KR102527527B1 - Composition for preventing, ameliorating or treating coronavirus infectious disease comprising herbal medicine extract as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating coronavirus infection comprising an Amomum tsao-ko extract as an active component. The Amomum tsao-ko extract has an excellent effect of protecting cells from coronavirus infection, and inhibiting the replication of the virus within the cell and Intracellular infection of viruses. Therefore, the composition of the present invention containing the Amomum tsao-ko extract as the active component can be usefully used as a treatment for coronavirus infection, health functional food for anti-coronavirus, quasi-drugs, cosmetics, or feed additives.

Description

Composition for preventing, ameliorating or treating coronavirus infectious disease comprising herbal medicine extract as effective component}

The present invention relates to a composition for preventing, improving or treating a coronavirus infection comprising an extract of herbal medicine, particularly an extract of excess as an active ingredient.

The new type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) has a single-stranded positive RNA genome consisting of about 30,000 base pairs, and the virus surface is surrounded by an envelope. After it was first reported in Wuhan, China at the end of December 2019, infections worldwide exploded. The virus spreads very efficiently between people, so the World Health Organization has declared it as a pandemic that can spread worldwide and is seeking countermeasures.

Normal Covid-19 patients show relatively mild symptoms such as elevated body temperature, coughing and chest pain, but some patients develop serious illnesses such as shortness of breath and pneumonia, leading to death.

To date, approved treatments for coronavirus infections include ribavirin, famciclovir, nitazoxanide, nafamostat, and chloroquine. In addition, remdesi Vir (remdesivir, GS-5734) and favipiravir are used for the treatment of coronavirus infections, but their effectiveness is low and the incidence of mutant viruses is high due to the nature of nucleic acid analog antiviral agents, so two or more antiviral agents are applied together. . Most of the antiviral drugs used for coronavirus infection have RNA polymerase inhibitory ability, but Mouse hepatitis virus, a type of coronavirus resistant to remdesivir, was discovered, and the existing antiviral The current situation is that there is no effect of the virus agent. In addition, since antiviral agents developed to date have severe side effects, great care is required in their application.

In addition, the most common treatment method at present is symptomatic therapy to relieve symptoms. Therefore, there is an increasing need for the development of new anti-coronavirus agents with excellent infection suppression effect and low toxicity for preventing and treating the outbreak of coronavirus.

On the other hand, Korean Patent Publication No. 2022-0013625 discloses an anti-coronavirus composition characterized in that it contains an extract of euphorbia japonica as an active ingredient, and Korean Patent Publication No. 2022-0018953 includes a bracken extract or a fraction thereof. Although an antiviral composition is disclosed, there is no disclosure at all about a composition for preventing, improving or treating coronavirus infection comprising the herbal extract of the present invention, in particular, the excess extract as an active ingredient.

The present invention was derived from the above needs, by providing an antiviral composition against coronavirus comprising an excess extract as an active ingredient, and confirming that the excess extract inhibits coronavirus infection and proliferation, The invention was completed.

In order to solve the above problems, the present invention provides an antiviral composition for coronavirus containing an extract of Amomum tsao-ko as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising an extract of Amomum tsao-ko as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving coronavirus infection comprising Amomum tsao-ko extract as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving coronavirus infection comprising Amomum tsao-ko extract as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving coronavirus infection comprising an extract of Amomum tsao-ko as an active ingredient.

In addition, the present invention provides a feed additive for preventing or improving coronavirus infection comprising Amomum tsao-ko extract as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating a coronavirus infection comprising an excess extract as an active ingredient, wherein the excess extract has an effect of protecting cells from coronavirus infection, replication of the virus in cells and cells of the virus It has the effect of suppressing my infection.

1 is a result of confirming the antiviral effect of the excess extract against SARS-CoV2 by CPE (Cytopathic effect) inhibition assay. (A) is a photograph of a plate stained with crystal violet for the antiviral effect on SARS-CoV2 according to the excess extract treatment by concentration and the result of quantification, and (B) is a positive control, SARS-CoV2 according to remdesivir treatment This is a picture of a plate stained with crystal violet for antiviral effect and the result of quantification thereof. NC is a normal group, and VC is a control group treated with SARS-CoV2. ***, **** mean that the cell lesion reduction rate of the excess extract treatment group or remdesivir treatment group increased statistically significantly compared to the control group (VC) treated with SARS-CoV2, and *** is p<0.001, **** is p<0.0001.
Figure 2 is the result of confirming the viral replication inhibitory effect on SARS-CoV2 of the excess extract. NC is a normal group, and VC is a control group treated with SARS-CoV2. **** means that the number of viral copies of the excess extract treatment group decreased statistically significantly compared to the control group (VC) treated with SARS-CoV2, p<0.0001.
Figure 3 is a fluorescence photograph (A) confirming the viral infection inhibitory effect on SARS-CoV2 of the excess extract and a result (B) of quantification thereof. NC is a normal group, and VC is a control group treated with SARS-CoV2. **** means that the viral infection of the excess extract treatment group was statistically significantly reduced compared to the control group (VC) treated with SARS-CoV2, p<0.0001.

In order to achieve the object of the present invention, the present invention provides an antiviral composition for coronavirus comprising an extract of Amomum tsao-ko as an active ingredient.

The coronavirus may be one or more selected from the group consisting of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS CoV) and Middle East respiratory syndrome (MERS) coronavirus, It is preferably type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), but is not limited thereto.

The extraction solvent of the excess extract is preferably water, C ₁ ~ C ₄ lower alcohol or a mixture thereof, more preferably water, but is not limited thereto.

Excess extract of the present invention has the effect of inhibiting the proliferation of type 2 severe acute respiratory syndrome coronavirus.

In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising an extract of Amomum tsao-ko as an active ingredient.

The pharmaceutical composition according to the present invention is formulated according to conventional methods into oral formulations such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent.

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and various compounds or mixtures including mineral oil and the like.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. is prepared by mixing In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

A suitable dosage of the pharmaceutical composition of the present invention will be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. can

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

In addition, the present invention provides a health functional food composition for preventing or improving coronavirus infection comprising Amomum tsao-ko extract as an active ingredient.

The composition may be prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient can be appropriately used depending on the purpose of its use (prevention or improvement). In general, when preparing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the total amount of raw materials. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of health functional food. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, and vitamin complexes, and includes all health foods in a conventional sense.

In addition, the health functional food composition of the present invention can be made into food, particularly functional food. The functional food of the present invention includes components commonly added during food preparation, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to active ingredients. The natural carbohydrate is a monosaccharide (eg, glucose, fructose, etc.), a disaccharide (eg, maltose, sucrose, etc.), an oligosaccharide, a polysaccharide (eg, dextrin, cyclodextrin, etc.) or a sugar alcohol (eg, For example, xylitol, sorbitol, erythritol, etc.) is preferable. As the flavoring agent, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent used in beverages and the like. The ratio of the components to be added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention provides a quasi-drug composition for preventing or improving coronavirus infection comprising Amomum tsao-ko extract as an active ingredient.

The quasi-drugs may include external skin preparations and personal hygiene products. For example, it may be a disinfectant cleanser, shower foam, gargreen, wet tissue, detergent soap, hand wash, or ointment, but is not limited thereto.

When the quasi-drug composition according to the present invention is used as a quasi-drug additive, the composition may be added as it is or used together with other quasi-drugs or quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention may be prepared in the form of, for example, a general emulsified formulation and a solubilized formulation. For example, it may have formulations such as emulsions, creams, ointments, sprays, oil gels, gels, oils, and aerosols such as lotions, but it may be used without limitation as long as it exhibits the effect of preventing or improving coronavirus infection of the present invention. there is.

In addition, the quasi-drug composition may appropriately contain oil, water, a surfactant, a moisturizer, a lower alcohol having 1 to 4 carbon atoms, a thickener, a chelating agent, a colorant, a preservative, or a flavoring agent, which are generally formulated in quasi-drug compositions, in each dosage form. Can be used in combination.

In addition, the present invention provides a cosmetic composition for preventing or improving coronavirus infection comprising an extract of Amomum tsao-ko as an active ingredient.

The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the extract, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and conventional adjuvants such as carriers.

The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it may be formulated into a flexible lotion (skin), nourishing lotion (milk lotion), nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, shampoo, pack, spray or powder. can

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier components. It can be.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar or tragacantha and the like may be used.

When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition, the present invention provides a feed additive for preventing or improving coronavirus infection comprising Amomum tsao-ko extract as an active ingredient.

The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. In the present invention, the term 'feed' may refer to any natural or artificial diet, one meal, etc., or a component of the one meal meal, suitable for or suitable for consumption by animals. The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feed such as proteins, inorganic materials, oils, mineral oils, oils, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

Hereinafter, the present invention will be described in more detail using preparation examples and examples. These Preparation Examples and Examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Preparation Example 1. Preparation of Excess Extract

The excess was purchased as a standard product from Kwang Myung Dang Pharmaceutical. Specifically, after adding 10 parts by weight of water (100 ° C) based on 1 part by weight of the excess dried raw material, refluxed extraction for 3 hours, and then filtered through a sieve (extraction cloth). The material passed through the 53 μm filter was concentrated under reduced pressure, and then lyophilized and used as a sample for the experiment.

Example 1. Confirmation of antiviral activity of excess extract through CPE (cytopathic effect) inhibition assay

After culturing Vero E6 cells in a 96-well plate, SARS-CoV2 was inoculated with m.o.i 0.01. After inoculation, the sample of Preparation Example 1 prepared by diluting with DMEM containing 2% (v/v) FBS and 1% (v/v) penicillin-streptomycin to match the treatment concentration was treated, and then treated for 5 days. cultured. After incubation, the cell culture medium was removed and treated with a mixed solution of methanol:acetone in a volume ratio of 1:1, followed by fixation at room temperature for 10 minutes. Thereafter, the solution was removed, and 0.4% (w/v) crystal violet was added to each well, followed by staining at room temperature for 10 minutes, and then the solution was removed and dried. After drying, methanol:acetic acid was mixed at a volume ratio of 1:1, and 100 μl of a 5-fold diluted solution with tertiary distilled water was added to each well, crystal violet was extracted for 2 hours, and absorbance was measured at 550 nm.

As a result, when the excess extract of the present invention was treated as shown in FIG. 1, the proliferation of SARS-CoV2 was inhibited in a concentration-dependent manner, and the IC ₅₀ value was 101.9 μg/ml, confirming that the excess extract was SARS-CoV2. The antiviral effect of protecting cells from viruses was excellent.

On the other hand, remdesivir used as a positive control showed a CPE reduction activity of 45 to 55% at 5 to 10 μM, and at 20 μM it was confirmed that it inhibited not only the proliferation of SARS-CoV2 but also cell proliferation. .

Example 2. Verification of viral replication inhibitory effect of excess extract

The effect of inhibiting viral replication of the excess extract was confirmed by qPCR. Specifically, after culturing Vero E6 cells in a 96-well plate, SARS-CoV2 was inoculated with m.o.i 0.01, treated with an excess extract of 100 or 250 μg/mL, and cultured for 24 hours. After culturing, RNA of the virus released outside the cell was extracted using Bioneer's AccuPrep Viral RNA Extraction kit. Subsequently, cDNA was synthesized using Toyobo's ReverTra Ace qPCR RT kit, PCR was performed with specific primers for the RBD of spike protein, and viral RNA copy number was measured using spike protein gene plasmid standard samples. Calculated.

As a result, as shown in FIG. 2, the excess extract was excellent in inhibiting the replication of SARS-CoV2 virus.

Example 3. Verification of viral infection inhibitory effect of excess extract

The inhibitory effect of SARS-CoV2 virus infection of the excess extract was confirmed by immunofluorescence analysis.

Specifically, Vero E6 cells were plated at 1×10 ⁴ cells/well, treated with excess extract of 100 or 250 μg/mL, and then treated with 2% (v/v) FBS and 1% (v/v) antibiotics. 1000-fold diluted SARS-CoV2 was treated with DMEM containing , and cultured for 24 hours. After incubation, the cells were fixed with a 4% (v/v) formaldehyde solution, reacted with a 2000-fold diluted primary antibody (N-protein), and then reacted with a 200-fold diluted secondary antibody. After the reaction, the cells were observed under a fluorescence microscope and the fluorescence intensity was analyzed.

As a result, intracellular infection of the SARS-CoV2 virus was effectively inhibited by treatment with the excess extract, as shown in FIG. 3 .

Claims (10)

An antiviral composition against type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) consisting of a water extract with excess active ingredient ( Amomum tsao-ko ). delete delete A pharmaceutical composition for preventing or treating Type 2 Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV2) infection consisting of a water extract with excess active ingredient ( Amomum tsao-ko ). [Claim 5] The pharmaceutical composition for the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection according to claim 4, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the excess water extract. composition. A health functional food composition for preventing or improving type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection consisting of an excess active ingredient ( Amomum tsao-ko ) water extract. The method of claim 6, wherein the composition is prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candy, syrups and beverages Type 2 Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV2) Health functional food composition for preventing or improving infectious diseases. A quasi-drug composition for preventing or improving type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection consisting of an extract of water with excess active ingredient ( Amomum tsao-ko ). A cosmetic composition for preventing or improving Type 2 Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV2) infection consisting of a water extract with excess active ingredient ( Amomum tsao-ko ). A feed additive for preventing or improving type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection consisting of water extract with excess active ingredient ( Amomum tsao-ko ).

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