KR102594706B1 - 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, ameliorating or treating coronavirus infection, which contains extract of Chrysanthemum chinensis as an active ingredient. The extract of Chrysanthemum primrose has the effect of protecting cells from coronavirus infection, preventing the replication of viruses in cells and viruses. Since it has an excellent effect of suppressing intracellular infection, the composition of the present invention containing the extract of Chrysanthemum chinensis as an active ingredient can be usefully used as a treatment for coronavirus infection, health functional food for anti-coronavirus, quasi-drugs, cosmetics, or feed additives. there is.

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 coronavirus infection containing herbal medicine extract, especially Chrysanthemum chinensis extract, as an active ingredient.

The new type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) is a virus that has a single-stranded positive RNA genome consisting of approximately 30,000 base pairs, and the viral surface is surrounded by an envelope. Since it was first reported in Wuhan, China at the end of December 2019, the number of infected people has rapidly increased worldwide. The virus spreads so efficiently between people that the World Health Organization has declared it a pandemic with the potential to spread around the world and is seeking responses.

Typical Covid-19 patients show relatively mild symptoms such as increased body temperature, cough, and chest pain, but some patients develop serious diseases such as difficulty breathing and pneumonia, leading to death.

To date, recognized treatments for coronavirus infections include ribavirin, famciclovir, nitazoxanide, nafamostat, and chloroquine. In addition, Remdesi Remdesivir (GS-5734) and favipiravir are being used to treat coronavirus infections, but their effectiveness is low and the incidence of variant viruses is high due to the nature of nucleic acid analog antivirals, so two or more types of antivirals are applied together. . Most antiviral drugs used for coronavirus infections have the ability to inhibit RNA polymerase, but mouse hepatitis virus, a type of coronavirus that is resistant to remdesivir, has been discovered, and existing antiviral drugs have been discovered. Viral drugs are ineffective. In addition, antiviral drugs developed to date have severe side effects, so great caution is required in their application.

Additionally, currently the most common treatment method is symptomatic therapy to relieve symptoms. Therefore, the need to develop new anti-coronavirus drugs with excellent infection control effects and low toxicity to prevent and treat coronavirus outbreaks is increasing.

Meanwhile, Korean Patent Publication No. 2022-0013625 discloses an anti-coronavirus composition characterized by containing an extract of Ulgoebul tree as an active ingredient, and Korean Patent Publication No. 2022-0018953 discloses an anti-coronavirus composition containing fern extract or a fraction thereof. Although an antiviral composition has been disclosed, there has been no disclosure at all regarding a composition for preventing, improving, or treating coronavirus infection containing the herbal medicine extract of the present invention, especially the Chrysanthemum chinensis extract, as an active ingredient.

The present invention has been developed in response to the above-mentioned needs, by providing an antiviral composition for coronavirus containing an extract of Chrysanthemum chinensis as an active ingredient, and confirming that the extract of the extract of Chrysanthemum chinensis inhibits coronavirus infection and proliferation. , completed the present invention.

In order to solve the above problems, the present invention provides an antiviral composition against coronavirus containing Melia azedarach fruit extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection, comprising extract of Melia azedarach fruit as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving coronavirus infection containing Melia azedarach fruit extract as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving coronavirus infection containing Melia azedarach fruit extract as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving coronavirus infection containing Melia azedarach fruit extract as an active ingredient.

In addition, the present invention provides a feed additive for preventing or improving coronavirus infection containing Melia azedarach fruit extract as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating coronavirus infection, which contains extract of Chrysanthemum chinensis as an active ingredient. The extract of Chrysanthemum primrose has the effect of protecting cells from coronavirus infection, preventing the replication of viruses in cells and viruses. It is effective in suppressing intracellular infection.

Figure 1 shows the results of confirming the antiviral effect of the Chrysanthemum chinensis extract against SARS-CoV2 through CPE (Cytopathic effect) inhibition analysis. (A) is a photo of a plate stained with crystal violet and the results of quantification of the antiviral effect against SARS-CoV2 according to the treatment of the extract of Chrysanthemum chinensis at different concentrations, and (B) is a photo of the antiviral effect against SARS-CoV2 according to treatment with remdesivir, which is a positive control. This is a photo of a plate stained with crystal violet and the results of quantifying the antiviral effect against CoV2. NC is the normal group, and VC is the control group treated with SARS-CoV2. ***, ***** mean that there was a statistically significant increase in the reduction rate of cell lesions in the Chrysanthemum chinensis extract-treated group or the remdesivir-treated group compared to the control group (VC) treated with SARS-CoV2, ** * means p<0.001, **** means p<0.0001.
Figure 2 shows the results confirming the inhibitory effect of Virus replication on SARS-CoV2 of Chunnyeonja extract. NC is the normal group, and VC is the control group treated with SARS-CoV2. ***** means that there was a statistically significant decrease in the number of viral copies in the group treated with Chrysanthemum chinensis extract compared to the control group (VC) treated with SARS-CoV2, p<0.0001.
Figure 3 is a fluorescent photograph (A) confirming the inhibitory effect of Virus infection inhibitory effect on SARS-CoV2 of the Chrysanthemum chinensis extract and the quantification result (B). NC is the normal group, and VC is the control group treated with SARS-CoV2. **** means that there was a statistically significant decrease in viral infection in the group treated with Chrysanthemum chinensis extract 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 against coronavirus containing extract of Melia azedarach fruit as an active ingredient.

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

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

The Chunryeonja 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 extract of Melia azedarach fruit as an active ingredient.

The pharmaceutical composition according to the present invention can be formulated and used in the form of oral dosage forms such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. You 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, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

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 contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in the pharmaceutical composition. It is prepared by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used.

The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. You 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 topically on the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

In addition, the present invention provides a health functional food composition for preventing or improving coronavirus infection containing Melia azedarach fruit extract as an active ingredient.

The composition may be manufactured 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 is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of active ingredient can be appropriately used depending on the purpose of use (prevention or improvement). Generally, when manufacturing a 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 raw materials. However, in the case of long-term intake for health purposes, the amount may be below 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 are no particular restrictions on the types of health functional foods. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. There are drinks, alcoholic beverages, and vitamin complexes, and it includes all health foods in the conventional sense.

Additionally, the health functional food composition of the present invention can be manufactured into food, especially functional food. The functional food of the present invention includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., For example, xylitol, sorbitol, erythritol, etc.) is preferable. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above 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 carbonating agents used in beverages. The ratio of the ingredients 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 containing Melia azedarach fruit extract as an active ingredient.

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

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

For example, the quasi-drug composition of the present invention may be manufactured in the form of a general emulsified formulation or solubilized formulation. For example, it may have a formulation such as lotion, cream, ointment, spray, oil gel, gel, oil, or aerosol, but can be used without limitation as long as it shows the effect of preventing or improving the coronavirus infection of the present invention. there is.

In addition, the quasi-drug composition contains oil, water, surfactants, moisturizers, lower alcohols with 1 to 4 carbon atoms, thickeners, chelating agents, colorants, preservatives or fragrances, etc., which are generally mixed in quasi-drug compositions, as appropriate for each dosage form. Can be used in combination.

In addition, the present invention provides a cosmetic composition for preventing or improving coronavirus infection containing Melia azedarach fruit extract as an active ingredient.

The cosmetic composition of the present invention contains ingredients commonly used in cosmetic compositions in addition to the extract, and includes conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

The cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing products. , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it can be manufactured in the form of 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. You can.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacantha, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier ingredients. 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 can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants 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, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar or tragacantha can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

In addition, the present invention provides a feed additive for preventing or improving coronavirus infection containing Melia azedarach fruit 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 mean any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals. The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, oils and fats, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more types.

Hereinafter, the present invention will be described in more detail using manufacturing examples and examples. These manufacturing examples and examples are only for illustrating 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 Cheonryeonja Extract

Cheonryeonja purchased a standard product from Gwangmyeongdang Pharmaceutical. Specifically, 10 parts by weight of water (100°C) was added to 1 part by weight of Cheonryeonja dried raw material, extracted under reflux for 3 hours, and then filtered through a strainer (extraction cloth). After passing through a 53㎛ filter, the product was concentrated under reduced pressure, then freeze-dried and used as a sample for the experiment.

Example 1. Confirmation of antiviral activity of Cheonryeonja extract through CPE (Cytopathic effect) inhibition analysis

After culturing Vero E6 cells in a 96-well plate, SARS-CoV2 was inoculated at an m.o.i of 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 then treated with a 1:1 volume ratio of methanol:acetone and fixed for 10 minutes at room temperature. Afterwards, the solution was removed, 0.4% (w/v) crystal violet was added to each well, and the cells were stained at room temperature for 10 minutes, then all of the solution was removed and dried. After drying, methanol:acetic acid was mixed at a 1:1 volume ratio, and 100 ㎕ of a solution diluted 5 times with distilled water was added to each well. Crystal violet was extracted for 2 hours, and the absorbance was measured at 550 nm.

As a result, as shown in Figure 1, when treated with 10 to 500㎍/㎖ of the Chrysanthemum chinensis extract of the present invention, the proliferation of SARS-CoV2 was inhibited in a concentration-dependent manner, and the IC ₅₀ value was confirmed to be 239.3㎍/㎖. Therefore, the extract of Chrysanthemum chinensis had an excellent antiviral effect in protecting cells from the SARS-CoV2 virus.

Meanwhile, remdesivir, used as a positive control, showed 45-55% CPE reduction activity at 10μM, and was confirmed to not only inhibit the proliferation of SARS-CoV2 but also cell proliferation at 20μM.

Example 2. Confirmation of virus replication inhibitory effect of Chunryeonja extract

The virus replication inhibitory effect of Chunryeonja extract was confirmed by qPCR. Specifically, after culturing Vero E6 cells in a 96-well plate, SARS-CoV2 was inoculated at an m.o.i of 0.01, treated with 500 ㎍/mL of Chrysanthemum chinensis extract, and cultured for 24 hours. After incubation, the RNA of the virus released out of the cells was extracted using Bioneer's AccuPrep Viral RNA Extraction kit. Then, cDNA was synthesized using Toyobo's ReverTra Ace qPCR RT kit, then PCR was performed with specific primers for the RBD of the spike protein, and the viral RNA copy number was measured using the spike protein gene plasmid standard sample. Calculated.

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

Example 3. Confirmation of the virus infection inhibition effect of Chunryeonja extract

The inhibitory effect of Chunryeonja extract on SARS-CoV2 virus infection was confirmed using immunofluorescence analysis. Specifically, Vero E6 cells were distributed in a 96-well plate at 1 × 10 ⁴ cells/well, treated with 500 ㎍/mL of Chrysanthemum chinensis extract, then 2% (v/v) FBS and 1% (v/v) SARS-CoV2 diluted 1000 times was treated with DMEM containing antibiotics and cultured for 24 hours. After incubation, the cells were fixed with a 4% (v/v) formaldehyde solution, reacted with a primary antibody (N-protein) diluted 2000 times, and then reacted with a secondary antibody diluted 200 times. After the reaction, the cells were observed under a fluorescence microscope, and the fluorescence intensity was analyzed.

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

Claims (10)

An antiviral composition against type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) containing water extract of Melia azedarach fruit as an active ingredient. delete delete A pharmaceutical composition for preventing or treating type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection, comprising water extract of Melia azedarach fruit as an active ingredient. The method of claim 4, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in addition to the water extract of Chrysanthemum chinensis for preventing or treating type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection. Pharmaceutical composition. A health functional food composition for preventing or improving type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection, containing water extract of Melia azedarach fruit as an active ingredient. The method of claim 6, wherein the composition is prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, and beverage. Severe acute respiratory syndrome type 2 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, containing water extract of Melia azedarach fruit as an active ingredient. A cosmetic composition for preventing or improving type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection, containing water extract of Melia azedarach fruit as an active ingredient. A feed additive for preventing or improving type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) infection, containing Melia azedarach fruit water extract as an active ingredient.