Antiviral agent comprising ecklonia cava extract or phlorotannin as active ingredient

Abstract

The present invention relates to an antiviral composition comprising an Ecklonia cava extract, a fraction of the Ecklonia cava extract, or phlorotannin as an active ingredient. More specifically, the Ecklonia cava extract, the fraction of the Ecklonia cava extract, or the phlorotannin of the present invention effectively inhibits the proliferation and replication of flavivirus and thus may be usefully employed as an antiviral agent, a pharmaceutical composition, a health functional food composition, a quasi-drug composition, or a feed additive composition for anti-flavivirus activity.

Description

Antiviral agent containing perilla extract or phlorotannin as an active ingredient

[0001] The present invention relates to an antiviral agent comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0002] Flaviviruses are RNA viruses belonging to the Flaviviridae family, which includes many pathogenic viruses such as dengue fever, Zika virus, yellow fever, Japanese encephalitis, and West Nile virus.

[0003] Flaviviruses are spherical particles, approximately 40-60 nm in diameter, and enveloped viruses. Their genome consists of single-stranded RNA, approximately 11 kb in length. The genome consists of a single open reading frame, which encodes structural and nonstructural proteins.

[0004] Flaviviruses are primarily transmitted by mosquitoes and can infect humans and animals. They primarily infect and multiply in cells within blood and tissues, causing a variety of diseases. Flaviviruses can infect a wide range of species and cause a variety of symptoms. For example, dengue fever can cause high fever, muscle pain, and bleeding, while yellow fever can cause fever, liver failure, and bleeding. West Nile virus can cause neurological symptoms such as encephalitis.

[0005] Zika virus was first isolated from monkeys in Uganda in 1947, and the first human infection case was reported in Tanzania in 1953. The first outbreak occurred in Yap Island in the Federated States of Micronesia in 2007, and it began to receive attention in 2013 when a large-scale outbreak occurred in French Polynesia. In 2015, the number of Zika virus infected patients began to rapidly increase in Central and South American countries, including Brazil, and neurological complications such as microcephaly and Guillain-Barré syndrome were reported, leading the WHO to declare a Public Health Emergency of International Concern (PHEIC) on February 1, 2016. Since then, the number of countries with Zika virus outbreaks has continued to expand, mainly in Central and South America, and the number of affected areas and patients is also gradually increasing in Southeast Asian countries.

[0006] While Zika virus symptoms are generally mild, a potentially life-threatening threat is that Zika virus infection during pregnancy can cause congenital brain malformations, including microcephaly. Microcephaly has been reported in 29 countries, including Brazil and Colombia, with Brazil reporting the highest number of cases, with over 2,000. Other congenital Zika virus-related symptoms include craniofacial malformations, ventriculomegaly, motor developmental delays, hearing and visual impairments, eye defects, arthrogryposis, and hearing loss. While complications, such as Guillain-Barré syndrome and neurological complications, can be extremely life-threatening, there is currently no vaccine or treatment for Zika virus infection.

[0007] Against the backdrop of the above, research on flaviviruses is still ongoing, and the development of effective preventive and therapeutic methods is an important task.

[0008] Under the aforementioned technical background, the inventors of the present invention, while conducting intensive research to develop a novel substance having antiviral activity against flaviviruses, confirmed that phlorotannins derived from seaweed or extracts of kelp have antiviral activity, and completed the present invention.

[0009] The purpose of the present invention is to provide an antiviral agent, pharmaceutical composition, health functional food composition, quasi-drug composition, feed additive composition, or a method for preventing, improving, or treating flavivirus infectious diseases, which comprises a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0010] To achieve the above purpose, an antiviral agent comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient is provided.

[0011] The above phlorotannin may be at least one selected from the group consisting of phloroglucinol, eckol, 8,8'-bieckol, eckstolonol, dieckol, and phlorofucofuroeckol A.

[0012] The above phlorotannin may be diechol.

[0013] The above extract may be extracted using water, a lower alcohol of C1 to C4, or a mixed solvent thereof.

[0014] The above phlorotannin may be isolated from a fraction of a perilla extract.

[0015] The above fraction may be an ethyl acetate fraction.

[0016] The above antiviral agent may have antiviral activity against flaviviruses.

[0017] The above antiviral agent may have antiviral activity against one or more flaviviruses selected from the group consisting of Zika virus, Dengue virus, Yellow fever virus, West Nile virus, and Japanese encephalitis virus.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating flavivirus infection, comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0019] In addition, a health functional food composition for preventing or improving flavivirus infection disease is provided, which comprises a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0020] In addition, a pharmaceutical composition for preventing or improving flavivirus infection disease is provided, which comprises a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0021] In addition, a feed additive composition for preventing or improving flavivirus infection disease is provided, which comprises a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0022] In addition, a method for preventing, improving or treating a flavivirus infection disease is provided, comprising a step of administering a composition containing a perilla extract, a fraction of the extract or phlorotannin to a subject in need thereof.

[0023] The present invention relates to an antiviral agent comprising a perilla frutescens extract, a fraction thereof, or phlorotannin as an active ingredient. In one embodiment of the present invention, the phlorotannin, diechol, has an effect of inhibiting the mRNA expression of the Zika virus NS1 protein. In addition, it significantly reduces the mRNA expression of IFIT1 and IFIT2, which are important elements of the antiviral immune response in host cells, and has an effect of inhibiting Zika virus plaque formation. In addition, the perilla frutescens extract or a fraction thereof has an effect of inhibiting the formation of Zika virus plaques, inhibiting the mRNA expression of the NS1 protein in a concentration-dependent manner, and significantly reducing the expression of TNF-α, IFIT1, and IFIT2 increased by Zika virus infection, and therefore can be usefully used as an antiviral agent for antiviral activity, a pharmaceutical composition, a health functional food composition, a quasi-drug composition, a feed additive composition, or a method for preventing, improving, or treating flavivirus infectious diseases.

[0024] Figure 1 shows the Zika virus infection inhibition efficacy of Dieckol and Zeaxanthin.

[0025] Figure 2 shows the inhibitory efficacy of Dieckol on the expression of inflammatory factors caused by Zika virus infection.

[0026] Figure 3 is an image and graph showing the inhibition effect of Zika virus plaque formation by Dieckol.

[0027] Figure 4 shows the cell viability of Ectocarcinoma extract (ECE).

[0028] Figure 5 is an image showing the plaque formation inhibitory effect of ECE extract.

[0029] Figure 6 is a graph showing the plaque formation inhibitory effect of ECE extract.

[0030] Figure 7 shows that ECE inhibits the mRNA expression of NS1 protein in a concentration-dependent manner.

[0031] Figure 8 shows the inhibitory effect of ECE on the expression of inflammatory factor TNF-α caused by Zika virus infection.

[0032] Figure 9 shows the inhibitory effect of ECE on the expression of inflammatory factor IFIT1 caused by Zika virus infection.

[0033] Figure 10 shows the inhibitory effect of Ecklonia cava extract (ECE) on the expression of inflammatory factor IFIT2 caused by Zika virus infection.

[0034] Hereinafter, the present invention will be described in more detail.

[0035]

[0036] The present invention provides an antiviral agent comprising a perilla leaf extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0037] In the present invention, the antiviral agent may have antiviral activity against flavivirus.

[0038] The term "antiviral" as used in the present invention refers to an activity that directly or indirectly interferes with or inhibits one or more steps of the viral infection cycle, specifically, the viral infection cycle consisting of virus penetration, virus replication, virus assembly, and virus release in a host cell. This includes any effect that non-specifically inhibits the increase in virus titer within a virus-infected organism, or non-specifically reduces the virus titer level.

[0039] In the present invention, the antiviral agent can exhibit a killing and proliferation inhibition effect on various species of the genus Flavivirus.

[0040] In the present invention, the antiviral agent may have antiviral activity against one or more flaviviruses selected from the group consisting of Zika virus, Dengue virus, Yellow fever virus, West Nile virus, and Japanese encephalitis virus.

[0041] In one embodiment of the present invention, the antiviral agent may have antiviral activity against Zika virus, but is not limited thereto.

[0042] In the present invention, Ecklonia cava can be purchased commercially or harvested directly.

[0043] In the present invention, the extract of the perilla frutescens may be obtained by extraction, fractionation, and separation from nature using extraction, fractionation, and separation methods known in the art. The extract defined in the present invention is obtained by extraction treatment from perilla frutescens using an appropriate solvent, and is a concept that includes, for example, a crude extract of perilla frutescens, a polar solvent-soluble extract, or a non-polar solvent-soluble extract.

[0044] In the present invention, the perilla extract can be extracted using various extraction solvents and extraction methods.

[0045] In the present invention, the kelp extract may be extracted with water, a lower alcohol having C1 to C4, or a mixed solvent thereof. The alcohol having C1 to C4 may be methanol, ethanol, propanol, isopropanol, butanol, etc. The extraction solvent may be a mixed solvent of water and the alcohol having C1 to C4. Ethanol may be preferably used, and more preferably 50 to 100% (v / v) ethanol may be used, more preferably 60 to 90% (v / v), 65 to 80% (v / v), 75 to 85% (v / v), and most preferably 70% (v / v) ethanol may be used, but is not limited thereto.

[0046] In addition, methods such as hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and pressing can be used as extraction methods.

[0047] After obtaining an extract using water or an organic solvent as described above, a liquid can be obtained by cooling, heating, and filtering at room temperature using a conventional method known in the art, or the solvent can be additionally evaporated, spray-dried, or freeze-dried.

[0048] The above-mentioned manufactured kelp extract can be obtained by further distillation and then fractionation, and specifically, it can be obtained as a further purified fraction using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. Preferably, fractionation can be performed using silica gel column chromatography, but the present invention is not limited thereto, and any method that can obtain an additional fraction using an appropriate solvent can be used.

[0049] In one embodiment of the present invention, the fraction may be, but is not limited to, an ethyl acetate fraction of a perilla ethanol extract.

[0050] In the present invention, the phlorotannin is a polyphenolic substance and a phloroglucinol derivative, which is a component contained in large quantities in brown algae. Representative substances of the phlorotannin include eckol, dieckol, and triphlorethol A, and the components have excellent effects such as antioxidant, anti-inflammatory, UV protection, whitening, antihypertensive, and anti-diabetic.

[0051] Phlorotannin included in the composition of the present invention has antiviral activity against flavivirus.

[0052] In one embodiment of the present invention, the phlorotannin may be at least one selected from the group consisting of phloroglucinol, eckol, 8,8'-bieckol, eckstolonol, dieckol, and phlorofucofuroeckol A, preferably eckol, 8,8'-bieckol, eckstolonol, dieckol, or phlorofucofuroeckol A, and more preferably dieckol, but is not limited thereto.

[0053] The above diet is C 36 H 22 O 18 It has the molecular formula of , can be derived from seaweed, can be synthesized using commercially available reagents, can be obtained by extracting and separating from commercially available products, natural products, especially seaweed, etc., and can be used without limitation. The structural formula of the above diechol is Dibenzo[b,e][1,4]dioxin-1,3,6,8-tetrol,4-[4-[6-(3,5-dihydroxyphenoxy)-4,7,9-trihydroxydibenzo[b,e][1,4]dioxin-2-yl]oxy]-3,5-dihydroxyphenoxy]-, and the chemical formula is as follows.

[0054] <Chemical formula>

[0055]

[0056] In one embodiment of the present invention, diechol, a compound represented by the chemical formula above, has antiviral efficacy against Zika virus.

[0057] In one embodiment of the present invention, diechol reduced plaque formation in Zika virus-infected cells, and reduced plaque formation by about 50% and 40%, respectively, compared to the control group at concentrations of 25 and 50 μM, which are non-toxic to the cells.

[0058] In addition, in one embodiment of the present invention, diechol significantly reduced the expression of IFIT1 (Interferon-induced proteins with tetratricopeptide repeats 1) and IFIT2 (Interferon-induced proteins with tetratricopeptide repeats 2) in host cells. This suggests that diechol inhibits Zika virus replication.

[0059] Additionally, in one embodiment of the present invention, the antiviral efficacy of the extract of perilla leaves inhibited the mRNA expression of the Zika virus NS1 protein and reduced the plaque formation of the Zika virus.

[0060] Additionally, the extract of perilla leaves significantly reduced the expression of TNF-α increased by Zika virus infection and significantly reduced the expression of IFIT1 and IFIT2 in host cells increased by Zika virus infection.

[0061]

[0062] In addition, the present invention provides a pharmaceutical composition for preventing or treating flavivirus infection, which comprises a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0063] In the present invention, the composition may have antiviral activity against one or more flaviviruses selected from the group consisting of Zika virus, Dengue virus, Yellow fever virus, West Nile virus, and Japanese encephalitis virus. Preferably, the composition has antiviral activity against Zika virus, but is not limited thereto.

[0064] In one embodiment of the present invention, the composition can be used as a pharmaceutical composition for preventing, improving, and treating Zika virus infectious diseases.

[0065] The term "Zika virus infection disease" used in the present invention means any disease or pathologic condition that is caused, progresses, or worsens directly or indirectly by infection with Zika virus.

[0066] In one embodiment of the present invention, the phlorotannin may be at least one selected from the group consisting of phloroglucinol, eckol, 8,8'-bieckol, eckstolonol, dieckol, and phlorofucofuroeckol A, preferably eckol, 8,8'-bieckol, eckstolonol, dieckol, or phlorofucofuroeckol A, and more preferably dieckol, but is not limited thereto.

[0067] In one embodiment of the present invention, the disease or pathological condition may be at least one selected from the group consisting of, but is not limited to, fever, headache, arthralgia, conjunctivitis, maculopapular rash, microcephaly, Guillain-Barré syndrome, meningitis, thrombocytopenic purpura, leukopenia, acute disseminated encephalomyelitis, and acute myelitis.

[0068] The term "treatment" used in the present invention means suppressing the development of a disease, illness, or symptom; alleviating a disease, illness, or symptom; or eliminating a disease, illness, or symptom. The composition of the present invention functions to suppress the development of a disease or its symptoms caused by a viral infection, or to eliminate or alleviate the disease, illness, or symptom by enhancing the survival rate of Zika virus-infected cells and thereby suppressing the effects of viral infection. Therefore, the composition of the present invention may be a composition for treating a Zika virus infection disease or Zika virus infection symptom, such as a perilla leaf extract, a fraction of the extract, or diechol itself, or may be used as a therapeutic adjuvant to suppress viral activity when administered together with another antiviral composition. The terms "treatment" or "therapeutic agent" include the meaning of "therapeutic adjuvant" or "therapeutic adjuvant."

[0069] The composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. Furthermore, the composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. Suitable formulations known in the art are preferably those disclosed in the literature (Remington's Pharmaceutical Science, recently published by Mack Publishing Company, Easton PA). Carriers, excipients and diluents that may be included in the above composition 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, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the above composition, 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., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to 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 sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.In addition, non-aqueous solvents and suspending agents may be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may be used, such as witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0070] The term "administration" as used in the present invention means providing a given composition of the present invention to a subject by any suitable method.

[0071] The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the subject, the degree of disease, the drug form, the route and period of administration, and can be appropriately selected by a person skilled in the art.

[0072] The pharmaceutical composition of the present invention can be administered to a subject via various routes. Any route of administration is contemplated, including oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebrovascular injection.

[0073] The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers to improve or treat diseases.

[0074] The above composition may be formulated as a parenteral dosage form. The parenteral dosage form may be an injection or a topical agent for skin.

[0075] Topical skin preparations may be creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, medicated bandages, lotions, or combinations thereof.

[0076]

[0077] In addition, the present invention provides a health functional food composition for preventing or improving flavivirus infection disease, which comprises a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0078] In the present invention, 'health functional food' refers to a food having a biological regulation function such as disease prevention or improvement, biological defense, immunity, or recovery after illness, and must be harmless to the human body when consumed over a long period of time.

[0079] In one embodiment of the present invention, the phlorotannin may be at least one selected from the group consisting of phloroglucinol, eckol, 8,8'-bieckol, eckstolonol, dieckol, and phlorofucofuroeckol A, preferably eckol, 8,8'-bieckol, eckstolonol, dieckol, or phlorofucofuroeckol A, and more preferably dieckol, but is not limited thereto.

[0080] In one embodiment of the present invention, the perilla extract, a fraction of the extract, or diechol may be added to a health functional food for the purpose of antiviral activity.

[0081] The above antiviral may have antiviral activity against one or more flaviviruses selected from the group consisting of Zika virus, Dengue virus, Yellow fever virus, West Nile virus, and Japanese encephalitis virus.

[0082] In one embodiment of the present invention, the antiviral may have antiviral activity against Zika virus, but is not limited thereto.

[0083] When the perilla extract of the present invention, fractions thereof, or diechol are used as food additives, they may be added as is or used in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of active ingredients mixed may be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment).

[0084] There are no specific restrictions on the types of foods mentioned above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, including all health foods in the conventional sense.

[0085] The health beverage composition of the present invention may, like conventional beverages, contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates described above may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame.

[0086] In addition to the above, the composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may include fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination.

[0087]

[0088] In addition, the present invention provides a pharmaceutical composition for preventing or improving flavivirus infection, comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

[0089] The composition may have antiviral activity against one or more flaviviruses selected from the group consisting of Zika virus, Dengue virus, Yellow fever virus, West Nile virus, and Japanese encephalitis virus.

[0090] In one embodiment of the present invention, the flavivirus may have antiviral activity against Zika virus, but is not limited thereto.

[0091] In one embodiment of the present invention, the phlorotannin may be at least one selected from the group consisting of phloroglucinol, eckol, 8,8'-bieckol, eckstolonol, dieckol, and phlorofucofuroeckol A, preferably eckol, 8,8'-bieckol, eckstolonol, dieckol, or phlorofucofuroeckol A, and more preferably dieckol, but is not limited thereto.

[0092] In one embodiment of the present invention, the perilla extract, a fraction of the extract, or diechol may be added to an over-the-counter drug for the purpose of antiviral activity against Zika virus.

[0093] The term “quasi-drug” refers to products that are used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases in humans or animals, and have a milder effect than pharmaceutical products. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products other than those used for pharmaceutical purposes, and include products used for treating or preventing diseases in humans or animals, and products that have a mild effect on the human body or do not act directly.

[0094] The over-the-counter pharmaceutical composition may be one or more selected from the group consisting of, but is not limited to, a coating agent, a disinfectant, a wet tissue, a body cleanser, a foam, a soap, a mask, an ointment, a cream, a lotion, an essence, and a spray.

[0095] Quasi-drug compositions can be administered orally or parenterally in various dosage forms. When formulated, they can be prepared using commonly used diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, or surfactants.

[0096]

[0097] In addition, the present invention provides a feed additive composition for preventing or improving flavivirus infection, comprising a perilla leaf extract, a fraction thereof, or phlorotannin as an active ingredient. Any content overlapping with that of an antiviral agent, pharmaceutical composition, or health functional food composition is omitted in consideration of the complexity of the present specification.

[0098] In addition, in another embodiment of the present invention, a method for preventing and treating a flavivirus infection disease is provided, comprising a step of administering the antiviral agent to a subject in need thereof.

[0099] In addition, the present invention provides a method for preventing, improving or treating a flavivirus infection disease, comprising a step of administering a composition containing a perilla extract, a fraction of the extract or phlorotannin to a subject in need thereof.

[0100] To facilitate understanding of the present invention, preferred examples are presented below. However, the following examples are provided solely to facilitate understanding of the present invention, and the scope of the present invention is not limited by the examples.

[0101]

[0102] Example 1. Preparation of perilla extract

[0103] Sea tangle was collected from the shore of Seongsan-eup, Seogwipo-si, Jeju Special Self-Governing Province, washed in clean water, freeze-dried, and then finely ground. 2 kg of sea tangle powder was mixed with 2 L of 70% ethanol and stirred for 24 hours using a homogenizer at room temperature and 150 rpm. The stirred mixture was centrifuged at 4°C and 6,800 rpm and then filtered under reduced pressure using a Watman No. 4 filter paper. The residue was then mixed with 2 L of 70% ethanol and the above process was repeated three times. The supernatant was evaporated of ethanol using a vacuum rotary evaporator, and the remaining supernatant was freeze-dried to obtain an extract used as a sample (ECE).

[0104]

[0105] Example 2. Separation and purification of diechol

[0106] The extract of perilla leaves obtained in Example 1 was suspended in distilled water and distributed into ethyl acetate. The ethyl acetate fraction was subjected to silica column chromatography using a chloroform-methanol (100:1→solvent system). Thereafter, the separated active fraction was subjected to Sephadex LH-20 column chromatography using an 80% methanol solvent system, and further purified using an HPLC system to obtain diechol with a purity of 98%.

[0107]

[0108] Experimental Example: Measurement of the antiviral efficacy of perilla extract and diechol against Zika virus.

[0109] 1.1. Materials

[0110] Zika virus was provided by the Korea Disease Control and Prevention Agency, National Institute of Health, and Vero E6 cells were purchased from ATCC. DPBS, DMEM, DMEM / F12 powder, 100X L-glutamine, and FBS used in cell experiments were purchased from Gibco. Penicillin / Streptomycin and Crystal violet were purchased from Sigma-Aldrich. TRIzol and DEPC-water were purchased from Ambion, and chloroform, isopropanol, and ethanol were purchased from EMSURE. High Capacity RNA to cDNA kit and Power SYBR Green PCR Master Mix were purchased from Applied Biosystems.

[0111]

[0112] 1.2. Zika virus and sample handling

[0113] 1x10 per well in 6-well plates 6After seeding Vero E6 cells, they were cultured until they formed a monolayer. The cells were then washed with DPBS and dispensed into DMEM supplemented with 2% FBS for sample treatment. Two hours after sample treatment, the cells were infected with Zika virus (MOI 0.01) and cultured at 37°C for 48 hours. Cells treated with DPBS instead of the samples were used as uninfected cells (mock).

[0114]

[0115] 1.3. Real-time PCR

[0116] Real-time PCR was performed to analyze mRNA expression for key molecules, including the Zika virus NS1 protein, in cells treated with the virus and samples. RNA was extracted using the Acid guanidinium thiocyanate-phenol-chloroform extraction method. Cells treated with the virus and samples were treated with 1 mL of TRIzol, mixed well, and collected in an e-tube. The cells were then treated with 200 μL of chloroform, vortexed for 15 seconds, incubated at room temperature for 3 minutes, and centrifuged at 12,000 rpm at 4°C for 15 minutes. Afterwards, 500 μL of the aqueous layer was mixed with 500 μL of isopropanol, inverted 5–6 times, incubated at room temperature for 10 minutes, and centrifuged at 12,000 rpm at 4°C for 10 minutes. After discarding the supernatant, the remaining pellet was centrifuged at 12,000 rpm and 4℃ for 10 minutes with 1 mL of 75% EtOH added. The supernatant was discarded once again, the remaining pellet was completely dried, and 25 μL of DEPC-water was added to dissolve it. The RNA obtained in this way was stored at -80℃ and used in the experiment. cDNA synthesis was performed using a High-Capacity RNA-to-cDNA kit. 2 μg of RNA was mixed with 2X RT buffer mix, 20X RT enzyme mix, and DEPC-water in the correct ratio to make a total of 20 μL, and the reaction was performed at 37℃ for 60 minutes and inactivated at 95℃ for 5 minutes. The cDNA was stored at -20℃ and used in the experiment. Real-time PCR was performed using a SYBR green-based detection method. The synthesized cDNA was prepared by diluting it to 1 / 40, and SYBR green and the target primer were prepared by mixing them in the appropriate ratio.Afterwards, 4 μL of diluted cDNA and 6 μL of SYBR green-primer mix were treated in a 96-well Real-time PCR reaction plate, sealed with adhesive film, and analyzed using Quantstudio 3. The base sequences of the primers used in the experiment are shown in Table 1 below.

[0117] Target geneSequenceDirectionSEQ ID number Zika virus NS15'-CRA CTA CTG CAA GYG GAA GG-3'Forward15'-GCC TTA TCT CCA TTC CAT ACC-3'Reverse2GAPDH5'-GCA AAT TCC ATG GCA CCG T-3'Forward35'-TCG CCC CAC TTG ATT TTG G-3'Reverse4IFIT15'- GGA TTC TGT ACA ATA CAC TAG AAA CCA-3'Forward55'- CTT TTG GTT ACT TTT CCC CTA TCC-3'Reverse6IFIT25'- ATC CCC CAT CGC TTA TCT CT-3'Forward75'- CCACCTCAATTAATCAGGCACT-3'Reverse8TNFα5'-CCA ACT GTC ACT CAT TGC TGA-3'Forward95'-TTC CAA GAA GGA GAC CAT GTT T-3'Reverse10

[0118] R is Purine (A or G), Y is Pyrimidine (C or T)

[0119]

[0120] 1.4. Plaque assay

[0121] A plaque assay was performed to determine the number of virus particles (plaque-forming units, PFU) in each supernatant treated with virus and samples. For the plaque assay, cells were seeded as a monolayer in 6-well plates, and each supernatant was serially diluted 10-fold in DMEM. The cells were washed twice with DPBS, and then aliquots of the diluted supernatant were added. The plates were gently shaken every 15 minutes to ensure good virus adsorption and incubated for 2 hours. The supernatant was removed, and 3 mL of DMEM / F12 containing agarose was added to each well. Once the medium solidified, the 6-well plates were wrapped in foil, placed upside down in a CO2 incubator, and incubated for 4 days. The plates were then fixed with 4% formaldehyde for 1 hour, and the fixative and agarose DMEM / F12 were removed. The cells were stained with 0.1% crystal violet solution, and virus particles were counted. PFU per mL was calculated according to the formula below.

[0122] PFU / mL = Number of plaques / (Dilution factor × Volume of diluted virus / well)

[0123]

[0124] 1.5. MTT assay

[0125] Cytotoxicity was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazoliumbromide (MTT) assay. Cells were seeded at 8 × 10 per well in 96-well plates. 4After 16 hours of culture, the samples were treated with different concentrations and cultured for 24 hours. Afterwards, MTT solution (10 μl, 3 mg / ml) was added and cultured for an additional 3 hours at 37°C. After removing the supernatant, formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader. The cytotoxicity of the samples was expressed as a percentage of the control group.

[0126]

[0127] 1.6. Statistical Analysis

[0128] Statistical analysis was performed using GraphPad Prism 10 software. All data are presented as means and standard errors. To test the significance of each sample, one-way analysis of variance (ANOVA) was performed between each experimental group, and post hoc analysis was performed using Dunnett and Turkey tests (P<0.05).

[0129]

[0130] 2. Experimental results

[0131] 2.1. Comparison of the anti-Zika virus efficacy of diechol and zeaxanthin

[0132] The anti-Zika virus efficacy of diechol was evaluated by the inhibition rate of Zika virus NS1 protein mRNA expression compared to the control group (PC group) treated with the virus alone, and comparative analysis was performed using zeaxanthin, which has been reported to have antiviral efficacy against Herpes simplex virus. As a result, referring to Figure 1, the experimental group treated with diechol showed approximately 20% of the mRNA expression amount compared to the PC group, and the experimental group treated with zeaxanthin actually showed an increase in mRNA expression amount, confirming that diechol showed superior anti-Zika virus efficacy than zeaxanthin.

[0133] In the case of diechol, mRNA expression for IFIT1 (Interferon-induced proteins with tetratricopeptide repeats 1) and IFIT2 (Interferon-induced proteins with tetratricopeptide repeats 2), which are known to be important elements of the antiviral immune response by inhibiting translation of viral proteins and replication of the virus, was evaluated again, and the results are shown in Fig. 2. Referring to Fig. 2, diechol significantly reduced the expression of IFIT1 and IFIT2, which was increased by Zika virus infection.

[0134]

[0135] 2.2. Verification of the anti-Zika virus efficacy of Diecol

[0136] The anti-Zika virus efficacy of diechol was examined by the inhibition rate of Zika virus plaque formation compared to the control group (PC group) treated with the virus alone. As a result, diechol was confirmed to reduce plaque formation following Zika virus inoculation. As a result of examining plaque concentration, referring to Figure 3, while approximately 100,000 PFU / ml of plaques were formed in cells treated with Zika virus, diechol at non-toxic concentrations of 25 and 50 μM reduced plaque formation by approximately 50% and 40%, respectively, compared to the control group.

[0137]

[0138] 2.3. Anti-Zika virus efficacy of Ecklonia cava extract (ECE)

[0139] The cytotoxicity of ECE against Vero E6 was analyzed using the MTT assay. As a result, ECE showed a concentration-dependent increase in cell viability compared to the control at concentrations of 6.25, 12.5, 25, and 50 μg / ml, confirming its absence of cytotoxicity (Fig. 4). The anti-Zika virus efficacy of ECE was evaluated by the inhibition rate of Zika virus plaque formation and the inhibition rate of Zika virus NS1 protein mRNA expression compared to the control. As a result, ECE was confirmed to reduce plaque formation following Zika virus inoculation (Fig. 5). As a result of confirming the concentration of plaque, the concentration of plaque formed in the control group (PC) treated only with Zika virus was found to be 48,500±3,536 PFU / ml, and the concentration of plaque formed in the experimental groups treated with 6.25, 12.5, 25, and 50 μg / ml of ECE were found to be 16,300±8,910, 14,600±1,980, 7,400±849, and 7,300±1,838 PFU / ml, respectively, confirming that ECE inhibits the formation of plaque in a concentration-dependent manner (Fig. 6). In addition, as a result of confirming the mRNA expression of the NS1 protein of Zika virus, in the control group treated with only Zika virus, the mRNA of the NS1 protein increased by about 35,000,000 times, whereas in the experimental group treated with 6.25, 12.5, 25, and 50 μg / ml of ECE, the mRNA expression levels were 23,590,443±247,364, 12,086,611±460,692, 7,174,704±110,221, and 4,074,311±162,853, confirming that ECE inhibits the mRNA expression of the NS1 protein in a concentration-dependent manner (Fig. 7).

[0140]

[0141] 2.4. Efficacy of Ectocarcinoma japonica extract (ECE) on the expression of inflammatory factors induced by Zika virus infection

[0142] Tumor necrosis factor-α (TNF-α) is known as a pro-inflammatory cytokine that regulates inflammatory responses, and the IFIT family is known to be an important component of the antiviral immune response. Therefore, the efficacy of ECE on TNF-α, IFIT1, and IFIT2 expressed by Zika virus infection was confirmed. As a result, ECE at concentrations of 6.25, 12.5, 25, and 50 μg / ml significantly reduced the expression of TNF-α increased by Zika virus infection (Fig. 8). In addition, ECE at concentrations of 12.5, 25, and 50 μg / ml significantly reduced the expression of IFIT1 and IFIT2 increased by Zika virus infection (Figs. 9 and 10).

[0143]

[0144] In summary, the above results show that the diechol according to the present invention reduces plaques formed upon Zika virus inoculation, inhibits the proliferation of Zika virus more effectively than zeaxanthin, which has been reported to have an antiviral effect, and inhibits the expression of IFIT1 and IFIT2, confirming that it has excellent antiviral activity against Zika virus. In addition, it was confirmed that the Ecklonia cava extract inhibits TNF-α and IFIT1 and IFIT2 expressed by Zika virus infection and reduces plaque formation. Therefore, the Ecklonia cava extract, fractions of the Ecklonia cava extract, or diechol according to the present invention can be usefully used as an antiviral composition without side effects for preventing flavivirus infection, improving related diseases, or treating them.

Claims

1. An antiviral agent comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

2. An antiviral agent, wherein the above phlorotannin is at least one selected from the group consisting of phloroglucinol, eckol, 8,8'-bieckol, eckstolonol, dieckol, and phlorofucofuroeckol A.

3. In paragraph 1, The above phlorotannin is an antiviral agent, which is diechol.

4. In paragraph 1, The above extract is an antiviral agent extracted using water, a lower alcohol of C1 to C4, or a mixed solvent thereof.

5. In paragraph 1, The above phlorotannin is an antiviral agent isolated from a fraction of a perilla extract.

6. In paragraph 1, The above antiviral agent is an antiviral agent having antiviral activity against flavivirus.

7. In paragraph 6, The above antiviral agent has antiviral activity against at least one flavivirus selected from the group consisting of Zika virus, Dengue virus, Yellow fever virus, West Nile virus, and Japanese encephalitis virus.

8. A pharmaceutical composition for preventing or treating flavivirus infection, comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

9. A health functional food composition for preventing or improving flavivirus infection disease, comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

10. A pharmaceutical composition for preventing or improving flavivirus infection, comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

11. A feed additive composition for preventing or improving flavivirus infection, comprising a perilla extract, a fraction of the extract, or phlorotannin as an active ingredient.

12. A method for preventing, improving or treating a flavivirus infection disease, comprising administering to a subject in need thereof a composition containing a perilla extract, a fraction of the extract or phlorotannin.

Images