CN114949044A - Antiviral traditional Chinese medicine composition and application thereof - Google Patents

Antiviral traditional Chinese medicine composition and application thereof Download PDF

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CN114949044A
CN114949044A CN202110199421.1A CN202110199421A CN114949044A CN 114949044 A CN114949044 A CN 114949044A CN 202110199421 A CN202110199421 A CN 202110199421A CN 114949044 A CN114949044 A CN 114949044A
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chinese medicine
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viruses
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辛远航
陈国瑞
李燕维
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Shanghai Guankrypton Pharmaceutical Technology Development Co ltd
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Shanghai Guankrypton Pharmaceutical Technology Development Co ltd
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/48Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/489Sophora, e.g. necklacepod or mamani
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K36/484Glycyrrhiza (licorice)
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    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/53Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539Scutellaria (skullcap)
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    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/63Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
    • A61K36/634Forsythia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/70Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • AHUMAN NECESSITIES
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    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
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    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention relates to an antiviral traditional Chinese medicine composition which can be used for preparing a medicine for resisting viruses or preventing and treating viral diseases (particularly respiratory tract infection mainly caused by viruses). The traditional Chinese medicine composition follows the theoretical formula of traditional Chinese medicine and is prepared from the following raw materials in parts by weight: 1-25 parts of radix sophorae flavescentis, 1-25 parts of wild buckwheat rhizome and leaf, 1-25 parts of liquorice, 1-25 parts of honeysuckle, 1-25 parts of scutellaria baicalensis and 1-25 parts of fructus forsythiae. The traditional Chinese medicine composition has the obvious effect of inhibiting common viruses which can cause respiratory tract infection, so that the traditional Chinese medicine composition can be used for preparing antiviral medicines or medicines for preventing and treating viral diseases (particularly respiratory tract infection mainly caused by viruses), disinfectants and the like. The invention also relates to the medical application of the traditional Chinese medicine composition.

Description

Antiviral traditional Chinese medicine composition and application thereof
Technical Field
The invention relates to the technical field of traditional Chinese medicines and the field of health maintenance, in particular to a traditional Chinese medicine composition with antiviral effect and viral disease prevention and treatment effect, and particularly with the effect of preventing and treating respiratory tract infection mainly caused by viruses. In addition, the invention also relates to the medical application and the health maintenance application of the traditional Chinese medicine composition.
Background
Many viruses are pathogens that cause common infectious diseases, including rabies, smallpox, polio, hepatitis, yellow fever, immunodeficiency, encephalitis, respiratory infections, influenza, and the like, and are extremely infectious and dangerous. Such as rubella and cytomegalovirus, can cause congenital abnormalities, and tumor viruses can cause tumors in humans and animals. The main pathogens of the common cold are rhinoviruses, and then parainfluenza viruses, adenoviruses, echoviruses, coxsackieviruses, respiratory syncytial viruses and the like, which are often easy to be combined with bacterial infection. Influenza (influenza for short) is an acute respiratory infectious disease caused by influenza virus, pathogens are influenza virus A, influenza virus B and influenza virus C3, the influenza virus has acute high fever, hypodynamia, general muscular soreness and mild respiratory symptoms clinically through droplet transmission, and pneumonia is easily complicated by middle-aged people and patients with chronic respiratory diseases or heart diseases. Influenza viruses, particularly type a, are highly variable and often cause outbreaks, epidemics or pandemics. It can be said that due to the severe disturbance of the natural environment, the rapid variation of viruses, and the unscientific eating of wild animals by human beings, new viral diseases, especially the outbreak and prevalence of respiratory tract infection epidemic caused by viruses, are increasing, and the human society faces a great threat. At present, most of domestic and foreign western medicines adopt ribavirin, chloroquine, redciclovir and the like for treating rhinovirus, coronavirus and the like, but the effect is very limited and the toxic and side effects are very large. Because of the rapid spread, rapid mutation, high drug resistance of many viruses and the continuous emergence of new viruses, the research and development of drugs and vaccines require a long period, so that the prevention and treatment of viral diseases often lack specific means, and the research and development of drugs for preventing and treating viral diseases, particularly respiratory tract infection mainly caused by viruses, are urgently needed.
The traditional Chinese medicine has the comprehensive effects of inhibiting virus replication, regulating the immune function, improving blood circulation, relieving fever and pain, resisting bacteria and diminishing inflammation and the like, can play a role in multiple aspects, and has the advantages of low drug resistance, less adverse reaction and side effect and the like. The existing research shows that the action mechanism is as follows: some heat-clearing and detoxifying traditional Chinese medicines have direct antiviral effects, such as cyrtomium rhizome, honeysuckle, wild chrysanthemum flower, dyers woad leaf and the like; some heat-clearing and detoxifying Chinese herbal medicines have the effects of inducing interferon production in a human body, regulating the immunity of the human body, inhibiting virus replication, and improving the lung ventilation function and microcirculation, so that the condition of a patient is relieved, the course of disease is shortened, and the death rate is reduced. In a word, the traditional Chinese medicine has unique advantages and broad prospects in the aspect of preventing and treating viral diseases, particularly viral respiratory tract infection. The traditional Chinese medicine prescription is a main form of clinical application of the traditional Chinese medicine, can play a synergistic effect through the compatibility and combination of different traditional Chinese medicines, and plays a role through multiple ways and multiple target points. Because of various Chinese medicines, the Chinese medicine can be determined only by searching good Chinese medicine formula and investing a great deal of energy for screening and verification, and is a focus of attention in academia.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine composition with antiviral effect and a traditional Chinese medicine composition with the effect of preventing and treating viral diseases, in particular to a traditional Chinese medicine composition capable of preventing and treating respiratory tract infection mainly caused by viruses, and provides the medical and health maintenance application of the traditional Chinese medicine composition.
Because the clinical application form of the traditional Chinese medicine is mainly to select a plurality of traditional Chinese medicines to form a compound, which is also the advantage of the traditional Chinese medicine in preventing and treating viral diseases, the demand for good traditional Chinese medicine formula is very urgent. However, because of a large variety of medicines, the screening of good traditional Chinese medicine formula has large workload and great difficulty. The inventor is engaged in the traditional Chinese medicine research for a long time, aiming at the etiology and pathogenesis analysis of common viral respiratory tract infection under the guidance of the theory of traditional Chinese medicine, and through in vitro and animal tests and clinical verification, finally finds out the antiviral traditional Chinese medicine composition with better curative effect, and can prepare the antiviral traditional Chinese medicine composition into a proper dosage form for directly inhibiting viruses or preventing and treating viral diseases, in particular to preventing and treating respiratory tract infection mainly caused by viruses. The traditional Chinese medicine formula mainly comprises radix sophorae flavescentis, wild buckwheat rhizome and leaf, liquorice, honeysuckle, scutellaria baicalensis and fructus forsythiae. Wherein radix Sophorae Flavescentis is dried root of Sophora flavescens Ait, bitter and cold. It has effects of clearing heat, eliminating dampness, killing parasite, and promoting urination, and is suitable for heart, liver, stomach, large intestine, and bladder channels. The stem and leaf of rhizoma Fagopyri Dibotryis (D.Don) Hara is stem and leaf of rhizoma Fagopyri Dibotryis (D.Don) Hara of Polygonaceae, has bitter, pungent and cool nature, and has effects of clearing heat and detoxicating, invigorating spleen and promoting diuresis, dispelling pathogenic wind and dredging collaterals. The licorice is the dry root of Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat or Glycyrrhiza glabra L of Leguminosae, has sweet and flat nature and flavor, and has the effects of tonifying spleen and qi, clearing away heat and toxic materials, eliminating phlegm and stopping cough, relieving spasm and pain, and harmonizing the effects of the other drugs. The honeysuckle is a dried flower bud or a flower with an initial bloom of Lonicera japonica Thunb, Lonicera hypoglauca Miq, Lonicera confusa DC or Lonicera dasystyla Rehd of Lonicera japonica of Caprifoliaceae, has sweet and cold properties and tastes sweet and cold, enters lung, heart and stomach meridians, and has the effects of clearing heat and removing toxicity, and cooling and dispersing wind heat. The Scutellariae radix is dried root of Scutellaria baicalensis Georgi of Labiatae, and is bitter in taste and cold in nature, and enters lung, gallbladder, spleen, large intestine and small intestine meridians. Clear heat and dry dampness, purge fire and remove toxicity. Fructus forsythiae is dry fruit of Vahl of Forsythia subsensis (Thunb.) of Oleaceae, is bitter in taste and slightly cold, enters lung, heart and small intestine channels, and has effects of clearing away heat and toxic materials, relieving swelling and resolving hard mass. Generally, the prescription has the effects of clearing away heat and toxic materials, ventilating and smoothing lung, reducing phlegm and harmonizing ying and weiqi, and can be clinically used for resisting viruses such as influenza virus, rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, adenovirus, coronavirus and the like, or preventing and treating diseases caused by the viruses, particularly respiratory infection. In consideration of the factors of market circulation, storage and the like of the traditional Chinese medicines, the traditional Chinese medicines applied in clinic are mostly dry products. If the fresh-keeping effect can be well achieved without rotting, the traditional Chinese medicine composition adopts the corresponding fresh products of the traditional Chinese medicines in the formula, and is also feasible and even better in effect.
According to an embodiment, the antiviral traditional Chinese medicine composition provided by the invention is prepared from the following raw material medicines in parts by weight: 1-25 parts of radix sophorae flavescentis, 1-25 parts of wild buckwheat rhizome and leaf, 1-25 parts of liquorice, 1-25 parts of honeysuckle, 1-25 parts of scutellaria baicalensis and 1-25 parts of fructus forsythiae. Preferably: 1 part of radix sophorae flavescentis, 1 part of wild buckwheat rhizome and leaf, 1 part of liquorice, 1 part of honeysuckle, 1 part of scutellaria baicalensis and 1 part of forsythia suspensa.
The traditional Chinese medicine composition can be prepared by extracting each single medicine by adopting a proper solvent and then mixing, or can be prepared by mixing each single medicine, extracting by adopting a proper solvent, adjusting to a specified concentration or drying and then using. The extraction solvent can be water or 5-95% ethanol water solution, or methanol, preferably water or 25-75% ethanol water solution. If the extract is used as a decoction, water is preferably used as a solvent for extraction; when used as another preparation, a 10% to 50% ethanol aqueous solution is preferable, and a 25% ethanol aqueous solution is most preferable, and water may be used as a solvent.
The traditional Chinese medicine composition (liquid, extract and powder) can be prepared into various oral, injection and external preparations by adopting the conventional preparation technology with the auxiliary material carrier which is acceptable in pharmacy, such as common formulations of aerosol, decoction, tablets, capsules, granules, mixture and the like, and can be applied to places or people in need in oral, rectal or parenteral, nasal inhalation, atomized inhalation and spraying modes. The pharmaceutically acceptable auxiliary material carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, and polyethylene glycol, etc.; propellants such as freon, hydrocarbons (propane, n-butane, isobutane); cosolvents such as propylene glycol, glycerol, ethanol, etc. In addition, other adjuvants such as flavoring agent, sweetener, etc. can be added for the preparation.
Experimental studies of the following examples of the present invention show that: the Chinese medicinal composition has the pharmacological action of remarkably inhibiting common viruses which can cause respiratory tract infection. The traditional Chinese medicine (medicinal plant) raw materials selected by the traditional Chinese medicine composition are mainly medicinal and edible varieties, have no toxic or side effect, high safety and low cost, and have remarkable advantages. Because viral upper respiratory tract infection is frequent and a specific prevention and treatment means is lacked, the traditional Chinese medicine composition has a good application prospect.
Detailed Description
The following detailed description of the present invention will be provided in conjunction with examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure. Various changes and modifications may be effected therein by one skilled in the art, and such equivalent changes and modifications also fall within the scope of the invention as defined in the appended claims.
Example 1 preparation of extract
Weighing 100g of radix sophorae flavescentis, 25g of stem and leaf of wild buckwheat rhizome, 10g of liquorice, 10g of honeysuckle, 10g of scutellaria baicalensis and 10g of fructus forsythiae respectively, adding 25% ethanol aqueous solution which is 10 times of the total weight of the medicinal materials, carrying out hot reflux extraction for 2 times, carrying out 1 hour each time, combining the extracting solutions, concentrating, and adjusting the concentration with distilled water to be equal to the concentration of the crude drug to be 1.0 g/mL. The extractive solution can be directly used as decoction or intermediate, and can be processed into appropriate dosage forms by adjusting concentration according to preparation requirement and adding appropriate adjuvants.
Example 2 preparation of extract
Weighing 25g of radix sophorae flavescentis, 25g of wild buckwheat rhizome and leaf, 100g of liquorice, 25g of honeysuckle, 100g of scutellaria baicalensis and 5g of fructus forsythiae respectively, adding 25% ethanol aqueous solution which is 10 times of the total weight of the medicinal materials, carrying out hot reflux extraction for 2 times, carrying out 1 hour each time, combining extracting solutions, concentrating, and adjusting the concentration by using distilled water to be equal to the concentration of the crude drug to be 1.0 g/mL. The extractive solution can be directly used as decoction or intermediate, and can be processed into appropriate dosage forms by adjusting concentration according to preparation requirement and adding appropriate adjuvants.
Example 3 preparation of extract
Weighing 5g of radix sophorae flavescentis, 5g of fagopyrum dibotrys stem and leaf, 50g of liquorice, 100g of honeysuckle, 25g of scutellaria baicalensis and 25g of fructus forsythiae respectively, adding 25% ethanol aqueous solution which is 10 times of the total weight of the medicinal materials, carrying out hot reflux extraction for 2 times, carrying out 1 hour each time, combining extracting solutions, concentrating, and adjusting the concentration with distilled water to be equal to the concentration of the crude drug to be 1.0 g/mL. The extractive solution can be directly used as decoction or intermediate, and can be processed into appropriate dosage forms by adjusting concentration according to preparation requirement, adding appropriate adjuvants.
Example 4 preparation of extract solution
Weighing 25g of radix sophorae flavescentis, 100g of fagopyrum dibotrys stem and leaf, 10g of liquorice, 50g of honeysuckle, 10g of scutellaria baicalensis and 5g of fructus forsythiae respectively, adding 25% ethanol aqueous solution which is 10 times of the total weight of the medicinal materials, carrying out hot reflux extraction for 2 times, carrying out 1 hour each time, combining extracting solutions, concentrating, and adjusting the concentration with distilled water to be equal to the concentration of the crude drug to be 1.0 g/mL. The extractive solution can be directly used as decoction or intermediate, and can be processed into appropriate dosage forms by adjusting concentration according to preparation requirement, adding appropriate adjuvants.
Example 5 preparation of extract
Weighing 5g of radix sophorae flavescentis, 5g of wild buckwheat rhizome and leaf, 5g of liquorice, 5g of honeysuckle, 5g of scutellaria baicalensis and 5g of fructus forsythiae respectively, adding 25% ethanol aqueous solution which is 10 times of the total weight of the medicinal materials, carrying out hot reflux extraction for 2 times, carrying out 1 hour each time, combining extracting solutions, concentrating, and adjusting the concentration by using distilled water to be equal to the concentration of the crude drug to be 1.0 g/mL. The extractive solution can be directly used as decoction or intermediate, and can be processed into appropriate dosage forms by adjusting concentration according to preparation requirement, adding appropriate adjuvants.
EXAMPLE 6 preparation of granules
The extract prepared in any one of embodiments 1 to 5 is concentrated and dried to obtain an extract, 20g of the extract is taken, 40g of dextrin is added, the extract is uniformly mixed, a proper amount of 60% ethanol is added to prepare a soft material, the soft material is sieved by a 24-mesh sieve to be granulated, the dry material is dried at 50 ℃ for 2 hours, the dry granules are sieved by a 30-mesh sieve to be granulated, and the obtained product is subpackaged.
EXAMPLE 7 preparation of capsules
The extract prepared in any one of embodiments 1 to 5 is concentrated and dried to obtain an extract, 20g of the extract is taken, 40g of dextrin is added, the mixture is uniformly mixed, a proper amount of 60% ethanol is added to prepare a soft material, the soft material is sieved by a 24-mesh sieve and granulated, the granulation is carried out at 50 ℃ for 2 hours, and the granules are dried and filled into capsules.
Example 8 preparation of mixture/oral liquid/syrup
The extract prepared in any one of embodiments 1 to 5 is concentrated, and then added with a suitable pharmaceutical excipient (white sugar, honey, phenylpropionic acid, ethylparaben, or the like) to adjust the concentration, thereby preparing a mixture, an oral liquid, or a syrup.
EXAMPLE 9 preparation of Aerosol
The extract prepared in any one of embodiments 1 to 5 is added with ethanol, essence, preservative and the like, the concentration is adjusted to 1 to 100mg/mL, and the mixture is filled into an aerosol bottle by taking propane and butane as propellants to prepare aerosol.
Example 10 determination of antiviral Activity of the Chinese medicinal composition of the present invention
Purpose of the experiment
The inhibiting effect of the traditional Chinese medicine composition on the common viruses causing respiratory tract infection is observed.
2 materials of experiment
2.1 reagents
DMEM medium (TBD10565, the balance of Tianjin, the world), double antibody (streptomycin mixed liquor, lot: BL-XB-0197, Boguan), phosphate buffer solution (PBS, PB2004Y, Tianjin sea), fetal bovine serum (FBS, 10099-141, Gibco USA), EDTA-0.25% pancreatin (BL-XB-0168, Boguan), thiazole blue dye solution (MTT, BL-SJ-0717, Boguan), ribavirin injection (Chengxin pharmaceutical industry GmbH, 1410206811); acyclovir for injection (Hubei noon pharmaceutical Co., Ltd., 1704031).
2.2 instruments
A two-beam ultraviolet-visible spectrophotometer (model TU-1901, Beijing Pujingyo general instruments, Inc.), a CKX-41 type inverted microscope (Olympus, Japan); HFsafe-1200TE type biosafety cabinet, MCO-15AC type CO 2 Cell constant temperature incubators (SANYO Electric biological co.ltd.japan), a TGL16M model bench top multitube automatic balance centrifuge (altin, shanghai), a BT25S electronic balance (one ten-thousandth, Sartorius, germany).
2.3 cell and Virus strains
Human malignant embryonic rhabdomyosarcoma cells (RD cells), rhesus embryonic kidney cells (MA104 cells), and human laryngeal carcinoma epithelial cells (Hep2 cells) were purchased from Shanghai Biomedicine science and technology, Inc. Hand-foot-and-mouth virus (EV71), herpes simplex virus type 1 (HSV-1), Respiratory Syncytial Virus (RSV), Coxsackie virus type B3 (CV-B3) and Coxsackie virus type B5 (CV-B5) were provided by the institute of basic medicine, college of medical sciences, Shandong province. Cell culture solution: taking 450mL of DMEM medium, adding 50mL of fetal bovine serum and 2mL of double antibody, mixing uniformly, and refrigerating at 4 ℃. Cell maintenance solution: the cell culture solution was diluted 5-fold with DMEM medium.
3 Experimental methods
3.1 preparation of test drug solutions
The extract of the Chinese medicinal composition of example 1-5 is filtered through a 0.22 μm microporous membrane and stored in a refrigerator at-20 deg.C for further use.
3.2 preparation of Positive control
The acyclovir injection is prepared by using 2% DMSO cell maintenance solution, and the concentration is 50 mg/mL -1 (ii) a The ribavirin injection adopts stock solution with the concentration of 50 mg/mL -1
3.3 cell recovery and culture
And taking out the cryopreservation tube for preserving the cells from the liquid nitrogen tank, immediately putting the tube into warm water at 37-42 ℃ for thawing, and enabling the cells to rapidly pass through a freeze thawing process. Transferring the cell suspension into a centrifuge tube in a superclean bench for centrifugation, wherein the centrifuge has set parameters of the rotation speed of 800 r.min -1 And the time is 5 min. After centrifugation, the supernatant is aspirated, cell culture medium is added, the precipitated cells are blown gently through a pipette, the newly obtained cell suspension is transferred into a culture flask and placed in CO 2 Constant temperature incubator (culture conditions of 37 ℃ and 5% CO) 2 And relative humidity 75%), and subculturing after the cells are fully paved at the bottom of the bottle.
3.4 amplification of viruses
Collecting cells in exponential growth phase and in good growth state, discarding culture medium, washing with PBS for 3 times, adding virus solution 0.5mL, and placing in CO 2 Culturing in a constant temperature incubator, and slightly shaking the culture flask every 15min to make the virus liquid fully contact with the cells. With another cell control, 0.5mL of cell maintenance solution was added and the cells were cultured as above. After 1 hour of culture, 4.5mL of cell maintenance medium was added and the culture was continued. After 24h, microscopic examination is carried out, and the number of cells infected with the lesion>At 90%, the culture was stopped. Repeatedly freezing and thawing the cells for 3-4 times, transferring the virus liquid to a centrifuge tube for centrifugation, and setting the centrifuge with the set parameters of the rotation speed of 1000 r.min -1 Taking supernatant fluid, quantitatively subpackaging in a freezing tube, sealing for 5min,and storing in an ultra-low temperature refrigerator at-80 ℃ for later use.
3.5 determination of viral virulence
The amplified virus was diluted 10-fold with a cell maintenance medium, and the cells were sequentially plated in 96-well cell culture plates confluent with a monolayer of cells, with a total of 10 concentration gradients, and each concentration was repeated 3 times, with cell controls. The culture was carried out according to the method under "3.4". Observing the lesion under microscopic examination after 24h, and determining the number of cells infecting the lesion>At 90%, the culture was stopped. 10 μ L of 5 mg/mL was added to each well -1 The MTT dye solution is continuously cultured for 4h, the dye solution is removed by suction, 100 mu L DMSO is added into each hole, the mixture is decolored for L0 min in a dark place at room temperature, and the A value of the mixture at 490nm is measured by a microplate spectrophotometer.
3.6 determination of cytotoxicity of extract
Diluting the Chinese medicinal composition extractive solution with cell maintenance solution at 2 × ratio, setting 10 concentration gradients, sequentially inoculating into 96-well cell culture plate with confluent monolayer of cells, repeating each concentration for 3 times at 100 μ L per well, and setting cell control. The culture was carried out according to the method under "3.4". After 24h, the cell state was observed by microscopic examination, and the A value was measured by the method under the item "3.5".
3.7 determination of antiviral Activity of extract
From nontoxic concentration, the Chinese medicinal composition extractive solution is diluted by cell maintenance solution at 2 × ratio for 10 concentrations, added into 96-well cell culture plate with monolayer cells, 100 μ L per well, and then 100 μ L of virus solution is added, each concentration is repeated for 3 times, and positive drug (ribavirin or acyclovir) control group, virus control group and cell control group are provided. The culture was carried out according to the method under "3.4". After 24h, cytopathic effects were observed, and when > 90% of the viral controls appeared, the A value was determined as under "3.5".
4 results of the experiment
4.1 Virus virulence assay results
Calculating the half infection concentration (TCID) of the virus liquid according to Reed-Muench formula 50 ) The cell survival rate is x 100% for each group a/normal cell a; (ii) the rate of cell morbidity is 1-cell survival; distance ═ (above 50% variability-50%)/(above 50% variability-below 50% morbidity rate) x 100%; TCID 50 Results are shown in table 1 for anti (lg greater than 50% lesion rate virus dilution + distance).
TABLE 1.4 Virus TCIDs 50 Measurement results
Figure BDA0002948021590000061
4.2 measurement of cytotoxicity of extract solution
Calculating half Toxic Concentration (TC) of the drug by applying Reed-Muench formula 50 ) The results are shown in Table 2.
TABLE 2 TC of extracts versus 3 cells 50 Measurement result (mg. mL) -1 )
Figure BDA0002948021590000071
4.3 antiviral Activity assay results
Calculating half Effective Concentration (EC) of the drug by applying Reed-Muench formula 50 ) And Therapeutic Index (TI), EC 50 Not ═ Antilg (lg greater than 50% lesion rate virus dilution-specific distance)]X the initial concentration (C) of the test sample liquid medicine; TI-half Toxic Concentration (TC) 50 ) Effective concentration at half maximum (EC) 50 ) The results are shown in tables 3 and 4. The inhibition effect of the traditional Chinese medicine composition on the viruses is obviously better than that of common medicines of ribavirin and acyclovir.
TABLE 3 EC of extracts against 5 viruses 50 Measurement results (mg. mL) -1 )
Figure BDA0002948021590000072
TABLE 4 TI assay results of 5 viruses in the extract
Figure BDA0002948021590000081

Claims (7)

1. An antiviral traditional Chinese medicine composition is characterized by being prepared from the following raw material medicines in parts by weight: 1-25 parts of radix sophorae flavescentis, 1-25 parts of wild buckwheat rhizome leaves, 1-25 parts of liquorice, 1-25 parts of honeysuckle, 1-25 parts of scutellaria baicalensis and 1-25 parts of fructus forsythiae.
2. The antiviral traditional Chinese medicine composition as claimed in claim 1, which is prepared from the following raw materials in parts by weight: 1 part of radix sophorae flavescentis, 1 part of wild buckwheat rhizome and leaf, 1 part of liquorice, 1 part of honeysuckle, 1 part of scutellaria baicalensis and 1 part of forsythia suspensa.
3. The antiviral traditional Chinese medicine composition according to claim 1 or 2, wherein the traditional Chinese medicine composition is an aerosol, a decoction, a tablet, a capsule, a granule or a mixture.
4. The antiviral traditional Chinese medicine composition of claim 3, wherein the traditional Chinese medicine composition is an aerosol.
5. The use of a Chinese medicinal composition as claimed in any one of claims 1 to 4 in the preparation of an antiviral medicament.
6. Use of the Chinese medicinal composition of any one of claims 1 to 4 in the preparation of an anti-coronavirus medicament.
7. The use of the Chinese medicinal composition of any one of claims 1 to 4 in the preparation of a medicament for the prevention and treatment of respiratory tract infection caused by viruses as the main component.
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Citations (2)

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