CN116898902A - Pharmaceutical composition for preventing and treating coronavirus infection - Google Patents
Pharmaceutical composition for preventing and treating coronavirus infection Download PDFInfo
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- CN116898902A CN116898902A CN202310188887.0A CN202310188887A CN116898902A CN 116898902 A CN116898902 A CN 116898902A CN 202310188887 A CN202310188887 A CN 202310188887A CN 116898902 A CN116898902 A CN 116898902A
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Abstract
The application discloses a pharmaceutical composition for preventing and treating coronavirus infection. The pharmaceutical composition of an embodiment of the application comprises mixed extracts of radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum or fractions thereof.
Description
Technical Field
The present application relates to a pharmaceutical composition for preventing and treating coronavirus infection, and more particularly, to a pharmaceutical composition for preventing and treating coronavirus infection comprising a Jiufeng detoxification soup extract as an active ingredient.
Background
Coronaviruses (coronaviruses) were first found in chickens in 1937 and then in animals such as pigs, dogs, birds, etc., and by 1965 in humans. Such a name is given to a pattern similar to a corona phenomenon in which a rear edge portion of a solar light bulb is shielded by a moon to emit white light when a person is on a full day.
Coronaviruses are known to induce mainly pneumonia and enteritis, or also nervous system infections and hepatitis in humans and animals. Coronaviruses belong to the family Coronaviridae (Coronaviridae), have a spherical outer membrane, and are sense ribonucleic acid (positive sense RNA) viruses of about 100nm-120nm in size (Master, 2006). Coronaviruses are formed from five structural proteins, namely the most coat spike protein (S), hemagglutinin-esterase (HE) protein, transmembrane (M) protein, small membrane (E) protein, and nucleocapsid (N) protein (Lai and Homes,2001.Fields Virology). Among them, spike proteins, which act as ligands binding to cellular receptors and induce fusion between host cells and viruses, are known as the most mutated proteins.
Conventionally, coronaviruses are considered to be hardly infectious to humans, but mainly infectious to pathogenic bacteria of animals such as dogs, pigs, and cattle. Even if a human is infected, only one of many viruses inducing respiratory symptoms induces intestinal diseases such as cold and diarrhea with low risk in children. Until the etiology of Middle East Respiratory Syndrome (MERS), which causes hundreds and thousands of deaths worldwide, has been ascertained to be a novel (variant) coronavirus, there is growing interest.
Middle east respiratory syndrome was originally discovered in sauter arabia in 2012, and is a disease that develops severe respiratory symptoms such as hyperpyrexia, cough, dyspnea, etc., and is accompanied by complications and death under severe conditions. Although no clear source and route of infection is identified, it is reported that infection is highly likely by contact with camels in the middle east and can be transmitted by close contact between humans. Patients mainly concentrated in the middle east and more than 100 infected persons appear in the korean universe starting from 5 months 2015.
As described above, it is known that the middle east respiratory syndrome and severe acute respiratory syndrome are each caused by a mutant coronavirus (middle east respiratory syndrome-coronavirus, severe acute respiratory syndrome-coronavirus, and severe acute respiratory syndrome coronavirus type 2) belonging to the genus B coronavirus (Beta coronavirus). Thus, the middle east respiratory syndrome-coronavirus, severe acute respiratory syndrome-coronavirus, and severe acute respiratory syndrome coronavirus type 2 share similar characteristics in terms of clinical symptoms, etiology, and infection. In the phylogenetic tree (phylogenetic tree), severe acute respiratory syndrome-coronavirus and severe acute respiratory syndrome coronavirus type 2 belong to a system B including bat-derived coronaviruses that are different from bat-severe acute respiratory syndrome-like coronaviruses, and middle east respiratory syndrome-coronavirus belongs to a system C including bat-derived coronaviruses (Trends Microbiol 24:490-502; https:// nextstreain.org/groups/blab/sams-like-covf_virus_type = SARS-CoV-2).
The middle east respiratory syndrome-coronavirus, severe acute respiratory syndrome-coronavirus, and severe acute respiratory syndrome coronavirus type 2 are all considered to originate from bats (anti-viral Res 101:45-56) as primary hosts for a variety of coronaviruses, and their clear routes of infection have not been clarified to date. On the other hand, it is believed that the above-mentioned viruses increase the possibility of co-infection of humans and animals (Nat Rev Microbiol 14:523-534) against the interspecies infections of the cat (palm civet) and the dromedary camel (dromedary camel), and nosocomial infections are the main cause of infection of the middle east respiratory syndrome, coronavirus, and severe acute respiratory syndrome, coronavirus, between humans (BMC Med 13:1-12).
On the other hand, although therapeutic agents for severe acute respiratory syndrome coronavirus type 2 are now being developed, they have been mainly developed centering on compounds or existing medicinal materials, and natural substance therapeutic agents have not been developed yet.
In particular, antiviral agents developed so far exhibit serious side effects, and their use requires much attention. Currently, these therapeutic agents have poor efficacy and side effects. Therefore, there is an increasing need to develop an excellent novel coronavirus therapeutic agent for preventing and treating the occurrence of coronaviruses, which has a remarkable infection-inhibiting effect and little toxicity.
Under such circumstances, the present inventors have continued efforts to develop a novel coronavirus therapeutic agent, and have confirmed that a mixed extract of saposhnikovia divaricata, burdock, forsythia suspensa, schizonepeta, notopterygium root, licorice root, platycodon root and gypsum, which has a superior effect of inhibiting the proliferation of severe acute respiratory syndrome coronavirus type 2, severe acute respiratory syndrome coronavirus and middle eastern respiratory syndrome coronavirus, has been accomplished.
Prior art literature
Patent literature
Patent document 1: korean laid-open patent publication No. 2022-0044082
Disclosure of Invention
Technical problem to be solved
The embodiment of the application provides an antiviral pharmaceutical composition for coronaviruses comprising mixed extracts of radix sileris, burdock, fructus forsythiae, herba schizonepetae, notopterygium root, liquorice, platycodon grandiflorum and gypsum or fractions thereof and a pharmaceutical composition for preventing and treating coronavirus infection.
Technical proposal for solving the problems
The application provides the following technical scheme.
The antiviral pharmaceutical composition for coronavirus according to an embodiment of the present application comprises a mixed extract of radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum or fractions thereof.
Also, according to an embodiment of the present application, there is provided an antiviral pharmaceutical composition for coronaviruses extracted by mixing the above-mentioned ledebouriella root, burdock fruit, forsythia fruit, schizonepeta, notopterygium root, licorice root, platycodon root and gypsum in a weight ratio of 500:500:835:250:250:500:500:1665.
In addition, according to an embodiment of the present application, there is provided an antiviral pharmaceutical composition for coronaviruses concentrated by adding 8 to 10 times of pure water to the mixed weight of the above-mentioned ledebouriella root, burdock fruit, forsythia fruit, schizonepeta, notopterygium root, licorice root, platycodon root and gypsum, extracting at 80 to 100 degrees for 2 to 3 hours, filtering, and concentrating the filtrate under reduced pressure at 60 degrees or less.
Also, according to an embodiment of the present application, there is provided an antiviral pharmaceutical composition for coronaviruses further comprising lactose hydrate, silica, magnesium stearate.
The application has the beneficial effects that:
the embodiment of the application comprises the mixed extracts or the fractions thereof of the divaricate saposhnikovia root, the burdock, the fructus forsythiae, the herba schizonepetae, the notopterygium root, the liquorice, the platycodon root and the gypsum, has antiviral effect on coronaviruses and has the effect of preventing and treating coronavirus infection.
Drawings
Fig. 1 to 4 are experimental result images showing the antiviral effect of the embodiment.
Fig. 5 and 6 are images showing experimental results of cell survival effects of examples.
Fig. 7 to 9 are experimental results images showing cytotoxicity and effective drug effect concentration of the examples.
Fig. 10 is an experimental result image showing the effective pharmacodynamic concentration of the positive control group.
Detailed Description
The present application will be further understood by the following examples. However, the following examples are merely illustrative of the present application, and the scope of the present application is not limited to the following examples. The embodiments of the present application are provided to more fully explain the present application to those skilled in the art to which the present application pertains. The present application is not limited to the embodiments set forth herein, but may be embodied in other forms.
The application relates to an antiviral pharmaceutical composition for coronavirus and a pharmaceutical composition for preventing and treating coronavirus infection, which comprises mixed extracts of radix sileris, burdock, fructus forsythiae, herba schizonepetae, notopterygium root, liquorice, balloonflower root and gypsum or fractional products thereof.
An embodiment of the present application may be an antiviral pharmaceutical composition comprising a Jiufeng Jiedu decoction extract as an active ingredient, and a pharmaceutical composition for preventing and treating coronavirus infection.
Jiufeng Jiedu decoction is one kind of otorhinolaryngological preparation for treating tonsillitis and other laryngopharynx swelling and pain.
The present inventors have confirmed a novel effect and use of a composition containing an extract of Jiufeng Jiedu decoction as an active ingredient, which has an antiviral effect against a novel coronavirus, and have developed a pharmaceutical composition for use in the prevention and treatment of coronavirus infection.
For example, the Jiufeng Jiedu decoction refers to a mixed extract or fraction thereof containing radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum.
The term "Fangfeng (Ledebouriella seseloides WOLFF.)" in the present application is prepared using roots and rhizomes of Fangfeng (Saposhnikovia divaricata Schiskin) of Umbelliferae, and has special odor, pungent, sweet and warm properties. It is effective in treating all wind diseases such as headache, aversion to cold, fever, general pain, and sore throat, and can be used for treating limb joint pain due to wind-cold-dampness, tetanus, muscle spasm, hemiplegia due to apoplexy, paralysis pain, skin pruritus, tinea, etc. Further, pharmacological actions are reported to be antipyretic, anti-inflammatory, sedative, immune-activating, antiallergic, antiulcer, antibacterial, skin-improving bacteria inhibitory, etc.
The term "burdock fruit" is the fruit of burdock Arctium lappa, and is a medicinal material with the effects of dispelling wind and heat, ventilating lung, eliminating swelling after acne and eruption and detoxifying.
Fructus Arctii refers to mature seeds of the biennial herb burdock (Arctium lappa Linne) belonging to the family Chrysanthemum (Compositae).
The term "fructus forsythiae" of the application is a fruit of golden bell flower (Forsythia Viridissima Lindley) or fructus forsythiae (Forsythia suspensa Vahi) belonging to the Oleaceae family (Oleaceae), which has special smell, bitter taste and slightly cool nature. Fructus forsythiae has effects of relieving fever and removing toxic substances, can be used for treating high fever and absentmindedness at the early stage of warm diseases, and can be used for treating abscess, macula, proctitis, lung abscess, lymphadenitis, pharyngolaryngitis, etc., and has diuretic and antiinflammatory effects. Also, it is known that it has pharmacological actions such as antibacterial action, anti-inflammatory action, blood pressure lowering, hemostatic action, liver treating action, antipyretic, antiemetic and diuretic actions.
The term "herba schizonepetae (Schizonepeta rhizome)" of the present application is a term annual herb of Labiatae, and whole herb is used as a medicinal material. The medicinal effect has antipyretic effect, so that the Chinese medicinal composition is used for sweating and relieving fever at the early stage of cold, is used for treating diseases such as sphagitis, skin diseases and apoplexy, and is fried to black to have hemostatic effect, and is applied to symptoms such as uterine bleeding, epistaxis, fecal bleeding, hematemesis and urinary hemorrhage.
The term "Notopterygium root" of the present application has strong dispersing effect, and thus, it is used in diseases such as exogenous headache, aversion to cold, fever, etc., and in diseases such as arthralgia, muscle spasm paralysis, shoulder pain, facial distortion, skin ulcer, aversion to cold, fever, pain, etc. in the early stage of sore and rash. The pharmacological effects include relieving fever, relieving pain, inhibiting dermatophytes, and increasing myocardial blood flow.
The term "licorice (Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra l.)" in the present application is a perennial herb of the order rosaceous, a dicotyledon plant, which has a special smell and a sweet taste. Licorice shows good curative effect by regulating toxicity of all medicines, and has effects of regulating cold and heat of viscera and pathogenic qi, dredging all blood vessels and strengthening musculoskeletal. The pharmacological effects have detoxification effects, are effective in hepatitis, urticaria, dermatitis, eczema and the like, and are known to have antitussive, expectorant, muscle relaxing, diuretic, antiinflammatory effects, and peptic ulcer inhibiting effects.
The term "platycodon grandiflorum" is a medicinal material prepared from root or peel of platycodon grandiflorum (Platycodon grandiflorum A. De Candolle) of platycodon grandiflorum, and has slightly odorous, bitter and flat taste. It is used in treating cough, expectoration, nasal obstruction, asthma, bronchitis, pleurisy, headache, aversion to cold, tonsillitis, etc. caused by laryngopharynx pain and common cold, and has the effects of eliminating phlegm, lowering blood sugar, lowering cholesterol, and improving bacteria inhibition.
The chemical component of the term "gypsum" according to the application is CaSO 4 ·2H 2 O. Diamond plate-shaped or columnar crystals are formed, occasionally gathered into a chrysanthemum shape, and double crystals in the shape of arrow tails are formed. In addition, there are many cases where a sheet-like, fibrous, block-like, dense agglomerate is formed, and especially, a parallel aggregate formed into a fibrous form is called fibrous gypsum, and a dense aggregate of fine particles is called anhydrite.
For example, the radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum may be mixed at a weight ratio of 400-600:400-600:800-850:200-300:200-300:400-600:1600-1700, and the radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum may be mixed at a weight ratio of 500:500:835:250:250:500:500:1665.
For example, the filtrate may be concentrated under reduced pressure at a temperature of 60 ℃ or less by adding 8 to 10 times of pure water to the mixture of the above-mentioned radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum, extracting at a temperature of 80 to 100 ℃ for 2 to 3 hours, and filtering.
Lactose hydrate, silica, magnesium stearate, for example, may also be included.
According to an embodiment of the present application, an antiviral effect against coronaviruses may be provided by including a mixed extract of ledebouriella root, burdock fruit, forsythia fruit, schizonepeta, notopterygium root, licorice root, platycodon root and gypsum or fractions thereof, which may have an effect for preventing and treating coronavirus infection.
By "preventing" is meant inhibiting or delaying all actions of coronavirus expression.
The term "treatment" as used herein refers to any action that alters the symptoms of an individual suspected of having a coronavirus infection or pathogenesis to improvement or cure.
The above pharmaceutical composition may comprise the active ingredient alone or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents to provide a pharmaceutical composition.
The term "pharmaceutically acceptable" as used herein refers to a property that exhibits no toxicity to cells or humans exposed to the combination.
Furthermore, the above pharmaceutical composition may be provided in admixture with a conventionally known coronavirus therapeutic substance. That is, the above-mentioned pharmaceutical composition may be administered in parallel with a known compound having an effect of preventing or treating coronavirus.
The term "administration" refers to the administration of a prescribed substance to an individual by an appropriate method, and "individual" refers to all animals including humans, mice, living mice, and domestic animals that have been or are able to be infected with coronavirus. Specifically, it may be, for example, a mammal including a human.
The administration route of the above-mentioned pharmaceutical composition includes oral, intravenous, intramuscular, intraarterial, intramedullary, epidural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal, but is not limited thereto.
The pharmaceutical composition may be administered orally or parenterally, and when administered parenterally, injection modes such as external or intraperitoneal injection, intrajob injection, subcutaneous injection, intravenous injection, intramuscular injection, and intrathoracic injection are preferably selected, but not limited thereto, and oral administration may be preferably selected from the viewpoint of selecting a more effective absorption route.
The preferred amount of the above-mentioned pharmaceutical composition to be administered varies depending on the state of the patient, the body weight, the severity of the disease, the pharmaceutical form, the route of administration and the period, and can be appropriately selected by the relevant practitioner. For example, the composition may be administered at 10mg/kg/day to 1000mg/kg/day, and suitably at 200mg/kg/day to 900mg/kg/day, but is not limited thereto. The administration may be performed once a day or divided into several administrations. The above-described amounts of administration are not limiting in any respect.
In the case of preparing the above pharmaceutical composition, it is prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, etc., which are generally used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations are prepared by mixing the above extract with one or more excipients, for example, with starch, calcium carbonate (calcium carbonate), sucrose (sucrose) or lactose (lactose), gelatin and the like. Besides the simple excipient, a lubricant such as magnesium stearate and talc is used. Liquid preparations for oral administration may contain various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water, liquid paraffin, which are common simple diluents, for example, suspending agents, internal solutions, emulsions, syrups, etc. Formulations for parenteral administration include sterilized aqueous solutions, nonaqueous solvents, suspending agents, oils, freeze-dried formulations, suppositories and the like. As the nonaqueous solvent and suspending agent, injectable esters such as propylene glycol (propylene glycol), vegetable oils such as polyethylene glycol and olive oil, and ethyl oleate can be used. As a base for suppositories, witepsol, polyethylene glycol, tween (tween) 61, cocoa butter, glycerol laurate, glycerol gelatin and the like can be used.
According to other embodiments of the present application, a health functional food for preventing and improving coronavirus infection is provided comprising a mixed extract of ledebouriella root, burdock fruit, forsythia fruit, schizonepeta, notopterygium root, licorice root, platycodon root and gypsum or fractions thereof.
The health functional food may further comprise one or more of carrier, diluent, excipient and additive, and is formulated into one dosage form selected from the group consisting of tablet, pill, powder, granule, powder, capsule and liquid. The food may be added with acteoside or its pharmaceutically acceptable salt, such as various foods, powder, granule, tablet, capsule, syrup, beverage, chewing gum, tea, vitamin complex, health food, etc.
The additive that may be contained in the health functional food may be one or more selected from the group consisting of natural carbohydrates, flavoring agents, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonating agents, and pulp.
Examples of the above natural carbohydrates are monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, and the like; and polysaccharides such as usual sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. The above flavoring agent may advantageously use natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside a, glycyrrhizic acid, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.). In addition, various nutrients, vitamins, minerals (electrolytes), synthetic flavors, natural flavors, colorants, thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. may be contained. In addition, the health functional food may contain pulp for preparing natural juice and vegetable beverage. These components may be used singly or in combination.
Preferably, specific examples of the carrier, excipient, diluent and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, methylcellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.
In the case of preparing the above health functional food, it can be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, etc., which are generally used.
The coronavirus is not particularly limited as long as it can be covered by one of ordinary skill in the art to which the present application pertains, and may be SARS-CoV-2Omicron, as appropriate.
As described above, although the exemplary embodiments of the present application have been described, the present application is not limited thereto, and it will be understood by those skilled in the art that various changes and modifications may be made without departing from the concept and scope of the appended claims.
Examples
Preparation examples antiviral pharmaceutical compositions concerning coronaviruses were prepared.
1. Preparing a dry extract
Respectively placing 500mg of radix Saposhnikoviae, 500mg of fructus Arctii, 835mg of fructus forsythiae, 250mg of herba Schizonepetae, 250mg of Notopterygii rhizoma, 250mg of Glycyrrhrizae radix, 500mg of radix Platycodi and 1685mg of Gypsum Fibrosum into an extractor to extract. In this case, the pure water was contained in an amount 8 times that of each raw material, and extraction was carried out at a temperature of 100℃for 3 hours. Next, the extract extracted in the extractor was filtered using 100 mesh (mesh) and moved to a thickener, and then concentrated under reduced pressure at a temperature of 60 ℃ or lower so that the drying loss was 50%. Then, the concentrated concentrate was dried under reduced pressure at a temperature of 70℃or lower and a pressure of 36 to 76 cmHg. Next, a step of pulverizing the dried product dried under reduced pressure using the pulverizing net of 1.2 was performed.
For each of the dried materials prepared by the above method, 10.48 weight percent of divaricate saposhnikovia root, 10.48 weight percent of burdock fruit, 17.51 weight percent of burdock fruit, 5.24 weight percent of fineleaf schizonepeta herb, 5.24 weight percent of notopterygium root, 5.24 weight percent of liquorice, 10.48 weight percent of platycodon root and 35.32 weight percent of gypsum are mixed to prepare the medicament taking the dried extract of the Jiufeng Jiedu decoction as the main component.
2. Preparation of pharmaceutical compositions
The dry extract prepared in the above preparation example 1, an excipient, a lubricant and a capsule were mixed to prepare a pharmaceutical composition in the form of a capsule.
Specifically, the lactose hydrate and silica as excipients were mixed with the dried extract and then filled into capsules with magnesium stearate as a lubricant, and the mixture was prepared according to the capsule preparation method in the general formulation rule of korean pharmacopoeia.
Experimental examples in vitro (in vitro) antiviral efficacy assessment
The efficacy of the pharmaceutical preparation of the above preparation in inhibiting SARS-CoV-2WT and SARS-CoV-2Omicron was confirmed by using Vero E6 cells as cells capable of confirming cytopathic effect (cytopathic effect) due to apoptosis after infection.
SARS-CoV-2WT (Betacoronavirus, scade, NCCP 43326/2020/Korea) used in the experiment was obtained from Korean national pathogen resource Bank as TCID 50 50 μl based dilution to 100TCID 50 50 μl was used, and antiviral efficacy against Vero E6 cells was evaluated using the dried extract as the pharmaceutical agent prepared by the above preparation example 1.
1. Viral infection and pharmaceutical treatment
In 96-well (well) cell culture plates, prepared Vero E6 cells were grown at 2X 10 4 After 24 hours (h) of incubation at cell/well concentration, vero E6 cell supernatants were removed and the cells were washed (washinging) using Dulcit Modified Eagle Medium (DMEM) containing 0% Fetal Bovine Serum (FBS).
Next, SARS-CoV-2 and SARS-CoV-2Omicron virus were adjusted to 100TCID 50 A concentration of 50. Mu.l was used to infect 50. Mu.l per well. The infected cells were cultured (incubation) in a cell culture apparatus at 37℃for 1 hour. Then, 150. Mu.l of Du's modified Igor's medium containing 2% fetal bovine serum and a drug diluted to a different concentration was used in place of the medium at 37℃and 5% CO 2 Is cultured under the condition of (2) for 4 days.
Also, the agents used in the experimental examples included a Filtration (Filtration) process using a 0.22 μm filter after diluting the dried extract prepared by the above preparation example 1 with a Phosphate Buffer Solution (PBS) containing 10% dimethyl sulfoxide (DMSO).
2. Antiviral efficacy was confirmed by Staining (Staining)
Confirmation of antiviral efficacy by staining was performed by staining cells with crystal violet (crystal violet).
Specifically, vero E6 cells infected with virus and treated with various concentrations of the agent in the above experimental example 1 were stained with 0.05% crystal violet in an amount of 1ml/well for 10 minutes and then washed, and the results were observed by measuring absorbance at a wavelength of 570 nm.
In the results using crystal violet staining, the stained portion of the cells was judged as a portion exhibiting virus inhibitory efficacy, and the unstained portion was judged as a portion not exhibiting inhibitory efficacy or exhibiting cytotoxicity.
Furthermore, radevir (RDV, remdesired) was used as a positive control group, and pure Vero E6 cells containing no virus and drug were used as a negative control group (100 TCID or NC).
2-1. One evaluation
In one evaluation, the above-mentioned agents were prepared to a concentration of 500mg/ml, and used with a phosphate buffer solution containing 10% dimethyl sulfoxide at 1/10 dilution, starting from 50mg/ml, and diluted 2-fold (2-fold) for experiments.
Specifically, the above-mentioned agent after filtration including the step of filtration in a 0.22 μm filter was compared with the pre-filtration and negative control group including no filtration step of the filter.
Referring to FIG. 1, it was confirmed that the antiviral efficacy of the agent was not exhibited for SARS-CoV-2 WT.
Referring to FIG. 2, it was confirmed that the antiviral effect against SARS-CoV-2Omicron was exhibited at a concentration of 6.25mg/ml to 12.5 mg/ml.
2-2. Second evaluation
In the second evaluation, the above-mentioned agent was prepared to a concentration of 500mg/ml, diluted with a phosphate buffer solution containing 10% dimethyl sulfoxide, and used, starting from 250mg/ml, and diluted in 3-fold dilution (3-fold dilution) to conduct an experiment.
Specifically, the case of using the above-described agent was compared with a positive control group and a negative control group using 50 μm of adefovir (RDV).
Referring to FIG. 3, it was confirmed that the antiviral efficacy of the agent was not exhibited for SARS-CoV-2 WT.
Referring to FIG. 4, in the case of SARS-CoV-2Omicron, cell survival was observed in the case of treating the drug at a concentration of 1.03mg/ml to 3.09mg/ml, and thus it was confirmed that the antiviral effect was exhibited.
3. Assessment of cytotoxicity
It was confirmed whether the agent prepared according to the preparation example induces cytotoxicity in Vero E6 cells. Specifically, vero E6 cells prepared in 96 well cell culture plates were grown at 2X 10 4 After 24 hours of cell/well incubation, the supernatant of Vero E6 cells was removed and the cells were washed with doe's modified eagle medium containing 0% fetal bovine serum.
Next, 150. Mu.l of a culture medium containing a drug diluted to a different concentration in Du's modified Igor's medium containing 2% fetal bovine serum was used for replacement, and the mixture was subjected to a dilution with 5% CO at 37 ℃C 2 After 4 hours of incubation under the conditions of (2) the crystal violet staining results were confirmed.
The dyeing method using crystal violet was the same as in experimental example 2 above.
3.1. One time evaluation 1
In one evaluation, the above-mentioned agents were prepared to a concentration of 500mg/ml, and used with a phosphate buffer solution containing 10% dimethyl sulfoxide at 1/10 dilution, starting from 50mg/ml, and diluted 2-fold for the experiment.
Specifically, the above-mentioned agent after filtration including the step of filtration in a 0.22 μm filter was compared with the pre-filtration and negative control group including no filtration step of the filter.
As shown in FIG. 5, the survival of cells was confirmed at a concentration of 12.5mg/ml or less, and the generation of a precipitate was confirmed in the case of using a drug for filtration.
3.2. Secondary evaluation
In the second evaluation, the above-mentioned agent was prepared to a concentration of 500mg/ml, diluted with a phosphate buffer solution containing 10% dimethyl sulfoxide, and used, starting from 250mg/ml, diluted in a 3-fold dilution manner to conduct an experiment.
Specifically, the case of using the above-mentioned drug was compared with the negative control group, and as shown in FIG. 6, cell survival was confirmed at a concentration of 1.03mg/ml or less until some cytotoxicity occurred at a concentration of 3.09 mg/ml.
3.3. Confirmation of dyeing site
The filtered above-mentioned agent including the step of filtering in the 0.22 μm filter was compared with the pre-filtration and negative control group including no filter step to confirm whether or not the precipitate was generated.
Specifically, CC 50 Shows cytotoxicity of the relevant agents, EC 50 The relative amounts of viruses inhibited by the agents are shown for effective pharmacodynamic concentrations of the agents.
As shown in fig. 7 and 8, it was confirmed that the cells were not stained with crystal violet at a high concentration, but the precipitate was found to be present without filtration.
3.4.EC 50 CC (CC) 50 Comprehensive evaluation of (2)
FIG. 9 is a comprehensive view showing cytotoxicity (CC 50 ) Concentration of effective drug (EC) 50 ) When the above-mentioned drug was used at a concentration of 0.343mg/ml to 250mg/ml, it was confirmed that a part of cytotoxicity (CC) was exhibited at a concentration of 3.09mg/ml 50 ) Shows effective drug effect concentration at 0.907mg/ml (EC 50 ) Further, the s.i. (selection index (Selectivity Index), CC of the above-described pharmaceutical agent can be confirmed 50 /EC 50 ) The value shows 3.41.
FIG. 10 is a graph showing the effective drug Effect Concentration (EC) of positive control groups using 0.1. Mu.M to 50. Mu.M of Rede-Sivir 50 ) It was confirmed that the effective drug effect concentration was exhibited at a concentration of 3.125. Mu.M.
Therefore, referring to the above experimental examples, it was confirmed that the pharmaceutical preparation prepared according to the above preparation example exhibited antiviral effects against SARS-CoV-2Omicron (NCCP 43408/2021/Korea).
Claims (8)
1. An antiviral pharmaceutical composition for coronaviruses, which is characterized by comprising a mixed extract of radix sileris, burdock, fructus forsythiae, herba schizonepetae, notopterygium root, liquorice, platycodon grandiflorum and gypsum or a fraction thereof.
2. The antiviral pharmaceutical composition according to claim 1, wherein the radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum are mixed at a weight ratio of 400-600:400-600:800-850:200-300:200-300:400-600:1600-1700.
3. The antiviral pharmaceutical composition according to claim 2, wherein the radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum are mixed at a weight ratio of 500:500:835:250:250:250:500:1665.
4. The antiviral pharmaceutical composition according to claim 2, wherein the purified water is added in an amount of 8 to 10 times the weight of the mixture of the radix Saposhnikoviae, fructus Arctii, fructus forsythiae, herba Schizonepetae, notopterygii rhizoma, glycyrrhrizae radix, radix Platycodi and Gypsum Fibrosum, and the mixture is extracted at a temperature of 80 to 100 ℃ for 2 to 3 hours, filtered, and the filtrate is concentrated under reduced pressure at a temperature of 60 ℃ or less.
5. The antiviral pharmaceutical composition according to claim 1, further comprising lactose hydrate, silica, magnesium stearate.
6. The antiviral pharmaceutical composition for coronaviruses according to claim 1, which has an antiviral effect against SARS-CoV-2 Omicron.
7. A pharmaceutical composition for preventing and treating coronavirus infection is characterized by comprising mixed extracts of radix sileris, burdock, fructus forsythiae, herba schizonepetae, notopterygium root, liquorice, platycodon grandiflorum and gypsum or fractions thereof.
8. A health functional food for preventing and improving coronavirus infection is characterized by comprising mixed extracts of radix sileris, burdock, fructus forsythiae, herba schizonepetae, notopterygium root, liquorice, platycodon grandiflorum and gypsum or fractions thereof.
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CN101023996A (en) * | 2007-03-26 | 2007-08-29 | 河南仲景药业股份有限公司 | Chinese medicine for treating exogenous epidemic disease and anemopyretic cold |
CN111494502A (en) * | 2020-04-23 | 2020-08-07 | 徐修信 | Traditional Chinese medicine composition for early novel coronary virus pneumonia and preparation method thereof |
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2023
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CN101023996A (en) * | 2007-03-26 | 2007-08-29 | 河南仲景药业股份有限公司 | Chinese medicine for treating exogenous epidemic disease and anemopyretic cold |
CN111494502A (en) * | 2020-04-23 | 2020-08-07 | 徐修信 | Traditional Chinese medicine composition for early novel coronary virus pneumonia and preparation method thereof |
CN111870657A (en) * | 2020-04-23 | 2020-11-03 | 江苏康缘药业股份有限公司 | Application of traditional Chinese medicine composition in preparation of medicine for treating or preventing coronavirus infection |
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