used for homogenization, and the half effective concentration (ECS0) was analyzed using GraphPad Prism 8.0.

[0071] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to MA-104 cells was measured by CellTiter-Blue assay. Specifically, MA-104 cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 48 hours, 20 μL of Reagent (G8081, Promega) was incubated at 37°C for 1-2 h, and the fluorescence signal was detected with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPadPrism 8.0.

[0072] Test Example 10 Anti-SARS-CoV virus (i.e. SARS) pharmacodynamics experiment

[0073] 1. Half effective concentration (EC50) test: Huh7 cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with SARS-CoV virus with a final MOI of 0.1, and different concentrations of drugs were added at the same time. The drug concentration range was 10.3 mg / mL to 0.02 mg / mL of a traditional Chinese medicine, and the concentration was diluted 2 times. After 2 hours of infection, PBS was washed 3 times and the medium containing the same concentration of drugs was added, and cultured for 48 hours. Observe under a microscope, collect cell nucleic acids, and use qRT-PCR to quantify viral nucleic acids. GAPDH was used for homogenization, and the half effective concentration (EC50) was analyzed using GraphPad Prism8.0.

[0074] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to Huh7 cells was measured by CellTiter-Blue assay. Specifically, Huh7 cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10.3 mg / mL~0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 48 hours, the supernatant was removed and washed with PBS, and 100 μL of new culture medium was added and 20 μL of each well was added. Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0075] Test Example 11 Anti-ZIKA virus pharmacodynamics experiment

[0076] 1. EC50 test: Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. The cells were infected with ZIKA virus at a final MOI of 0.01, and different concentrations of drugs were added at the same time. The drug concentration range was 10.3 mg / mL to 0.02 mg / mL of a traditional Chinese medicine, and the concentration was diluted 2 times. After 2 hours of infection, the PBS was washed 3 times and the medium containing the same concentration of drugs was added, and cultured for 48 hours. Observe under a microscope, collect cell nucleic acids, and use qRT-PCR to quantify viral nucleic acids. GAPDH was used for homogenization, and the EC50 was analyzed using GraphPadPrism 8.0.

[0077] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs to Vero E6 cells was measured by CellTiter-Blue assay. Specifically, Vero E6 cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10.3 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 48 hours, the supernatant was removed and washed with PBS, and 100 μL of new culture medium was added and 20 μL of each well was added. Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0078] Test Example 12 Anti-PV-1 virus (i.e. PV) pharmacodynamics experiment

[0079] 1. EC50 test: RD cells were pre-seeded in 96-well plates (2×104 cells / well) and cultured overnight. Cells were infected with PV-1 virus at a final MOI of 0.1, and different concentrations of drugs (three traditional Chinese medicines) were added at the same time. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. The cells were cultured for 24 hours. Observed under a microscope, cell nucleic acids were collected, and viral nucleic acids were quantified by qRT-PCR. GAPDH was used for homogenization, and the EC50 was analyzed using GraphPad Prism 8.0.

[0080] 2. Half cytotoxic concentration (CC50) test: The cytotoxicity of drugs (three traditional Chinese medicines) to RD cells was measured by CellTiter-Blue assay. Specifically, RD cells were pre-seeded in 96-well plates (2×104 cells / well), and different concentrations of drugs were added after overnight culture. The drug concentration range was 10 mg / mL to 0.02 mg / mL, and the concentration was diluted 2 times. After incubation for 24 hours, 20 μL of Reagent (Promega, G8081), incubated at 37°C for 1-2 h, and then detected the fluorescence signal with an excitation wavelength of 554 nm and an emission wavelength of 593 nm. The half cytotoxic concentration (CC50) was analyzed using GraphPad Prism 8.0.

[0081] 3. Results of antiviral pharmacodynamics experiments

[0082] like Figure 2As shown, the horizontal axis is the drug concentration, and the vertical axis is the percentage of cell activity and the percentage of virus inhibition. When the drug concentration is low, the cell activity percentage is high and the virus inhibition percentage is low. As the drug concentration increases, the cell activity percentage gradually decreases and the virus inhibition percentage gradually increases. When the drug concentration reaches a certain value, the cell activity percentage drops to the lowest and the virus inhibition percentage reaches the highest. CC50 is the drug concentration that reduces cell activity by 50%, and EC50 is the drug concentration that makes virus inhibition reach 50%. Table 1 shows the analysis results of the half-effective concentration and half-cytotoxic concentration of the compound Chinese medicine powder of the present invention for 12 different viruses.

[0083] Table 1 Pharmacodynamics test results of the compound Chinese medicine powder of the present invention against 12 viruses

[0084]

[0085] like Figure 3 As shown, the abscissa is the Chinese name of the main flavonoid components, and the ordinate is the relative concentration of the main flavonoid components. The components with a relatively high content include 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 6-deoxy-α-L-mannosyl-(1->2)-[6-deoxy-α-L-mannosyl-(1->6)]-β-D-pyranoside, narcissin, lycoside I, isorhamnetin-3-O-neohesperidin, and hydroxygenkwain. Most of these components have the effects of sedation, antibacterial, antiviral, antioxidant, anti-inflammatory, inhibiting bacterial growth, preventing the formation and spread of tumors, reducing inflammation, and scavenging free radicals.

Claims

1. A compound Chinese medicine powder, Features Made from the following ingredients: snake slough, toad skin and cockscomb.

2. The compound Chinese medicine powder according to claim 1, Features The invention is prepared from the following raw materials in parts by weight: 0.5 to 4.5 parts of snake slough, 0.5 to 4.5 parts of toad skin and 3 to 7 parts of cockscomb.

3. The compound Chinese medicine powder according to claim 1, Features The invention is prepared from the following raw materials in parts by weight: 2.5 parts of snake skin, 2.5 parts of toad skin and 5 parts of cockscomb.

4. The compound Chinese medicine powder according to claim 1, Features: The powder is an oral powder.

5. A method for preparing the compound Chinese medicine powder according to claim 1, Features: Wash and dry snake sloughs and toad skins respectively, mix and grind them into fine powder; soak cockscombs and then boil them, filter them after boiling, and concentrate the filtrate into an extract; add the fine powder into the extract and mix evenly, then dry the extract to make a dry extract, which can be obtained by grinding.

6. The preparation method according to claim 5, Features Follow these steps: <1> Select dried snake skin and toad skin, soak them in clean water, rinse them with clean water, drain them and set aside; <2> Dry the cleaned snake slough and toad skin, crush them and sieve them to obtain fine powder for later use; <3> Put cockscomb into a container and add appropriate amount of water to soak; <4> Adding Celosia to clear water and boiling, after boiling is completed, filtering out the medicinal liquid to obtain the first medicinal juice for later use; adding clear water to the medicinal residue after the first boiling, continuing boiling, after boiling is completed, filtering out the medicinal liquid to obtain the second medicinal juice for later use; <5> The decoctions after the two decoctions are mixed evenly; the mixed decoction is allowed to stand for clarification; the clarified decoction is filtered, and the clear decoction after filtration is concentrated to form an extract for later use; <6> Step <1> Slowly add the fine powder <5> The extract is mixed evenly, and then the extract is dried to prepare a dry extract; <7> The mixed dry extract is dried and crushed into a fine powder.

7. The preparation method according to claim 4, Features step <1> Medium: Soak for 1 hour.

8. The preparation method according to claim 4, Features step <2> Medium: Dry at 60℃ and sieve through 100 mesh.

9. The preparation method according to claim 4, Features: step <3> Soak for 30 minutes; Steps <4> Each time, add 8 times the amount of water to the raw material, and boil for 2 hours each time; Step <5> The relative density of the extract is 1.36 at 80°C.

10. Use of the compound Chinese medicine powder according to claim 1 in the preparation of antiviral drugs, Features: The viruses are P2V virus, RV virus, CV-A9 virus, CV-B3 virus, RSV virus, VACV_TT virus, EV71 virus, AdV virus, SARS virus, PEDV virus, ZIKA virus, and PV virus.

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