WO2022098133A1

WO2022098133A1 - Streptomyces sp. strain having antiviral efficacy and antiviral composition comprising same

Info

Publication number: WO2022098133A1
Application number: PCT/KR2021/015945
Authority: WO
Inventors: 남상집; 임병관
Original assignee: 이화여자대학교 산학협력단; 중원대학교 산학협력단
Priority date: 2020-11-06
Filing date: 2021-11-04
Publication date: 2022-05-12

Abstract

The present invention relates to a Streptomyces sp. strain having an antiviral efficacy and an antiviral composition including same.

Description

Streptomyces sp. strain having antiviral efficacy and antiviral composition comprising the same

The present invention relates to a Streptomyces sp. strain having antiviral efficacy and an antiviral composition comprising the same.

Viruses are one of the representative causes of numerous incurable diseases that threaten human health. Entero virus, Chikungunya Virus, SARS virus, MERS virus and Zika Intractable RNA viruses such as Zika virus pose a serious risk to mankind. Most of the epidemics have slightly different symptoms, but the period of the epidemic is roughly fixed, and huge capital is invested worldwide to detect and prevent or treat it.

In Korea, enterovirus is prevalent in young children during spring and summer. In particular, children under the age of three are more easily exposed to the virus as more and more children under the age of three are left in daycare facilities. Enteroviruses are well known as the cause of enteritis, hand, foot and mouth disease, herpangina (stomy herpes), spinal polio, aseptic meningitis in infants, and encephalitis. Enteroviruses are fatal enough to cause death in severe cases, so there is an urgent need to develop a virus treatment that can suppress their proliferation, but there is currently no treatment or preventive agent that can effectively suppress these viruses.

One object of the present invention is to provide an antiviral composition comprising a compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof.

Another object of the present invention is to provide a method for producing an antiviral composition and a strain capable of producing the composition.

Another object of the present invention is a Streptomyces genus strain having antiviral efficacy, a culture of the strain, a lysate of the strain, an extract of the cell, an extract of the culture, and an extract of the lysate. It is to provide an antiviral composition comprising one or more selected from.

Another object of the present invention, Streptomyces sp. strain or the step of extracting fractional extraction of the culture solution of the strain with an organic solvent; And to provide a method for preparing a compound having Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof, comprising the step of separating and purifying the fractionated extract by chromatography.

Another object of the present invention, Streptomyces sp. strain or the step of extracting fractional extraction of the culture solution of the strain with an organic solvent; And a method for preparing an antiviral composition comprising the step of separating and purifying the fractionated extract by chromatography, comprising the compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof is to provide

Antiviral agents can inhibit viruses through various mechanisms, among which the following two mechanisms are known that can be used to effectively inhibit viruses.

The first is to inhibit S-adenosylhomocysteine (hereinafter, SAH) hydrolase. SAH hydrolase is a tetramer-type enzyme that uses NAD+ as a coenzyme. It reversibly hydrolyzes SAH into adenosine (Ado) and homocysteine (Hcy), and proteins, lipids in vivo. , it is a very important enzyme for the methylation of substances in the body, such as histamine and norepinephrine, as well as nucleic acids. Inhibition of this SAH hydrolase causes the accumulation of SAH, and the excess SAH sequentially suppresses S-adenosylmethionine (AdoMet)-dependent transmethylase and capping of viral mRNA so that the protein necessary for viral replication is released. By preventing it from being made properly, it results in an antiviral effect. Since most animal DNA viruses as well as RNA viruses require methylation enzymes (viral mRNA guanosine N7-methytransferases, O-2′-methytransferases) for mRNA capping, SAH hydrolase is considered an essential element in the development of broad-spectrum antiviral agents. do. That is, the development of RNA virus therapeutics and SAH hydrolase inhibitors are considered to have a high correlation.

Second, it inhibits viral RNA polymerase. RNA viruses are replicated by inserting the substrate Nucleoside-5'-triphosphate (NTP) into the RNA chain by RNA polymerase. Therefore, a substance that inhibits RNA polymerase can also act as an antiviral agent, and it is converted into triphosphate in the body to selectively inhibit viral RNA polymerase or is directly inserted into the viral RNA chain to cause chain termination ), it is believed that effective antiviral agents can be developed.

Currently commercially available antiviral agents are mostly nucleoside derivatives such as iododeoxyuridine (IDU), acyclovir (ACV), azidothymidine (AZT), or proteins such as interferon (IFN), but these substances are cytotoxic. , hepatotoxicity, hyper-tolerance, and other side effects. In addition, in the case of oseltamivir phosphate (so-called Tamiflu) developed as a treatment for human influenza viruses A and B and avian influenza viruses, when administered to the elderly, vomiting, bronchitis, Side effects such as headache, cough, and dizziness have been reported. In addition, it is reported that the appearance of strains resistant to existing drugs is increasing. Due to these side effects or limited efficacy, existing antiviral substances have many limitations in effectively treating viral diseases. Therefore, there is a continuous need for the development of safe new antiviral substances with few side effects such as cytotoxicity, and research to search for substances with antiviral activity from safe natural products with few side effects to avoid the side effects of existing chemically synthesized drugs. is needed

The present inventors made diligent efforts to discover substances exhibiting effective antiviral efficacy, and as a result, 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)- from Marine Streptomyces among microorganisms isolated from marine sediment , isolated the methyl ester, and found that the compound can effectively inhibit the proliferation of viruses, thereby completing the present invention.

According to an example of the present invention, the antiviral efficacy of the Streptomyces sp. strain fraction isolated from the marine sediments of Antarctica was verified, and 4-benzoxazolecarboxyl, a compound of Formula 1, isolated from the Streptomyces sp. strain through detailed verification The antiviral effect of acid, 2-(2-hydroxyphenyl)-, methyl ester was confirmed. Specifically, 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester was added to the cell culture medium at a concentration of 100-0.0001 μg/ml to HeLa cells cultured in a 96-well cell culture dish, and the virus was treated. was infected with 10 ⁵ pfu/ml for 18 hours, and then 10 μl of Cell Counting Kit 8 (CCK-8), a reagent for detecting proliferation of cells, was added and incubated for 2 hours more. Then, by measuring the absorbance at 450 nm using a Microplate reader (Molecular device, USA), the survival of the cells was confirmed to confirm the antiviral efficacy. As shown in FIG. 5 expressing the above results, the survival rate of HeLa cells increased dependently on the treatment concentration of 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester after virus infection, and from this, 4-benzo The excellent antiviral efficacy of sazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester was confirmed. In addition, direct antiviral efficacy was confirmed by confirming the production of viral membrane protein and the activity of the host protein-cleaving enzyme (FIG. 3).

Hereinafter, the present invention will be described in more detail.

An example of the present invention relates to an antiviral composition comprising a compound having the following formula (1), a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof:

[Formula 1]

"4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester" of the present invention includes, without limitation, the substance represented by Formula 1 and derivatives, isomers, etc. that can be recognized in the art as similar thereto Unless otherwise specified below, as an active (active) ingredient included in the composition of the present invention, 4-benzoxazole carboxylic acid of Formula 1, 2-(2-hydroxyphenyl)-, methyl All esters as well as pharmaceutically acceptable salts, hydrates, solvates, isomers (preferably optical isomers) or derivatives thereof are included, and all of these should be construed as being included in the scope of the present invention.

The active ingredient of the present invention, “4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester” may exist in the form of a salt, particularly a pharmaceutically acceptable salt. As the salt, salts commonly used in the art may be used without limitation. The term “pharmaceutically acceptable salt” of the present invention refers to a concentration having an effective action that is relatively non-toxic and harmless to a patient, and the side effects caused by this salt are 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl) -, means any and all organic or inorganic addition salts of said compounds which do not impair the beneficial efficacy of the methyl esters.

In addition, as an active ingredient included in the composition of the present invention, pharmaceutically acceptable salts thereof, as well as solvates such as hydrates that can be prepared therefrom, and possible derivatives and isomers, preferably optical isomers, are included without limitation. do. Hydrates, solvates, derivatives and isomers of 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester can be prepared using methods known in the art to form 4-benzoxazolecarboxylic acid, 2-( 2-hydroxyphenyl)-, methyl ester.

The antiviral composition according to the present invention is a compound of Formula 1, 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or a derivative as an active ingredient, and the composition may be a pharmaceutical composition, a cosmetic composition, a composition for external application for skin, a health functional food, or a quasi-drug composition, and these may be injections, solutions, tablets, capsules, ointments, ointments, creams, Essence, foam, lotion, nourishing lotion, soft water, softening lotion, pack, emulsion, makeup base, soap, detergent, bath agent, sun cream, sun oil, suspension, emulsion, paste, gel, lotion, powder, soap, surfactant It may have a formulation selected from the group consisting of containing cleansing, oil, foundation, powder foundation, emulsion foundation, wax foundation, patch and spray. For example, the composition may be a pharmaceutical composition having antiviral efficacy.

In the present invention, "antiviral" efficacy may be, for example, prevention, treatment, or improvement of enterovirus infection, but is not limited thereto, and includes any activity that prevents, treats or ameliorates general viral infection.

"4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester" contained in the composition of the present invention, a pharmaceutically acceptable salt, hydrate, solvate, isomer (preferably, optical isomer) thereof ) or derivatives may inhibit virus proliferation or growth.

The "inhibition of virus proliferation" means inhibiting S-adenosylhomocysteine (hereinafter referred to as SAH) hydrolase in the body or in vitro, such as inhibiting viral RNA polymerase (polymerase) of the virus in vivo or in vitro It may include any mechanism that inhibits proliferation.

The "inhibition of virus growth" refers to inhibiting the attachment of a virus to a host cell, inhibiting the initiation of invasion into the host cell, attacking the invading virus and promoting its degradation, inhibiting the proliferation of the virus, and Similarly, it may include both mechanisms that lower the activity of the virus in the cell and suppress the infectivity.

Said 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, a pharmaceutically acceptable salt, hydrate solvate, isomer (preferably optical isomer) or derivative thereof is a virus, in particular an enterovirus. Because it inhibits proliferation and exhibits excellent antiviral efficacy, enterovirus infection and related diseases, such as enteritis, hand, foot and mouth disease, stomatitis, stomatitis herpes, polio, spinal polio, meningitis, infantile aseptic meningitis, encephalitis One or more diseases selected from the group consisting of can be prevented, improved, alleviated or treated.

In a specific example according to the present invention, it was confirmed that 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester exhibited more than twice the antiviral activity compared to the control group. Therefore, as an active ingredient according to the present invention, a claim comprising 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, a pharmaceutically acceptable salt, hydrate, solvate, derivative or isomer thereof The virus composition exhibits excellent virus proliferation inhibitory activity, and is therefore useful as a drug for preventing or treating a viral infection.

Accordingly, another embodiment of the present invention relates to a composition for preventing or treating enterovirus infection, comprising the compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof. . The infection may be one or more selected from the group consisting of enteritis, hand, foot and mouth disease, stomatitis, herpetic stomatitis, polio, spinal polio, meningitis, infantile aseptic meningitis, and encephalitis. The composition may be a pharmaceutical composition, a health functional food, a cosmetic composition, or a quasi-drug composition.

The 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, pharmaceutically acceptable salt, hydrate solvate, isomer (preferably However, the effective amount of the optical isomer) or derivative will vary depending on the form in which the pharmaceutical composition having antiviral activity is commercialized, the method in which the compound is applied to the body, and the length of time it stays in the body. For example, when the pharmaceutical composition having the antiviral activity is commercialized as a drug for the treatment of anterovirus infection, it may include a pharmaceutically acceptable salt, hydrate solvate, isomer (preferably optical isomer) or derivative thereof. There will be. Although not limited thereto, the antiviral composition according to the present invention includes 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, pharmaceutically acceptable salts, hydrates, solvates, isomers ( Preferably, the optical isomer) or derivative based on the total weight of the pharmaceutical composition having the antiviral activity, 0.0001 to 15% by weight, for example, 0.001 to 10% by weight, for example 0.001 to 5% by weight. In addition, when the pharmaceutical composition having antiviral activity according to the present invention contains 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, and methyl ester in the form of an extract and/or fraction containing them, the Based on the total weight of the pharmaceutical composition having antiviral activity, 0.001 to 50% by weight, for example, 0.01 to 30% by weight, or 0.1 to 10% by weight, for example, 1 to 5% by weight may be included. However, since the above ratio varies depending on the dosage form in which the pharmaceutical composition of the present invention is prepared, its specific application site (face, neck, etc.) or its desired application amount, the above ratio limits the scope of the present invention in any aspect. should not be construed as

The composition having antiviral activity according to the present invention may be a pharmaceutical composition. The pharmaceutical composition having antiviral activity according to the present invention is 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, a pharmaceutically acceptable salt, hydrate, solvate, derivative or isomer thereof. In addition, a cosmetically acceptable carrier or additive may be included. For example, fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, emulsifiers, fillers, sequestering agents, chelating agents, preservatives, Vitamins, blocking agents, wetting agents, oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, etc., but are not limited thereto, and may include all known substances applicable to the pharmaceutical composition. In addition, the pharmaceutical composition having antiviral activity according to the present invention may be formed in one or more formulations selected from the group consisting of injections, ointments, external solutions, patches, capsules, and tablets, but is not limited thereto, and all known formulations can be formed. On the other hand, when the composition having antiviral efficacy of the present invention having the above formulation is formed in an ointment, paste, cream or gel state, animal oil, vegetable oil, wax, paraffin, starch, gum tragacanth, cellulose derivative , polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, and the like may be further included. When the antiviral composition according to the present invention is formed in the form of a solution or emulsion, it may further include a solvent, a solubilizer, an emulsifier, and the like. The solvent, solubilizer, and emulsifier include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, sorbate. fatty acid esters of heartburn, and the like. When the antiviral composition according to the present invention is formed in a suspension form, a liquid diluent such as water, ethanol, propylene glycol, etc., ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, etc. Suspension agent , microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, gum tragacanth and the like may be further included.

The pharmaceutical composition according to the present invention may be used alone or in combination with other pharmaceutically active compounds. The pharmaceutical composition may be in the form of a known oral or parenteral administration formulation. Accordingly, the antiviral composition of this embodiment is a filler in addition to the 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, a pharmaceutically acceptable salt, hydrate, solvate, derivative or isomer thereof. , extenders, binders, wetting agents, disintegrants, diluents such as surfactants, excipients and/or carriers may be further included. For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose, lactose. , or gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration may include various excipients, for example, wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition having antiviral efficacy according to the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral or parenteral administration (intraperitoneal, rectal, intravenous, intramuscular or subcutaneous injection, or transdermal administration). In this case, the parenteral route is preferably transdermal administration, and among them, it may be topical application. The preferred dosage of the antiviral composition according to the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. For example, for a desirable effect, the antiviral pharmaceutical composition according to the present invention may be administered in an amount of 0.1 to 100 mg/kg once to several times a day, but is not limited thereto. The parenteral administration dose of the antiviral pharmaceutical composition according to the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but in the case of external application, 1.0 to 3.0 ml per day is administered once to It can be applied by dividing it into several times, but is not limited thereto.

When the antiviral composition according to the present invention is a cosmetic composition, for example, a cleansing foam, cleansing cream, or cleansing water, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl Taurate, sarcosinate-based compound, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, ethoxylated glycerol fatty acid ester, etc. may be further included. .

The 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester (compound having Formula 1) may include purchased ones, those isolated from microorganisms, synthesized ones, and the like, For example, it may be extracted from microorganisms and separated, for example, isolated from a Streptomyces sp. strain. Specifically, the compound may be isolated from a Streptomyces sp. strain or a culture of the strain. The Streptomyces sp. strain may be, for example, Streptomyces griseolus SCO-774 having an accession number KCTC14340BP.

The Streptomyces griseolus ( Streptomyces griseolus ) SCO-774 strain is a strain having 4-benzoxazole carboxylic acid, 2- (2-hydroxyphenyl) -, methyl ester producing ability, 4-benzoxazole carboxylic acid, 2 -(2-hydroxyphenyl)-, is the first strain confirmed to have the ability to produce methyl ester. The composition having antiviral activity according to the present invention is at least one selected from the group consisting of the cells of the strain, the culture of the strain, the lysate of the strain, the extract of the cell, the extract of the culture, and the extract of the lysate. may include.

The compound may be obtained by fractional extraction of the strain or a culture of the strain. As a method of extracting the compound having Formula 1 from the Streptomyces sp. strain having the antiviral effect, a known microbial extraction method may be used without limitation. In addition, the extraction solvent used for extraction may also be extracted by appropriately selecting a solvent known in the art according to the characteristics of the microorganism, including water or organic solvent, for example, water, ethyl acetate, methanol, ethanol, propanol , isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and may be extracted with one or more solvents selected from the group consisting of dichloromethane, for example, may be extracted with an ethyl acetate solvent.

The compound may be obtained from a small fraction obtained by fractionally extracting the strain or a culture of the strain to obtain a fractional extract, and further fractionating the fractionated extract. Specifically, the compound may be obtained from a small fraction obtained by fractionally extracting the strain or a culture of the strain to obtain an organic solvent fraction extract from the organic solvent layer, and further fractionating the organic solvent fraction extract. For example, the compound is a small fraction obtained by fractionally extracting the strain or a culture of the strain to obtain an organic solvent fraction extract from the organic solvent layer, and further fractionating the organic solvent fraction extract using reverse phase column chromatography. may be obtained from A small fraction of the extract may be prepared using a fractionation solvent capable of obtaining a large amount of 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester as active ingredients, for example, water and A fraction fractionated with a mixed solvent of an alcohol (eg, a lower alcohol having 1-3 carbon atoms), for example, may be water and a methanol fraction. For example, the small fraction may be obtained by further fractionating the fractional extract with 20, 40, 50, 60, 70, 80, or 100% solution of methanol.

The compound may be obtained by isolating and purifying the small fraction. Specifically, the compound may be obtained by separating and purifying the small fraction by, for example, HPLC. That is, the compound may be obtained from a second small fraction obtained by fractionally extracting the strain or a culture of the strain, and separating and purifying a small fraction obtained by further separating the fractional extract. The fraction, subfraction, or secondary subfraction may contain useful secondary metabolites, including Formula 1, to exhibit excellent antiviral effects.

Another example of the present invention is a strain of Streptomyces having an antiviral effect, a culture of the strain, a lysate of the strain, an extract of the cell, an extract of the culture, and an extract of the lysate. It relates to an antiviral composition comprising at least one selected from. The extract of the culture may be a fractional extract of the culture. The composition may include a fractional extract of the culture. The extract of the culture may include an organic solvent layer, a fraction obtained by separating the organic solvent layer, or both of the fractional extracts of the culture. The dried product and the concentrate mean obtained by drying or concentrating the culture in a conventional manner.

The composition may include the compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof. The compound having Formula 1 may be isolated from the Streptomyces sp. strain or a culture of the strain. The strain may be isolated from Antarctic marine sediment. For example, the compound having Formula 1 may be isolated from the extract of the SCO774 strain of the genus Streptomyces. As an example, the SCO774 strain of Streptomyces or a culture of the strain may be isolated from an extract extracted with ethyl acetate.

Another example of the present invention relates to a method for obtaining Formula 1 comprising the step of obtaining an extract by adding ethyl acetate to Streptomyces sp.,. The method may further include the step of removing the organic solvent from the extract. As an example, the strain may be a Streptomyces sco774 strain having accession number KCTC14340BP.

The composition may have antiviral efficacy against enterovirus. 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester of Formula 1 isolated from the Streptomyces sp. strain has antiviral efficacy, specifically, excellent antiviral efficacy against anterovirus appeared to exist. The 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester can prevent, treat, and improve various infections that can be caused by anteroviruses by inhibiting the growth of anteroviruses.

Accordingly, another example of the present invention is a Streptomyces strain having an antiviral effect, a culture of the strain, a lysate of the strain, an extract of the cell, an extract of the culture, and an extract of the lysate. It relates to a composition for preventing or treating enterovirus infection, comprising at least one selected from the group consisting of. For example, the strain may be a Streptomyces sco774 strain having an accession number KCTC14340BP.

The infection may be one or more selected from the group consisting of enteritis, hand, foot, and mouth disease, stomatitis, stomatitis herpes, polio, spinal polio, meningitis, and encephalitis.

The culture may include a culture medium with a cell-free culture, or a Streptomyces sp. strain, including the culture of the strain and not including the strain. The culture medium may be a known appropriate medium for culturing the strain, but in order to provide optimal conditions for the production of useful secondary metabolites, the culture medium is Marine broth (BD), SYP, seawater contained culture solution (SYP SW: 10 g/L starch, 2 g/L yeast extract, 4 g/L peptone) and the like. The medium form of the marine broth may be both a solid medium and a liquid medium, preferably a liquid medium. The SYP SW medium may be both a solid medium and a liquid medium, preferably a liquid medium. The culture period for obtaining a culture may be about 4 days or more, for example, 4 to 20 days, for example, 6 to 15 days in which the production of effective secondary metabolites is active.

As the medium for the marine broth, starch (Soluble starch), yeast extract (Yeast extract), peptone (Peptone), sea salt (sea salt) is used. In the case of solid medium, 4 g of starch, 1.6 g of yeast extract, 0.3 g of peptone, 7.2 g of agar, 13.9 g of sea salt per 400 mL of distilled water water) is added to 400 mL. For liquid medium, add 4L of distilled water to 40g of starch, 16g of yeast extract, 8g of peptone, and 139g of sea salt based on 4L.

Another example of the present invention is composed of a Streptomyces spp. strain having antiviral efficacy, a culture of the strain, a lysate of the strain, an extract of the cell, an extract of the culture, and an extract of the lysate It relates to a composition for preparing a compound having Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof, comprising at least one selected from the group.

Another example of the present invention is composed of a Streptomyces spp. strain having antiviral efficacy, a culture of the strain, a lysate of the strain, an extract of the cell, an extract of the culture, and an extract of the lysate It relates to a composition for preparing an antiviral composition comprising one or more selected from the group. The antiviral composition may be an antiviral composition against enterovirus.

Another example of the present invention relates to a method for producing 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester using a Streptomyces sp. strain. The preparation method, the step of obtaining an extract by adding an extraction solvent to the composition comprising the Streptomyces sp. strain; and isolating and purifying the extract. By the obtaining method according to the present invention, 4-benzoxazole carboxylic acid useful as an active ingredient of an antiviral composition, 2-(2-hydroxyphenyl)-, methyl ester can be easily obtained.

Alternatively, an example of the present invention comprises the steps of fractional extraction of a Streptomyces sp. strain or a culture solution of the strain with an organic solvent; And it relates to a method for preparing a compound having Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof, comprising the step of separating and purifying the fractionated extract by chromatography.

Alternatively, an example of the present invention relates to a method for preparing an antiviral composition, comprising the step of culturing a Streptomyces sp. strain having antiviral efficacy. The manufacturing method comprises the steps of fractionally extracting the culture of the strain; and separating the fractional extract. The preparation method may further include the step of isolating the fractional extract to obtain a compound having the formula (1).

Alternatively, an example of the present invention comprises the steps of fractional extraction of a Streptomyces sp. strain or a culture solution of the strain with an organic solvent; And comprising the step of separating and purifying the fractionated extract by chromatography, comprising the compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof, Preparation of an antiviral composition it's about how

The extraction solvent may be selected without limitation as long as it is a solvent capable of extracting 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester from Streptomyces sp. strain, but an organic solvent, for example, At least one organic solvent selected from the group consisting of ethyl acetate, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and dichloromethane may be used, for example, ethyl acetate can

The extraction may be replaced by a method using an extraction apparatus such as supercritical extraction, high pressure extraction or ultrasonic extraction, or a method using celite or polystyrene or polyamide adsorption resin, but is not limited thereto. The amount of solvent or extraction temperature at the time of extraction can be appropriately changed by those skilled in the art, but it is preferable to extract by adding 1 to 3 times the amount of the solvent by volume of the amount of the culture solution during extraction, and it is preferable to perform extraction at room temperature. The number of extractions is preferably 1 to 3 times, and drying under reduced pressure is preferably performed using a rotary vacuum evaporator, but is not limited thereto. The drying temperature under reduced pressure may be, for example, 20 to 40 °C, for example 30 °C, but is not limited thereto.

Additionally, in the method for preparing 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester according to the present invention, before obtaining an extract by adding the extraction solvent, the strain is obtained by culturing the Streptomyces strain. It may further comprise the step of preparing a composition comprising.

In addition, in the method for preparing 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester according to the present invention, an extract is obtained by adding the extraction solvent, and an extraction solvent, preferably an organic solvent, is obtained from the extract. It may further include the step of removing. Removal of the organic solvent may be performed using methods known in the art without limitation.

For the separation and purification method, a known method may be used without limitation, for example, chromatography may be used. The "chromatography" may be used without limitation chromatography according to size, charge, hydrophobicity or affinity, for example, flash chromatography (TLC), may be reversed-phase liquid chromatography. The chromatography may be performed using at least one selected from the group consisting of water and alcohol as a developing solvent.

Numerical values described in the present specification above should be interpreted as including equivalent ranges unless otherwise specified.

The composition for antiviral according to the present invention is a safe material with low cytotoxicity derived from a natural product, and can prevent, treat, and improve viral infection by effectively inhibiting virus proliferation. It can be usefully used in manufacturing.

1 is a view showing the morphology of a Streptomyces SCO774 strain in a marine solid medium according to an example of the present invention isolated from marine sediments. The back side of the plate is shown, and the lower left is an enlarged view of the strain part.

2 is a diagram showing the antiviral efficacy of each fraction by fractionating the extract of the SCO774 strain of Streptomyces genus according to an example of the present invention.

Figure 3 is a view showing the verification of the membrane protein production of the virus and the activity of the viral protein cleavage enzyme according to the fraction treatment of the SCO774 strain extract of Streptomyces genus according to an example of the present invention by western blot analysis.

4 is a view showing the HPLC separation result of the F6 fraction of the Streptomyces sp. strain extract according to an example of the present invention. After fractionation of the entire extract of the Streptomyces sp. strain according to an embodiment of the present invention, 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl )-, a chromatogram of pure separation of methyl ester. A peak in which 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester was separated at UV 254 nm was indicated by an arrow.

5 is a view showing the antiviral efficacy of the secondary fraction of the Streptomyces sp. strain extract according to an example of the present invention. The F6 fraction, which has excellent antiviral efficacy, was separated by HPLC, and the concentration-dependent effect of each separated material for inhibiting the growth of anterovirus was confirmed. It can be seen that the substance F6-7 has the best antiviral effect, and this substance was identified as 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester.

Figure 6a shows ¹ H NMR (300 MHz) data of 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, which is a compound isolated from a Streptomyces sp. strain according to an example of the present invention; It is a drawing.

6b is a view showing LC-MS results of 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, which are compounds isolated from a Streptomyces sp. strain according to an example of the present invention.

6c is a view showing the UV spectrum results of 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester, which are compounds isolated from a Streptomyces sp. strain according to an example of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

실시예 1. 균주 분리 및 동정Example 1. Isolation and identification of strains

(1) 해양퇴적토 내 균주의 분리(1) Isolation of strains in marine sediments

After drying the sediment collected from the Antarctic marine sediment sample on a clean bench, the dried matter is suspended in sterilized seawater and diluted by half, then ISP 1 SW (ISP 1 powder 1.6g/L, sea salt 34.74g/L, 1 Primary distilled water 400ml), ISP 4 SW (ISP 4 powder 3.7g/L, sea salt 34.74g/L, primary distilled water 400ml) inoculated on a solid medium, and inoculated with a single strain generated while culturing at 27℃ Marine broth A pure strain was isolated and selected by inoculation again on a solid medium. The isolated strain was named SCO774, and the SCO-774 strain had a morphological pattern similar to that of strains belonging to the genus Streptomyces sp. as shown in FIG. 1 . 1 is a view showing the morphology of SCO774 strain isolated from marine sediment in Marine solid medium, the upper left is the front side of the medium plate, the upper right is the back side of the medium plate, and the lower left is It is an enlarged view of the strain part.

(2) 균주의 동정(2) Identification of strains

For the species identification of the SCO774 strain isolated from marine sediment, 1 ml of the strain cultured at 27°C for 4 days in a marine broth liquid medium is taken and “Tissue Genomic DNA Isolation kit” (Cosmogenetech co, Ltd.. Seoul, South) Korea) to extract genomic DNA according to the manufacturer's protocol. For species analysis, PCR was performed using primers 518F and 805R for 16S rRNA gene amplification. The PCR product was purified using a “PCR purification kit” (Cosmogenetech co, Ltd. Seoul, South Korea), and the base sequence was analyzed using capillary electrophoresis (Applied Biosystems 3730XL). The primer sequences used for PCR are shown in Table 1.

종류Kinds	서열 (5’ -> 3’)sequence (5' -> 3')	서열번호SEQ ID NO:
518F518F	CCAGCAGCCGCGGTAATACCCAGCAGCCGCGGTAATAC	1 One
27F27F
AGAGTTTGATCMTGGCTCAGAGAGTTTGATCMTGGCTCAG	22
805R 805R		GACTACCAGGGTATCTAATCGACTACCAGGGTATCTAATC	33
1492R 1492R		TACGGYTACCTTGTTACGACTTTACGGYTACCTTGTTACGACTT	44

The 16S rRNA gene sequence obtained from the SCO-774 strain was compared with the information of the strains using BLAST search of the GenBank/EMBL/DDBJ database. As a result of identification, the SCO-774 strain of the present invention was identified as a Streptomyces griseolus strain belonging to the genus Streptomyces. The nucleotide sequence of the 16S rRNA genes of the SCO-774 strain of Streptomyces genus according to the present invention is shown in Table 2 (SEQ ID NO: 5):

구분division	16S rRNA16S rRNA	서열번호SEQ ID NO:
SCO-774SCO-774	AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATGTCGCAAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGTGGAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATGTCGCAAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGTGG	55

Streptomyces griseolus ( Streptomyces griseolus ) SCO774 strain was deposited with the Korea Research Institute of Bioscience and Biotechnology on October 21, 2020 and was given an accession number KCTC14340BP.

실시예 2. 균주 추출물의 항바이러스 효능 검증 (1)Example 2. Verification of antiviral efficacy of strain extract (1)

(1) 균주 추출물의 1차 분획(1) Primary fraction of strain extract

The Streptomyces griseolus SCO774 strain was placed in a 1 liter culture solution (10 g/L starch, 2 g/L yeast extract, 4 g/L peptone) containing seawater in a 2.5L culture vessel at 27°C, 130 for 7 days. Cultured under rpm conditions. Into the Streptomyces SCO774 strain culture medium, 1 L of ethyl acetate was added to separate the two layers according to the polarity, and an ethyl acetate layer was obtained therefrom. Thereafter, the organic solvent was removed from the ethyl acetate layer using a vacuum rotary concentrator to obtain a concentrated ethyl acetate extract.

The ethyl acetate extract (4.0 g) was fractionated with a mixed solvent of water and methanol, and a total of 8 fractions (F1 to F7) were fractionated by reverse phase column chromatography using C-18 silica following a stepwise gradient with a methanol concentration of 20 to 100%. and FW).

(2) 항바이러스 효능 검증(2) Verification of antiviral efficacy

The antiviral efficacy and cytotoxicity of the Streptomyces griseolus SCO774 strain extract was confirmed using HeLa cells. Fractions F6 and F7 of the Streptomyces griseolus SCO774 strain extract were added to the cell culture medium at a concentration of 0.01 ug/ml, 0.1 ug/ml, 1ug/ml, or 10ug/ml, respectively, to HeLa cells cultured in a 96-well cell culture dish After infecting with Entero virus at 10 ⁵ pfu/ml for 18 hours, 10 μl of Cell Counting Kit 8 (CCK-8), a reagent for detecting proliferation of cells, was added and incubated for 2 hours more. . Then, by measuring the absorbance at 450 nm using a Microplate reader (Molecular device, USA), the survival of the cells was confirmed to confirm the antiviral efficacy. The results of confirming the antiviral efficacy of the Streptomyces griseolus SCO774 strain extract are shown in FIG. 2 . In FIG. 2, negative is a negative control group not infected with the virus, and positive is a positive control group infected with only the virus without drug treatment. As shown in FIG. 2 , the fraction of the SCO774 strain extract had an enterovirus growth inhibitory effect and showed an antiviral effect on enterovirus.

In order to find out more about the antiviral effect of the F6 fraction, which showed the best antiviral efficacy, the activity of viral protein cleavage enzymes related to virus proliferation and the production of membrane proteins were verified by western blot analysis.

Specifically, the F6 fraction and virus were treated on HeLa cells for 18 hours, and then the cells were lysed using PBS lysis buffer (1x phosphate buffer saline, 1% Triton-X 100) to extract proteins. The extracted protein was heated at 98°C for 10 minutes, loaded on a 10% SDS-PAGE gel, and electrophoresed at 100 volt for 3 hours. Protein transfer was performed on the electrophoresed gel using PVDF membrane. After transfer, the membrane was blocked with 5% skim milk, and the anti-enterovirus VP1, eIF4G1, and GAPDH antibodies were reacted at 4°C for 18 hours, followed by confirmation using ECL solution.

As shown in FIG. 3 , the production of membrane proteins indicating virus proliferation was decreased according to the concentration of F6 fraction treatment, and in particular, the destruction of eIF4G1, a transcription initiator of cells cleaved by a virus-producing protein cleavage enzyme, which is evidence of direct virus proliferation, was It can be seen that the excellent antiviral effect is suppressed.

실시예 3. 균주 추출물의 항바이러스 효능 검증 (2)Example 3. Verification of antiviral efficacy of strain extract (2)

(1) 균주 추출물의 2차 분획(1) Secondary fraction of strain extract

Among the fractions of the Streptomyces griseolus SCO774 strain extract showing excellent antiviral efficacy in Example 2, the organic solvent was removed from the F6 fraction, the sixth fraction fractionated with 80% MeOH, to obtain 184.9 mg of a residue. The residue was dissolved in 6ml methanol, and high performance liquid chromatography (HPLC) was performed on the solution to separate 7 substances. The HPLC separation result of the F6 fraction of the Streptomyces griseolus SCO774 strain extract is shown in FIG. 4 . The conditions of the high performance liquid chromatography used were as follows:

- Column: Phenomenex Luna C-18 (2), 250×10.0 mm, 5 mm, 100 Å, UV = 254 nm)

- Solvent conditions: acetonitrile (CH ₃ CN): water (H 2 O) = 30: 70

- Solvent rate: 2.0 mL/min

(2) 항바이러스 효능 검증(2) Verification of antiviral efficacy

The antiviral efficacy and cytotoxicity of (2) of Example 2 and the secondary fraction of the Streptomyces griseolus SCO774 strain extract were confirmed and shown in FIG. 5 . 5, the F6-7 peak (4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester) among the secondary fractions of the SCO774 strain extract showed the highest antiviral effect.

The F6-7 peak in the F6 fraction of the Streptomyces griseolus SCO774 strain extract is indicated by an arrow in FIG. 4 . The F6-7 fraction was isolated at a retention time of 12.5 to 13 minutes.

실시예 4. 화합물의 동정Example 4. Identification of compounds

The active ingredient of the F6-7 fraction showing the highest antiviral efficacy in Example 3 was identified through NMR, LC-MS and ultraviolet spectrum (IR), and the results are shown in FIGS. 6a, 6b and 6c, respectively.

As shown in the ¹ H NMR result of Figure 6a, It can be seen that ¹ H NMR contains a benzene ring between 6.0 ppm and 8.0 ppm, and it can be seen that 1,2 substituents are included through the coupling pattern.

In addition, as shown in the LC-MS result of FIG. 6b , it could be confirmed that [M+H] was 270 by mass, and therefore it was a natural product with a molecular weight of 269, and finally, through the UV spectrum comparative analysis of FIG. 6c It was confirmed that 4-benzoxazole carboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester (Formula 1) in the SCO-774 strain extract. Figure 6c is the UV form of 4-benzoxazolecarboxylic acid, 2-(2-hydroxyphenyl)-, methyl ester.

In this specification, details that can be sufficiently recognized and inferred by those of ordinary skill in the art of the present invention are omitted, and the technical spirit or essential configuration of the present invention is changed in addition to the specific examples described in the present specification More various modifications are possible within the range not to do so. Therefore, the present invention may be practiced in a manner different from that specifically described and illustrated in this specification, which will be understood by those of ordinary skill in the art of the present invention.

[Accession number]

Name of depositary institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14340BP

Deposit date: 20201021

Claims

Streptomyces strain having antiviral efficacy, the culture of the strain, the lysate of the strain, the extract of the cell, the extract of the culture, and the extract of the lysate comprising at least one selected from the group consisting of , antiviral composition.
The composition of claim 1, wherein the composition has antiviral efficacy against enteroviruses.
The composition of claim 1, wherein the extract of the culture is a fractional extract of the culture.
According to claim 3, wherein the fractional extract is water, ethyl acetate, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and at least one selected from the group consisting of dichloromethane The composition, which is extracted with a solvent.
The composition of claim 1, wherein the extract of the culture includes an organic solvent layer, a fraction obtained by separating the organic solvent layer, or both of the fractional extracts of the culture.
The composition of claim 1, wherein the extract of the culture comprises a second fraction obtained by separating and purifying the first fraction obtained by separating the fractional extract of the culture.
The composition according to any one of claims 1 to 6, wherein the composition comprises a compound having the following formula (1), a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof:

[Formula 1]
The composition of claim 1, wherein the strain is Streptomyces SCO774 having accession number KCTC14340BP.
Streptomyces strain having antiviral efficacy, the culture of the strain, the lysate of the strain, the extract of the cell, the extract of the culture, and the extract of the lysate comprising at least one selected from the group consisting of , a composition for preventing or treating enterovirus infection.
The composition of claim 9, wherein the infection is at least one selected from the group consisting of enteritis, hand, foot and mouth disease, stomatitis, herpetic stomatitis, polio, spinal polio, meningitis, and encephalitis.
The composition of claim 9, wherein the composition is a pharmaceutical composition, a health functional food, or a quasi-drug composition.
The composition of claim 9, wherein the composition comprises a compound having the following formula (1), a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof:

[Formula 1]
The composition of claim 9, wherein the strain is Streptomyces sco774 having accession number KCTC14340BP.
Streptomyces spp. strain having antiviral efficacy.
The strain according to claim 14, wherein the strain has antiviral efficacy against enteroviruses.
The strain according to claim 14, wherein the strain produces a compound having the following formula (1):

[Formula 1]
The strain according to claim 14, wherein the strain is Streptomyces sco774 having accession number KCTC14340BP.
A method for producing an antiviral composition comprising the step of culturing a Streptomyces sp. strain having antiviral efficacy.
The method of claim 18, further comprising: fractionally extracting the culture of the strain; and

Further comprising the step of isolating the fractional extract, the manufacturing method.
The method according to claim 19, further comprising the step of obtaining a compound having the following formula (1) by separating the fractionated extract:

[Formula 1]
The method according to claim 19, wherein the composition has antiviral efficacy against enteroviruses.
The method according to claim 19, wherein the strain is Streptomyces sco774 having accession number KCTC14340BP.
Streptomyces genus strain having an antiviral effect, the culture of the strain, the lysate of the strain, the extract of the microbial cell, the extract of the culture, and the extract of the lysate containing at least one selected from the group consisting of extracts , A composition for preparing a compound having the following formula (1):

[Formula 1]
An antiviral composition comprising a compound having the following formula (1), a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof:

[Formula 1]
25. The composition of claim 24, wherein the compound is isolated from a Streptomyces sp. strain or a culture of the strain.
The composition of claim 25, wherein the compound is obtained by fractional extraction of the strain or a culture of the strain.
The method of claim 26, wherein the fractional extraction is at least one selected from the group consisting of water, ethyl acetate, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, methylene chloride, hexane, cyclohexane, and dichloromethane. A composition using a solvent.
The composition of claim 25, wherein the compound is obtained from a small fraction obtained by fractionally extracting the strain or a culture of the strain, and further separating the fractional extract.
The composition of claim 25, wherein the compound is obtained from a second small fraction obtained by fractionally extracting the strain or a culture of the strain, and separating and purifying a small fraction obtained by further separating the fractional extract.
The composition of claim 24, wherein the composition has antiviral efficacy against enteroviruses.
A composition for preventing or treating enterovirus infection, comprising a compound having the following formula (1), a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof:

[Formula 1]
The composition of claim 31, wherein the infection is at least one selected from the group consisting of enteritis, hand, foot and mouth disease, stomatitis, stomatitis herpeticum, polio, spinal polio, meningitis, infantile aseptic meningitis, and encephalitis.
The composition of claim 31, wherein the composition is a pharmaceutical composition, a health functional food, or a quasi-drug composition.
Fractional extraction of a Streptomyces sp. strain or a culture solution of the strain; and

isolating and purifying the fractional extract,

A method for preparing a compound having the following formula (1), a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof:

[Formula 1]
35. The method of claim 34, further comprising the steps of: fractionally extracting the Streptomyces sp. strain or a culture solution of the strain to obtain a fractionated extract in an organic solvent layer;

further fractionating the fractionated extract to obtain small fractions; and

A manufacturing method comprising the step of isolating and purifying the small fraction.
Fractional extraction of a Streptomyces sp. strain or a culture solution of the strain; and

isolating and purifying the fractional extract,

A compound having the following formula (1), including a pharmaceutically acceptable salt, hydrate, solvate, isomer, or derivative thereof,

Method for preparing an antiviral composition:

[Formula 1]
37. The method of claim 36, further comprising the steps of: fractionally extracting the Streptomyces sp. strain or a culture solution of the strain to obtain a fractional extract in an organic solvent layer;

further fractionating the fractionated extract to obtain small fractions; and

A manufacturing method comprising the step of isolating and purifying the small fraction.