WO2023008666A1 - Virus-treating composition - Google Patents
Virus-treating composition Download PDFInfo
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- WO2023008666A1 WO2023008666A1 PCT/KR2021/018563 KR2021018563W WO2023008666A1 WO 2023008666 A1 WO2023008666 A1 WO 2023008666A1 KR 2021018563 W KR2021018563 W KR 2021018563W WO 2023008666 A1 WO2023008666 A1 WO 2023008666A1
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- composition
- virus
- truncated
- canavalin
- present
- Prior art date
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
- C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a composition for treating viruses.
- Concanavalin A (ConA), derived from Canavalia ensiformis, is a protein belonging to the lectin family that binds to sugars such as mannose or glucose at monosaccharide binding sites. Under physiological conditions, ConA is a tetramer and selectively binds to cell surface glycoproteins containing ⁇ -mannopyranosyl and ⁇ -glucopyranosyl residues. This feature has extensive applications in biology and biomedicine, and is often used as a binding agent for pathogen-physiological reactions, such as Denguevirus (DENV), Hepatitis C Virus (HCV), and Herpes Virus. (Herpes Virus; HSV), Human Immunodeficiency Virus (HIV), Influenza A Virus (IAV), and murine RNA tumor virus. It has a characteristic.
- DEV Denguevirus
- HCV Hepatitis C Virus
- IAV Influenza A Virus
- canavalin present in soybean binds to the virus to recognize the possibility of virus elimination and neutralization, and through the soybean fermentation process, toxic substances such as concanavalin A are removed and anti-bacterial It was purified as a useful protein sequence with viral properties, and a strategy for utilizing its antiviral properties is proposed.
- the present invention removes toxicity through a soybean fermentation process and obtains a purified end-truncated canavalin with a useful protein sequence having antiviral properties, and a composition comprising it as an active ingredient is to provide
- the present invention relates to a method for preparing a composition comprising end-truncated canavalin according to the present invention, which can remove toxic proteins and secure useful proteins through a soybean fermentation process.
- composition for virus elimination, neutralization, or both comprising soy-derived end-truncated canavalin as an active ingredient.
- the composition may be used for collecting samples for virus testing.
- the composition may collect a virus sample by detaching, neutralizing, or both of the virus from the site where the virus is attached.
- the site to which the virus is attached may be at least one of a living cell, a living tissue, a body fluid, and a substrate.
- composition for preventing or treating viral infection comprising soy-derived end-truncated canavalin as an active ingredient.
- the composition may prevent or treat infection by a virus by detaching, neutralizing, or both of the virus at the site where the virus is attached.
- the terminal-truncated canavalin may be C-terminal truncated canavalin.
- the terminal-truncated canavalin may include a C-terminal truncated sequence among the canavalin amino acid sequences.
- the end-truncated canavaline may have a binding inhibitory function between the viral spike receptor binding domain and the receptor.
- the end-truncated canavaline may prevent binding of the viral spike receptor binding domain and the receptor or detach the binding.
- the composition may be a powder, solution, gel, paste, suspension or aerosol.
- the composition may be an oral or nasal composition.
- the composition may be an oral irrigation solution or a nasal irrigation solution.
- the oral washing liquid may be gargle or toothpaste
- the nasal washing liquid may be a nasal spray.
- the virus may be a respiratory virus.
- the respiratory virus is at least one selected from the group consisting of influenza virus, coronavirus, syncytial virus, adenovirus, human bocavirus, and rhinovirus. It may contain more than one.
- the end-truncated canavalin is derived from soy beans or fermented soybeans, and the end-truncated canavalin may be biotoxic-free.
- proteolysis may produce useful protein, terminal-truncated canavalin.
- the step of fermenting may be to ferment at 30 ° C to 40 ° C after inoculating Bacillus subtilus.
- the step of fermenting may be fermentation for one or more days in a dissolved oxygen atmosphere of 30% to 50%.
- the present invention through a soybean fermentation process, removes proteins such as concanavalin A (ConA) having toxicity present in soybeans, and through a purification process, terminal-truncated canavalin, a useful protein having antiviral properties, is It is possible to secure and utilize this to provide an antiviral composition having virus elimination, neutralization and / or both.
- ConA concanavalin A
- the present invention utilizes natural materials such as soybeans to prevent respiratory viruses such as coronaviruses from attaching to receptors, remove viruses that are already attached, and reduce virus infectivity. It is possible to provide a composition that can be made in a neutralized state without this, and quasi-drugs and pharmaceuticals using the same.
- the present invention can provide a pretreatment composition for collecting virus samples for virus diagnosis and examination by detaching and/or neutralizing viruses attached in vitro and/or in vivo using natural materials such as soybeans. there is.
- FIG. 1 is a schematic diagram showing a chromatogram purified by using Size Exclusion Chromatography for protein fraction obtained by fermenting soybeans according to the present invention according to an embodiment of the present invention.
- FIG. 2 shows basic data for calculating the mass value by obtaining the intact mass value of the TCan protein according to the present invention using a mass spectrometer, according to an embodiment of the present invention.
- FIG. 3 shows the LSSQDKPFNL sequence obtained from the N-terminal sequencing method in the entire protein sequence of canavalin, and the truncation portion in the entire protein sequence is indicated (LSSQDKPFNL portion bold), It shows the sequence part (underlined part) of the whole protein corresponding to the mass value calculated using the protein mass value information of FIG. 2.
- FIG. 4 schematically shows the inhibitory effect of the TCan composition according to the present invention on the binding between hACE2 receptor and RBD (SARS-CoV-2 spike receptor binding domain) according to an embodiment of the present invention.
- 5a shows changes in the expression level of inflammatory cytokines, and relates to interferon gamma (IFN- ⁇ ), according to an embodiment of the present invention.
- IFN- ⁇ interferon gamma
- 5b shows changes in the expression level of inflammatory cytokines, and relates to interleukin (IL)-17, according to an embodiment of the present invention.
- IL interleukin
- 5c shows changes in the expression level of inflammatory cytokines, and relates to IL-4, according to an embodiment of the present invention.
- Mean ⁇ SD from data (n 3 per group); **p ⁇ 0 01 and ****p ⁇ 0 0001 vs. naive control (NC) group; ##p ⁇ 0 01 and ## ##p ⁇ 0 0001 vs. con A group.
- Figure 5d shows changes in the expression level of inflammatory cytokines, and relates to IL-5, according to an embodiment of the present invention.
- Mean ⁇ SD from data (n 3 per group); **p ⁇ 0 01 and ****p ⁇ 0 0001 vs. naive control (NC) group; ##p ⁇ 0 01 and ## ##p ⁇ 0 0001 vs. con A group.
- Figure 5e shows changes in the expression level of inflammatory cytokines, according to an embodiment of the present invention, and relates to IL-13 in splenocytes of C57BL/6 mice injected with Con A and BE.
- Figure 6a shows the effect of Con A and BE injection on the survival rate of C57BL/6 mice, VC (vehicle control); Hourly survival was recorded up to 24 hours after injection of Con A at concentrations of 40 mg/kg, 80 mg/kg and 160 mg/kg (body weight, BW)).
- Figure 6b shows the effect of Con A and BE injection on the survival rate of C57BL/6 mice, VC (vehicle control); Hourly survival was recorded up to 24 hours after injection of BE at concentrations of 75 mg/kg, 160 mg/kg and 200 mg/kg (BW body weight).
- Figure 7a shows the results of changes in liver biomarker (AST) levels in response to Con A and BE instillation in mice according to an embodiment of the present invention, and the biomarker concentrations of Con A and BE administered mice Measured in serum (intravenous or intratracheal infusion, 5-15 or 10-40 mg/kg BW, respectively).
- AST liver biomarker
- Figure 7b shows the results of changes in liver biomarker (ALT) levels in response to Con A and BE instillation in mice, according to an embodiment of the present invention, and the biomarker concentrations of Con A and BE administered mice Measured in serum (intravenous or intratracheal infusion, 5-15 or 10-40 mg/kg BW, respectively).
- ALT liver biomarker
- BW body weight
- Figure 7c shows the results of changes in liver biomarker (TBIL) levels in response to Con A and BE instillation in mice, according to an embodiment of the present invention, and the biomarker concentrations of Con A and BE administered mice Measured in serum (intravenous or intratracheal infusion, 5-15 or 10-40 mg/kg BW, respectively).
- TBIL liver biomarker
- FIG. 7d shows the results of changes in liver biomarker (GGT) levels in response to Con A and BE instillation in mice, according to an embodiment of the present invention, and the biomarker concentrations of Con A and BE administered mice Measured in serum (intravenous or intratracheal instillation, 5-15 or 10-40 mg/kg BW, respectively).
- GGT liver biomarker
- the present invention relates to compositions comprising truncated canavaline and methods for their preparation.
- the present invention relates to a composition comprising end-truncated canavalin.
- the composition is derived from soybean, which is a natural material, purified from fermented soybean, and exhibits antiviral properties. It may include end-truncated canavalin, which is a useful protein with The end-truncated canavaline can prevent viruses from attaching to receptors or can detach viruses attached to receptors and/or convert them to a non-infectious neutral state. By using the interaction between the virus and the terminal-truncated canavaline, it can be used as a composition for preventing and/or treating viral infection, cleaning a virus, and a pretreatment composition for obtaining a sample for a virus test.
- the truncated canavalin is C-terminal truncated canavalin, and may include a C-terminal truncated sequence among canavalin amino acid sequences.
- the C-terminal truncated sequence can be identified by LSSQDKPFNL.
- the end-truncated canavalin may interfere with and inhibit the binding of the receptor-binding domain of the virus to the receptor, and surround and detach the already bound virus.
- it may provide a binding inhibitory function between the viral spike receptor binding domain and the receptor.
- binding of the viral spike receptor binding domain to the receptor may be prevented or the binding may be dissociated.
- it can neutralize viruses to lower infectivity.
- composition for preventing and/or treating viral infection comprising the soy-derived end-truncated canavalin as an active ingredient.
- the composition can prevent and/or treat infection by a virus by detaching, neutralizing, or both of the virus at the site where the virus is attached.
- the composition can prevent and/or treat viral infections by preventing or interfering with attachment of viruses in cells (in vivo), human tissues, human organs, and the like, or by neutralizing viruses.
- infection can be prevented and/or treated by rinsing the mouth with a gargle or spraying into the nasal cavity to prevent attachment of the virus to the human oral mucosa or nasal mucosa, or to detach and neutralize the attached virus.
- infection can be prevented and/or treated by preventing the attachment of viruses to mucous membranes in the human body in addition to the oral cavity and oral mucosa, or detaching and neutralizing the attached viruses.
- the composition may be a composition for desorption, neutralization or both of the virus.
- the composition surrounds and captures the surface of a virus attached to a bodily fluid, specimen, cell (ex vivo and/or in vivo), substrate for specimen collection, human tissue, etc. to desorb and/or neutralize the virus.
- a virus sample may be obtained by detaching and/or neutralizing viruses contained or attached to at least one of the oral surface, nasal surface, saliva, nasal mucus, and urine, and collecting them.
- it can be washed with a gargle to remove viruses attached to the oral mucosa of the human body, and washed to remove viruses attached to the human oral mucosa and the like in addition to the oral mucosa.
- the bodily fluid is a liquid discharged from a living body (or human body), and the bodily fluid may be at least one of saliva, nasal mucus, and urine.
- the bodily fluid when present in or on the surface of biological tissues and/or cells, may be obtained as a specimen together with detached and/or neutralized cells upon application of the composition.
- the obtained sample may be introduced into a virus diagnostic kit or PCR and used for virus testing.
- the composition is non-toxic to the body, and, for example, suppresses or frees T cell division, so that the induction of toxicity to the body can be prevented.
- the virus is an enveloped virus, a non-enveloped virus, or both, and may be, for example, a respiratory virus.
- the respiratory virus includes influenza virus (eg, influenza virus A (IVA) and influenza virus B (IVB)), coronavirus, syncytial virus (RSV, eg, respiratory cell Syncytial virus A (RSVA) and respiratory syncytial virus B (RSVB), adenovirus, human bocavirus, rhinovirus, human metapneumovirus (human metapneumovirus, hMPV), Middle East respiratory virus (MERS-CoV), SARS-associated coronavirus (SARS-coV), human parainfluenza virus 1 (HPIV 1), human parainfluenza virus 2 (human parainfluenza virus 2: HPIV 2) and human parainfluenza virus 3 (human parainfluenza virus 3: HPIV 3).
- influenza virus eg, influenza virus A (IVA) and influenza virus B (IVB)
- RSV respiratory cell
- the truncated canavalin is present in the composition in an amount of 1 ppm to 1000; 10 ppm to 1000 ppm; 10 ppm to 500 ppm; or 10 ppm to 200 ppm.
- the composition may be in the form of a solid, a liquid, or a mixture of the two, for example, a powder, solution, gel, paste, emulsion, colloidal dispersion, suspension or aerosol form.
- a powder, solution, gel, paste, emulsion, colloidal dispersion, suspension or aerosol form can be
- the composition may be an oral or nasal composition, which may be classified into medical or non-medical use depending on the purpose.
- the composition may be a gargle, an oral wash such as toothpaste, a nasal wash such as a nasal spray, and the like, and may provide an antiviral function for preventing and treating viral infections through nasal and/or oral washing. . That is, it is possible to obtain a virus sample by washing the oral cavity with gargling to provide a preventive and/or therapeutic effect against viruses, and by collecting desorbed and neutralized viruses present in the gargle solution after gargling the mouth.
- powder, liquid, etc. may be administered in the nasal cavity by inhalation, spraying, or injection using a device such as a tube, and washed to provide a preventive and / or therapeutic effect against viruses or to collect desorbed and / or neutralized viruses. can
- composition may be divided into oral administration or nasal administration according to the route of administration to provide antiviral functions such as prevention and treatment of viral infection.
- the composition may be a bodily fluid pretreatment composition for obtaining a virus sample in the oral cavity or nasal cavity, and, for example, by rinsing the mouth through gargling and spitting it out, the virus is detached and/or neutralized. specimens can be obtained.
- viruses attached to cells, substrates, etc. may be desorbed and/or neutralized outside the body, and virus samples may be collected for various purposes such as virus testing and diagnosis.
- the composition may include a carrier component, a base component, a fragrance, an additive, a solvent, etc. applied in the technical field of the present invention according to the formulation, if it does not deviate from the object of the present invention, , which is not specifically mentioned in this specification.
- the present invention relates to a method for producing a composition according to the present invention, and according to an embodiment of the present invention, the method for producing the composition includes fermenting beans, bean hulls or both; and separating the end-truncated canavalin from the fermented product.
- the manufacturing method removes ConA toxic protein, which is toxic in soybeans, for example, soybeans, through a fermentation process, provides fermentation conditions capable of securing useful antiviral proteins, and provides a composition containing the useful protein. is to do
- proteolysis can form useful protein, terminal-truncated canavalin. That is, in the step of fermenting, after mixing soybeans, soybean hulls, or both in a solvent, and inoculating Bacillus subtilus, the temperature is 10 ° C to 45 ° C; 20° C. to 40° C.; a temperature between room temperature and 40° C. or between 30° C. and 40° C.; 30% to 50% dissolved oxygen atmosphere; or at least 10 hours in both conditions; more than 1 day; or 1 to 5 days; or for 1 to 4 days;
- the fermentation process may proceed.
- the beans are small beans, and other beans other than small beans may be applied.
- the step of fermenting is pH 5 to 8; pH 6 to 7.5 or preferably in the neutral region.
- the solvent includes water, an organic solvent, or both, and for example, the organic solvent includes at least one of alcohol having 1 to 6 carbon atoms, butylene glycol, and propylene glycol.
- the organic solvent includes at least one of alcohol having 1 to 6 carbon atoms, butylene glycol, and propylene glycol.
- the step of separating the end-truncated canavalin from the fermented product may remove toxic substances from the fermented product and isolate useful protein sequences to obtain the end-truncated canavalin.
- the fermentation can be used to isolate end-truncated canavalin using size exclusion chromatography.
- Sword bean (Canavalia gladiata) was cultured under dissolved oxygen (20-40% saturation) and pH 7.0 conditions and at 33 ° C (or in purified water (96.059% to less than 100%)) for 4 days. subtilis was proteolysed. After protein hydrolysis, the product was centrifuged (12,000 ⁇ g) at 4° C. for 15 minutes, and the supernatant was collected and lyophilized (hereinafter referred to as BE (fermented soybean extract)).
- On-line top-down MS was performed on a waters ACQUITY UPLC I-Class system connected to a Synapt G2-S QTOF mass spectrometer (Waters Corp., USA). 2 ⁇ l of purified TCan was injected onto a 2.1 ⁇ 50 mm ACQUITY UPLC BEH300 C4 column (particle size 1.7 ⁇ m). The gradient started at 5% B (0.1% formic acid in acetonitrile) and was delivered in 400 ⁇ l, rising to 95% B in 6.7 min and 5% B in 9.0 min.
- the mass spectrometer is ESI-Positive MSE continuum data acquisition mode, capillary voltage 3.0 kV, cone voltage 50 V, desolvation gas 800 L/hr, source temperature 120 °C, desolvation temperature 300 °C worked in Data were collected at 1 spectra/second from 500-4,000 m/z.
- a maximum entropy algorithm (MaxEnt1) was used for mass deconvolution. Deconvolution was performed in the range of 1,500-2,600 Da centered on the expected mass and target resolution of 0.5 Da by repeating MaxEnt1 100 times.
- the electrospray spectrum of TCan is shown in FIG. 2 .
- Protein samples were transferred from an SDS-PAGE gel to a PVDF membrane and the protein bands transferred on the membrane were analyzed using the LC 492 Protein Sequencing System (Applied Biosystems Instruments, USA). The results are shown in Figure 3.
- Example 1 Evaluation of binding inhibitory ability of TCan composition between hACE2 receptor and RBD
- the ability to inhibit hACE2 receptor and SARS-CoV-2 spike receptor binding domain (RBD) binding was evaluated using the COVID-19 Neutralizing Antibody ELISA kit (Cat.# KA6111, abnova).
- the hACE2 receptor and RBD included in the kit were used, and evaluation was performed according to the instructions provided by the manufacturer.
- the effect of the TCan composition neutralizing RBD to inhibit hACE2 binding was performed by the following method.
- the RBD and TCan compositions were reacted at 37 °C for 2 hours. Then, the hACE2 receptor was added to the coated plate to induce binding with RBD for 2 hours.
- the effect of the TCan composition to detach the prebond between RBD and hACE2 was performed in the following way.
- RBD was added to the hACE2 receptor-coated plate to induce their binding at 37 °C for 2 hours. These bonds were then washed twice for 1 minute with the TCan composition.
- the effect of the TCan composition reacting with hACE2 to prevent the binding of RBD to hACE2 was evaluated by the following method.
- the TCan composition was added to the hACE2 receptor-coated plate and reacted at 37° C. for 1 hour. After washing with a washing buffer, RBD was added to induce binding with hACE2 at 37 °C for 2 hours.
- the TCan composition showed an inhibitory effect on the binding between RBD and hACE2 in a concentration-dependent manner.
- concentration (IC 50 ) of the TCan composition that inhibits the degree of binding between RBD and the hACE2 receptor by 50% was 4.569 ppm for the prevention method, 8.935 ppm for the neutralization method, and >35.89 ppm for the detachment method. Confirmed. Accordingly, it is possible to provide a TCan composition that inhibits the binding between RBD and the hACE2 receptor.
- Con A is well known to induce a mitotic response in spleen cells that activates the immune system, recruits lymphocytes, and induces cytokine production.
- T helper (Th) cytokine ((IFN-g for Th1, IL-17 for Th17, and IL-4, IL-5, and IL-13 for Th2 cells) were investigated The results are shown in Figures 5a, 5b, 5c, 5d and 5e.
- FIGS. 5A, 5B, 5C, 5D and 5E show that Con A induces significantly higher production of all Th cytokines compared to BE, IFN-g, IL-17, IL-5 and The production of IL-13 increased in BE-treated splenocytes during the experimental period, but the production was significantly lower than that of the Con A-treated group.
- FIGS. 6A and 6B survival rate was monitored hourly in C57BL/6 mice divided into 7 groups: vehicle control (VC); Con A treated groups at concentrations of 40, 80 and 160 mg/kg body weight (BW) (Con A 40, Con A 80, and Con A 160 groups) and BW treated groups at concentrations of 75, 160 and 200 mg/kg BW (BE 75, BE 160, BE 200 groups) (treatment time: 24 h.).
- vehicle control VC
- BW body weight
- the VC and BE-treated groups showed a survival rate of 100%, whereas the Con A-treated group showed a reduced survival rate.
- the survival rate of mice in the Con A treatment group starts to decrease from 17 hours after treatment, and the survival rate of mice in the Con A 160 treatment group is not observed until 24 hours after treatment.
- 60% survival was observed in the mice of the Con A 40 and Con A 80 groups after 24 hours of treatment.
- Con A activates T cells to secrete cytokines that cause liver damage.
- levels of serum AST, ALT, TBIL, and GGT which are biomarkers of liver function, were evaluated 24 hours after administration of Con A and BE to mice. The results are shown in Figs. 7a, 7b, 7c and 7d. Figures 7a, 7b, 7c and 7d show that the mode of administration affects liver biomarker levels. Intratracheal instillation of both Con A and BE-treated mice did not significantly affect the levels of liver biomarkers, but high concentrations of Con A (15 mg/kg BW) administered intravenously did not significantly affect all biomarkers (p-value ⁇ 0.05). It can be seen that the expression of
- useful protein sequences having antiviral properties and removing toxicity through the fermentation process of soybean were purified.
- mass spectrometry and N-terminal sequencing were used to secure intact mass values, and truncated canavalin (Tcan, Truncated Canavalin Tcan) was confirmed through N-terminal sequencing.
- Tcan Truncated Canavalin Tcan
- Tcan was analyzed by ELISA method. That is, Tcan, which has an antiviral useful effect in the fermented soybean extract, was secured, and Tcan showed strong binding to hACE2, preventing RBD from binding.
- the RBD region of the spike protein of the SARS-CoV-2 virus cannot bind to the receptor, so it can be used for pharmaceuticals and quasi-drugs such as gargles and nasal washes that can prevent viral infection in the future.
- the RBD region of the spike protein of the SARS-CoV-2 virus cannot bind to the receptor, so it can be used for pharmaceuticals and quasi-drugs such as gargles and nasal washes that can prevent viral infection in the future.
- TCan detaching RBD bound to hACE2 it can be used for pharmaceuticals and quasi-drugs such as gargles and nasal washes that can effectively wash out viruses already attached to the oral and nasal cavities, and body fluid pretreatment solutions for sample collection. .
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Abstract
The present invention relates to a virus-treating composition and to a composition containing bean-derived terminus-truncated canavalin for detachment, neutralization, or detachment/neutralization of viruses. Also, the present invention further relates to a composition for prevention or treatment of viral infections.
Description
본 발명은, 바이러스 처리용 조성물에 관한 것이다.The present invention relates to a composition for treating viruses.
작두콩(Canavalia ensiformis)에서 유래된 콘카나발린 A(concanavalin A; ConA)은, 단당류 결합 부위에서 만노오스 또는 글루코오스 등의 당과 결합을 하는 렉틴과에 속하는 단백질이다. 생리적 조건에서의 ConA는 사합체(tetramer)이며, α-만노피라노실(alpha-mannopyranosyl) 및 α-글루코피라노실(alpha-glucopyranosyl) 잔기를 포함하는 세포 표면의 당단백질에 선택적으로 결합한다. 이러한 특징은 생물학 및 생물의학에서 광범위하게 응용되고 있으며, 병원체-생리학적에서 반응을 위한 결합제로서 종종 사용되며, 뎅기열바이러스(Denguevirus, DENV), 간염C형 바이러스(Hepatitis C Virus; HCV), 헤르페스 바이러스 (Herpes Virus; HSV), 인간 면역 결핍 바이러스(Human Immunodeficiency Virus; HIV), 인플루엔자 A형 바이러스(Influenza A Virus; IAV), 마우스성 RNA 종양 바이러스(murine RNA tumour virus)와 같은 외피단백질 바이러스를 결합하는 특징이 있다. Concanavalin A (ConA), derived from Canavalia ensiformis, is a protein belonging to the lectin family that binds to sugars such as mannose or glucose at monosaccharide binding sites. Under physiological conditions, ConA is a tetramer and selectively binds to cell surface glycoproteins containing α-mannopyranosyl and α-glucopyranosyl residues. This feature has extensive applications in biology and biomedicine, and is often used as a binding agent for pathogen-physiological reactions, such as Denguevirus (DENV), Hepatitis C Virus (HCV), and Herpes Virus. (Herpes Virus; HSV), Human Immunodeficiency Virus (HIV), Influenza A Virus (IAV), and murine RNA tumor virus. It has a characteristic.
최근 비-외피단백질 바이러스인 노로바이러스와의 강한 결합력이 있는 것으로 보고되었으나, 콘카나발린 A는, T세포의 분열을 유발하는 독성 문제를 가지고 있어, 인체 적용에 어려움이 있다.Recently, it has been reported that there is strong binding force with norovirus, a non-enveloped protein virus, but concanavalin A has a toxicity problem of causing division of T cells, making it difficult to apply to the human body.
이에 본 발명은, 콘카나발린 A 이외에 작두콩에 존재하는 카나발린(Canavalin)이 바이러스에 결합하여 바이러스 탈리 및 중화 가능성을 인지하였고, 콩 발효 공정을 통해 콘카나발린 A와 같은 독성 물질을 제거하고 항바이러스 특성을 갖는 유용단백질 서열로 정제하였으며, 이의 항바이러스 특성을 활용하기 위한 전략을 제시하는 것이다.Therefore, in the present invention, in addition to concanavalin A, canavalin present in soybean binds to the virus to recognize the possibility of virus elimination and neutralization, and through the soybean fermentation process, toxic substances such as concanavalin A are removed and anti-bacterial It was purified as a useful protein sequence with viral properties, and a strategy for utilizing its antiviral properties is proposed.
본 발명은, 상기 언급한 문제점을 해결하기 위해서 콩 발효 공정을 거쳐 독성을 제거하고 항바이러스 특성을 갖는 유용단백질 서열로 정제된 말단-절단된 카나발린을 획득하고, 이를 유효성분으로 포함하는 조성물을 제공하는 것이다.In order to solve the above-mentioned problems, the present invention removes toxicity through a soybean fermentation process and obtains a purified end-truncated canavalin with a useful protein sequence having antiviral properties, and a composition comprising it as an active ingredient is to provide
본 발명은, 콩 발효 공정을 통해서 독성 단백질을 제거하고 유용단백질을 확보할 수 있는, 본 발명에 의한 말단-절단된 카나발린을 포함하는 조성물의 제조방법에 관한 것이다. The present invention relates to a method for preparing a composition comprising end-truncated canavalin according to the present invention, which can remove toxic proteins and secure useful proteins through a soybean fermentation process.
그러나, 본 발명이 해결하고자 하는 과제는 이상에서 언급한 것들로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 해당 분야 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the problem to be solved by the present invention is not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
본 발명의 일 실시예에 따라, 콩 유래 말단-절단된 카나발린을 유효성분으로 포함하는, 바이러스의 탈리, 중화 또는 이 둘을 위한, 조성물에 관한 것이다. According to one embodiment of the present invention, it relates to a composition for virus elimination, neutralization, or both, comprising soy-derived end-truncated canavalin as an active ingredient.
본 발명의 일 실시예에 따라, 상기 조성물은, 바이러스 검사를 위한 검체 수집에 이용되는 것일 수 있다. According to one embodiment of the present invention, the composition may be used for collecting samples for virus testing.
본 발명의 일 실시예에 따라, 상기 조성물은, 바이러스가 부착된 부위에서 바이러스를 탈리, 중화 또는 이 둘에 의해서 바이러스 검체를 수집하는 것일 수 있다.According to one embodiment of the present invention, the composition may collect a virus sample by detaching, neutralizing, or both of the virus from the site where the virus is attached.
본 발명의 일 실시예에 따라, 상기 바이러스가 부착된 부위는, 생체 세포, 생체 조직, 체액 및 기재 중 적어도 하나 이상인 것일 수 있다.According to one embodiment of the present invention, the site to which the virus is attached may be at least one of a living cell, a living tissue, a body fluid, and a substrate.
본 발명의 일 실시예에 따라, 콩 유래 말단-절단된 카나발린을 유효성분으로 포함하는, 바이러스의 감염 예방 또는 치료를 위한, 조성물에 관한 것이다.According to one embodiment of the present invention, it relates to a composition for preventing or treating viral infection, comprising soy-derived end-truncated canavalin as an active ingredient.
본 발명의 일 실시예에 따라, 상기 조성물은, 바이러스가 부착된 부위에서 바이러스를 탈리, 중화 또는 이 둘에 의해서 바이러스에 의한 감염을 예방 또는 치료하는 것일 수 있다.According to one embodiment of the present invention, the composition may prevent or treat infection by a virus by detaching, neutralizing, or both of the virus at the site where the virus is attached.
본 발명의 일 실시예에 따라, 상기 말단-절단된 카나발린은, C-말단 절단된 카나발린인 것일 수 있다.According to one embodiment of the present invention, the terminal-truncated canavalin may be C-terminal truncated canavalin.
본 발명의 일 실시예에 따라, 상기 말단-절단된 카나발린은, 카나발린 아미노산 서열 중 C-말단 절단된 서열을 포함하는 것일 수 있다.According to one embodiment of the present invention, the terminal-truncated canavalin may include a C-terminal truncated sequence among the canavalin amino acid sequences.
본 발명의 일 실시예에 따라, 상기 말단-절단된 카나발린은, 바이러스 스파이크 수용체 결합 도메인과 수용체의 결합 저해 기능을 갖는 것일 수 있다.According to one embodiment of the present invention, the end-truncated canavaline may have a binding inhibitory function between the viral spike receptor binding domain and the receptor.
본 발명의 일 실시예에 따라, 상기 말단-절단된 카나발린은, 바이러스 스파이크 수용체 결합 도메인과 수용체의 결합을 예방하거나 또는 결합을 탈리시키는 것일 수 있다.According to one embodiment of the present invention, the end-truncated canavaline may prevent binding of the viral spike receptor binding domain and the receptor or detach the binding.
본 발명의 일 실시예에 따라, 상기 조성물은, 분말, 용액, 젤, 페이스트, 서스펜션 또는 에어로졸인 것일 수 있다.According to one embodiment of the present invention, the composition may be a powder, solution, gel, paste, suspension or aerosol.
본 발명의 일 실시예에 따라, 상기 조성물은, 경구용 또는 비강용 조성물인 것일 수 있다.According to one embodiment of the present invention, the composition may be an oral or nasal composition.
본 발명의 일 실시예에 따라, 상기 조성물은, 경구 세척액 또는 비강 세척액인 것일 수 있다.According to one embodiment of the present invention, the composition may be an oral irrigation solution or a nasal irrigation solution.
본 발명의 일 실시예에 따라, 상기 경구 세척액은, 가글 또는 치약이고, 상기 비강 세척액은, 비강 스프레이인 것일 수 있다.According to one embodiment of the present invention, the oral washing liquid may be gargle or toothpaste, and the nasal washing liquid may be a nasal spray.
본 발명의 일 실시예에 따라, 상기 바이러스는, 호흡기 바이러스인 것일 수 있다.According to one embodiment of the present invention, the virus may be a respiratory virus.
본 발명의 일 실시예에 따라, 상기 호흡기 바이러스는, 인플루엔자바이러스, 코로나바이러스, 세포융합 바이러스, 아데노바이러스 (adenovirus), 인간 보카바이러스(human bocavirus) 및 리노바이러스(rhinovirus)로 이루어진 군에서 선택된 적어도 하나 이상을 포함하는 것일 수 있다.According to an embodiment of the present invention, the respiratory virus is at least one selected from the group consisting of influenza virus, coronavirus, syncytial virus, adenovirus, human bocavirus, and rhinovirus. It may contain more than one.
본 발명의 일 실시예에 따라, 상기 말단-절단된 카나발린은, 작두콩 또는 콩 발효물에서 유래되고, 상기 말단-절단된 카나발린은, 생체 독성-프리(free)인 것일 수 있다.According to one embodiment of the present invention, the end-truncated canavalin is derived from soy beans or fermented soybeans, and the end-truncated canavalin may be biotoxic-free.
본 발명의 일 실시예에 따라, 콩, 콩껍질 또는 이 둘을 발효시키는 단계; 및 발효물에서 말단-절단된 카나발린을 분리하는 단계; 를 포함하는, 조성물의 제조방법에 관한 것이다.According to one embodiment of the present invention, fermenting beans, bean hulls or both; and isolating the end-truncated canavalin from the fermented product; It relates to a method for producing a composition comprising a.
본 발명의 일 실시예에 따라, 상기 발효시키는 단계 중 단백질 가수분해에 의해서 유용단백질인 말단-절단된 카나발린이 생성되는 것일 수 있다.According to one embodiment of the present invention, during the fermentation step, proteolysis may produce useful protein, terminal-truncated canavalin.
본 발명의 일 실시예에 따라, 상기 발효시키는 단계는, 바실러스 서브틸러스를 접종한 이후에 30 ℃내지 40 ℃에서 발효시키는 것일 수 있다.According to one embodiment of the present invention, the step of fermenting may be to ferment at 30 ° C to 40 ° C after inoculating Bacillus subtilus.
본 발명의 일 실시예에 따라, 상기 발효시키는 단계는, 30 % 내지 50 %의 용존 산소 분위기에서 1 일 이상 동안 발효시키는 것일 수 있다.According to one embodiment of the present invention, the step of fermenting may be fermentation for one or more days in a dissolved oxygen atmosphere of 30% to 50%.
본 발명은, 콩 발효 공정을 통해서 콩에 존재하는 독성을 갖는 콘카나발린 A(concanavalin A; ConA)와 같은 단백질을 제거하고 정제 공정을 거쳐 항바이러스 특성을 갖는 유용단백질인 말단-절단된 카나발린을 확보하고, 이를 활용하여 바이러스 탈리, 중화 및/또는 이 둘을 갖는 항바이러스 조성물을 제공할 수 있다.The present invention, through a soybean fermentation process, removes proteins such as concanavalin A (ConA) having toxicity present in soybeans, and through a purification process, terminal-truncated canavalin, a useful protein having antiviral properties, is It is possible to secure and utilize this to provide an antiviral composition having virus elimination, neutralization and / or both.
또한, 본 발명은, 콩과 같은 천연소재를 활용하여 코로나바이러스와 같은 호흡기 바이러스가 수용체(receptor)에 부착되지 못하도록 방해하여 예방할 수 있는 용도로 사용하고, 이미 붙어 있는 바이러스를 떼어내며, 바이러스를 감염력이 없는 중화된 상태로 만들 수 있는, 조성물 및 이를 이용한 의약외품 및 의약품을 제공할 수 있다. In addition, the present invention utilizes natural materials such as soybeans to prevent respiratory viruses such as coronaviruses from attaching to receptors, remove viruses that are already attached, and reduce virus infectivity. It is possible to provide a composition that can be made in a neutralized state without this, and quasi-drugs and pharmaceuticals using the same.
또한, 본 발명은, 콩과 같은 천연소재를 활용하여 생체 외 및/또는 생체 내에서 부착된 바이러스를 탈리 및/또는 중화시켜 바이러스 진단 및 검사를 위한 바이러스 검체를 수집하기 위한 전처리 조성물을 제공할 수 있다.In addition, the present invention can provide a pretreatment composition for collecting virus samples for virus diagnosis and examination by detaching and/or neutralizing viruses attached in vitro and/or in vivo using natural materials such as soybeans. there is.
도 1은, 본 발명의 일 실시예에 따라, 본 발명에 의한 작두콩을 발효한 분회 단백질을 Size Exclusion Chromatography를 이용하여 정제한 크로마토 그램을 도식화하여 나타낸 것이다.1 is a schematic diagram showing a chromatogram purified by using Size Exclusion Chromatography for protein fraction obtained by fermenting soybeans according to the present invention according to an embodiment of the present invention.
도 2는, 본 발명의 일 실시예에 따라, 본 발명에 의한 TCan 단백질을 질량분석기를 이용하여 intact 질량값을 얻어 질량값을 계산하기 위한 기초 데이터를 나타낸 것이다.2 shows basic data for calculating the mass value by obtaining the intact mass value of the TCan protein according to the present invention using a mass spectrometer, according to an embodiment of the present invention.
도 3은, 본 발명의 일 실시예에 따라, 카나발린의 전체 단백질 서열에서 N-terminal sequencing 방법으로부터 LSSQDKPFNL 서열을 확보하여 전체 단백질 서열에서 절단(truncation) 부분을 표시(LSSQDKPFNL 부분의 볼드체)하였고, 도 2의 단백질 질량값 정보를 활용하여 계산된 질량값에 대응되는 전체 단백질의 서열 부분(밑줄 친 부분)을 표시하여 나타낸 것이다.Figure 3, according to an embodiment of the present invention, the LSSQDKPFNL sequence obtained from the N-terminal sequencing method in the entire protein sequence of canavalin, and the truncation portion in the entire protein sequence is indicated (LSSQDKPFNL portion bold), It shows the sequence part (underlined part) of the whole protein corresponding to the mass value calculated using the protein mass value information of FIG. 2.
도 4는, 본 발명의 일 실시예에 따라, 본 발명에 의한 TCan 조성물의 hACE2 수용체와 RBD(SARS-CoV-2 spike receptor binding domain) 결합을 저해하는 효과를 도식화하여 나타낸 것이다.FIG. 4 schematically shows the inhibitory effect of the TCan composition according to the present invention on the binding between hACE2 receptor and RBD (SARS-CoV-2 spike receptor binding domain) according to an embodiment of the present invention.
도 5a는, 본 발명의 일 실시예에 따라, 염증성 사이토카인의 발현량 변화를 나타낸 것으로, 인터페론 감마(IFN-γ)에 관한 것이다. (데이터에서 평균 ± SD(n = 3 per group); **p < 0·01 및 ****p < 0·0001 vs. naive control (NC) group; ##p < 0·01 및 ####p < 0·0001 vs. con A group이다.)5a shows changes in the expression level of inflammatory cytokines, and relates to interferon gamma (IFN-γ), according to an embodiment of the present invention. (Mean ± SD from data (n = 3 per group); **p < 0 01 and ****p < 0 0001 vs. naive control (NC) group; ##p < 0 01 and ## ##p < 0 0001 vs. con A group.)
도 5b는, 본 발명의 일 실시예에 따라, 염증성 사이토카인의 발현량 변화를 나타낸 것으로, 인터루킨(IL)-17에 관한 것이다. (데이터에서 평균 ± SD(n = 3 per group); **p < 0·01 및 ****p < 0·0001 vs. naive control (NC) group; ##p < 0·01 및 ####p < 0·0001 vs. con A group이다.)5b shows changes in the expression level of inflammatory cytokines, and relates to interleukin (IL)-17, according to an embodiment of the present invention. (Mean ± SD from data (n = 3 per group); **p < 0 01 and ****p < 0 0001 vs. naive control (NC) group; ##p < 0 01 and ## ##p < 0 0001 vs. con A group.)
도 5c는, 본 발명의 일 실시예에 따라, 염증성 사이토카인의 발현량 변화를 나타낸 것으로, IL-4에 관한 것이다. (데이터에서 평균 ± SD(n = 3 per group); **p < 0·01 및 ****p < 0·0001 vs. naive control (NC) group; ##p < 0·01 및 ####p < 0·0001 vs. con A group이다.)5c shows changes in the expression level of inflammatory cytokines, and relates to IL-4, according to an embodiment of the present invention. (Mean ± SD from data (n = 3 per group); **p < 0 01 and ****p < 0 0001 vs. naive control (NC) group; ##p < 0 01 and ## ##p < 0 0001 vs. con A group.)
도 5d는, 본 발명의 일 실시예에 따라, 염증성 사이토카인의 발현량 변화를 나타낸 것으로, IL-5에 관한 것이다. (데이터에서 평균 ± SD(n = 3 per group); **p < 0·01 및 ****p < 0·0001 vs. naive control (NC) group; ##p < 0·01 및 ####p < 0·0001 vs. con A group이다.)Figure 5d shows changes in the expression level of inflammatory cytokines, and relates to IL-5, according to an embodiment of the present invention. (Mean ± SD from data (n = 3 per group); **p < 0 01 and ****p < 0 0001 vs. naive control (NC) group; ##p < 0 01 and ## ##p < 0 0001 vs. con A group.)
도 5e는, 본 발명의 일 실시예에 따라, 염증성 사이토카인의 발현량 변화를 나타낸 것으로, Con A 및 BE 주입된 C57BL/6 마우스의 비장세포에서 IL-13에 관한 것이다.(데이터에서 평균 ± SD(n = 3 per group); **p < 0·01 및 ****p < 0·0001 vs. naive control (NC) group; ##p < 0·01 및 ####p < 0·0001 vs. con A group이다.)Figure 5e shows changes in the expression level of inflammatory cytokines, according to an embodiment of the present invention, and relates to IL-13 in splenocytes of C57BL/6 mice injected with Con A and BE. (In the data, mean ± SD (n = 3 per group); **p < 0 01 and ****p < 0 0001 vs. naive control (NC) group; ##p < 0 01 and ####p < 0 0001 vs. con A group.)
도 6a는, 본 발명의 일 실시예에 따라, C57BL/6 마우스의 생존율에 대한 Con A 및 BE 주입 효과를 나타낸 것으로, VC(vehicle control); 40 mg/kg, 80 mg/kg 및 160 mg/kg (체중, BW)) 농도에서 Con A를 주입한 후 최대 24시간까지 매시간 생존을 기록한 것이다.Figure 6a, according to an embodiment of the present invention, shows the effect of Con A and BE injection on the survival rate of C57BL/6 mice, VC (vehicle control); Hourly survival was recorded up to 24 hours after injection of Con A at concentrations of 40 mg/kg, 80 mg/kg and 160 mg/kg (body weight, BW)).
도 6b는, 본 발명의 일 실시예에 따라, C57BL/6 마우스의 생존율에 대한 Con A 및 BE 주입 효과를 나타낸 것으로, VC(vehicle control); 75 mg/kg, 160 mg/kg 및 200 mg/kg (BW 체중) 농도에서 BE를 주입한 후 최대 24시간까지 매시간 생존을 기록한 것이다.Figure 6b, according to an embodiment of the present invention, shows the effect of Con A and BE injection on the survival rate of C57BL/6 mice, VC (vehicle control); Hourly survival was recorded up to 24 hours after injection of BE at concentrations of 75 mg/kg, 160 mg/kg and 200 mg/kg (BW body weight).
도 7a는, 본 발명의 일 실시예에 따라, 마우스에서 Con A 및 BE 점적주입에 대한 반응으로 간 바이오마커(AST) 수준의 변화 결과를 나타낸 것으로, 바이오마커 농도는 Con A 및 BE 투여 마우스의 혈청에서 측정되었습니다(정맥 내 또는 기관 내 점적주입, 각각 5-15 또는 10-40 mg/kg BW). (평균 ± SD (n = 5 mice per group), ****p < 0·0001 vs. VC, AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level GGT, g-glutamyl transferase이다.)Figure 7a shows the results of changes in liver biomarker (AST) levels in response to Con A and BE instillation in mice according to an embodiment of the present invention, and the biomarker concentrations of Con A and BE administered mice Measured in serum (intravenous or intratracheal infusion, 5-15 or 10-40 mg/kg BW, respectively). (Mean ± SD (n = 5 mice per group), **** p < 0 0001 vs. VC, AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level GGT, g-glutamyl transferase.)
도 7b는, 본 발명의 일 실시예에 따라, 마우스에서 Con A 및 BE 점적주입에 대한 반응으로 간 바이오마커(ALT) 수준의 변화 결과를 나타낸 것으로, 바이오마커 농도는 Con A 및 BE 투여 마우스의 혈청에서 측정되었습니다(정맥 내 또는 기관 내 점적주입, 각각 5-15 또는 10-40 mg/kg BW). (평균 ± SD (n = 5 mice per group), ****p < 0·0001 vs. VC, AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level GGT, g-glutamyl transferase이다.)Figure 7b shows the results of changes in liver biomarker (ALT) levels in response to Con A and BE instillation in mice, according to an embodiment of the present invention, and the biomarker concentrations of Con A and BE administered mice Measured in serum (intravenous or intratracheal infusion, 5-15 or 10-40 mg/kg BW, respectively). (Mean ± SD (n = 5 mice per group), **** p < 0 0001 vs. VC, AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level GGT, g-glutamyl transferase.)
도 7c는, 본 발명의 일 실시예에 따라, 마우스에서 Con A 및 BE 점적주입에 대한 반응으로 간 바이오마커(TBIL) 수준의 변화 결과를 나타낸 것으로, 바이오마커 농도는 Con A 및 BE 투여 마우스의 혈청에서 측정되었습니다(정맥 내 또는 기관 내 점적주입, 각각 5-15 또는 10-40 mg/kg BW). (평균 ± SD (n = 5 mice per group), ****p < 0·0001 vs. VC, AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level GGT, g-glutamyl transferase이다.)Figure 7c shows the results of changes in liver biomarker (TBIL) levels in response to Con A and BE instillation in mice, according to an embodiment of the present invention, and the biomarker concentrations of Con A and BE administered mice Measured in serum (intravenous or intratracheal infusion, 5-15 or 10-40 mg/kg BW, respectively). (Mean ± SD (n = 5 mice per group), **** p < 0 0001 vs. VC, AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level GGT, g-glutamyl transferase.)
도 7d는, 본 발명의 일 실시예에 따라, 마우스에서 Con A 및 BE 점적주입에 대한 반응으로 간 바이오마커(GGT) 수준의 변화 결과를 나타낸 것으로, 바이오마커 농도는 Con A 및 BE 투여 마우스의 혈청에서 측정되었습니다(정맥 내 또는 기관 내 점적주입, 각각 5-15 또는 10-40 mg/kg BW).(평균 ± SD (n = 5 mice per group), ****p < 0·0001 vs. VC, AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level GGT, g-glutamyl transferase이다.)Figure 7d shows the results of changes in liver biomarker (GGT) levels in response to Con A and BE instillation in mice, according to an embodiment of the present invention, and the biomarker concentrations of Con A and BE administered mice Measured in serum (intravenous or intratracheal instillation, 5-15 or 10-40 mg/kg BW, respectively). (Mean ± SD (n = 5 mice per group), **** p < 0 0001 vs .VC, AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin level GGT, g-glutamyl transferase.)
이하 첨부된 도면을 참조하여 본 발명의 실시예들을 상세히 설명한다. 본 발명을 설명함에 있어서, 관련된 공지 기능 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 것이다. 또한, 본 명세서에서 사용되는 용어들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 각 도면에 제시된 동일한 참조 부호는 동일한 부재를 나타낸다.Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted. In addition, the terms used in this specification are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Like reference numerals in each figure indicate like elements.
명세서 전체에서, 어떤 부재가 다른 부재 "상에" 위치하고 있다고 할 때, 이는 어떤 부재가 다른 부재에 접해 있는 경우뿐 아니라 두 부재 사이에 또 다른 부재가 존재하는 경우도 포함한다.Throughout the specification, when a member is said to be located “on” another member, this includes not only a case where a member is in contact with another member, but also a case where another member exists between the two members.
명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification, when a certain component is said to "include", it means that it may further include other components rather than excluding other components.
본 발명은, 말단-절단된 카나발린을 포함하는 조성물 및 이의 제조방법에 관한 것이다.The present invention relates to compositions comprising truncated canavaline and methods for their preparation.
본 발명은, 말단-절단된 카나발린을 포함하는 조성물에 관한 것으로, 본 발명의 일 실시예에 따라, 상기 조성물은, 천연소재인 콩에서 유래된 것으로, 콩 발효물에서 정제되고 항바이러스 특성을 갖는 유용단백질인 말단-절단된 카나발린을 포함할 수 있다. 상기 말단-절단된 카나발린은, 바이러스가 수용체에 부착되는 것을 방지하거나 수용체에 부착된 바이러스를 떼어내거나 및/또는 감염력이 없는 중화상태로 전환시킬 수 있다. 이러한 바이러스와 말단-절단된 카나발린의 상호작용을 이용하여 바이러스 감염 예방 및/또는 치료를 위한 조성물, 바이러스 세척, 바이러스 검사를 위한 검체 획득을 위한 전처리 조성물 등으로 활용할 수 있다.The present invention relates to a composition comprising end-truncated canavalin. According to one embodiment of the present invention, the composition is derived from soybean, which is a natural material, purified from fermented soybean, and exhibits antiviral properties. It may include end-truncated canavalin, which is a useful protein with The end-truncated canavaline can prevent viruses from attaching to receptors or can detach viruses attached to receptors and/or convert them to a non-infectious neutral state. By using the interaction between the virus and the terminal-truncated canavaline, it can be used as a composition for preventing and/or treating viral infection, cleaning a virus, and a pretreatment composition for obtaining a sample for a virus test.
본 발명의 일 예로, 상기 말단-절단된 카나발린은, C-말단 절단된 카나발린이며, 카나발린 아미노산 서열 중 C-말단 절단된 서열을 포함할 수 있다. 예를 들어, 도 3에 나타낸 바와 같이, C-말단 절단된 서열은 LSSQDKPFNL로 확인할 수 있다.As an example of the present invention, the truncated canavalin is C-terminal truncated canavalin, and may include a C-terminal truncated sequence among canavalin amino acid sequences. For example, as shown in Figure 3, the C-terminal truncated sequence can be identified by LSSQDKPFNL.
본 발명의 일 예로, 상기 말단-절단된 카나발린은, 바이러스의 수용체 결합 도메인과 수용체의 결합을 방해 및 저해하고, 이미 결합된 바이러스를 둘러싸서 탈리시킬 수 있다. 예를 들어, 바이러스 스파이크 수용체 결합 도메인과 수용체의 결합 저해 기능을 제공할 수 있다. 또한, 바이러스 스파이크 수용체 결합 도메인과 수용체의 결합을 예방하거나 또는 결합을 탈리시킬 수 있다. 또한, 바이러스를 중화시켜 감염력을 낮출 수 있다.As an example of the present invention, the end-truncated canavalin may interfere with and inhibit the binding of the receptor-binding domain of the virus to the receptor, and surround and detach the already bound virus. For example, it may provide a binding inhibitory function between the viral spike receptor binding domain and the receptor. In addition, binding of the viral spike receptor binding domain to the receptor may be prevented or the binding may be dissociated. In addition, it can neutralize viruses to lower infectivity.
본 발명의 일 실시예에 따라, 상기 콩 유래 말단-절단된 카나발린을 유효성분으로 포함하는, 바이러스의 감염 예방 및/또는 치료를 위한, 조성물에 관한 것이다.According to one embodiment of the present invention, it relates to a composition for preventing and/or treating viral infection, comprising the soy-derived end-truncated canavalin as an active ingredient.
본 발명의 일 예로, 상기 조성물은, 바이러스가 부착된 부위에서 바이러스를 탈리, 중화 또는 이 둘에 의해서 바이러스에 의한 감염을 예방 및/또는 치료할 수 있다. 또한, 상기 조성물은, 세포(생체 내), 인체 조직, 인체 기관 등에서 바이러스가 부착되는 것을 방지 및 방해하거나 바이러스를 중화시켜 바이러스 감염을 예방하고 및/또는 치료할 수 있다. 예를 들어, 가글로 구강을 세척하거나 비강 내에 스프레이하여 인체 구강 점막 또는 비강 점막에 바이러스의 부착을 방지하거나 부착된 바이러스를 탈리시키고 중화시켜 감염을 예방하고 및/또는 치료할 수 있다. 또한, 구강 및 구강 점막 외에 인체 내 점막 등에 바이러스의 부착을 방지하거나 부착된 바이러스를 탈리시키고 중화시켜 감염을 예방하고 및/또는 치료할 수 있다.As an example of the present invention, the composition can prevent and/or treat infection by a virus by detaching, neutralizing, or both of the virus at the site where the virus is attached. In addition, the composition can prevent and/or treat viral infections by preventing or interfering with attachment of viruses in cells (in vivo), human tissues, human organs, and the like, or by neutralizing viruses. For example, infection can be prevented and/or treated by rinsing the mouth with a gargle or spraying into the nasal cavity to prevent attachment of the virus to the human oral mucosa or nasal mucosa, or to detach and neutralize the attached virus. In addition, infection can be prevented and/or treated by preventing the attachment of viruses to mucous membranes in the human body in addition to the oral cavity and oral mucosa, or detaching and neutralizing the attached viruses.
본 발명의 일 실시예에 따라, 상기 조성물은, 바이러스의 탈리, 중화 또는 이 둘을 위한, 조성물일 수 있다.According to one embodiment of the present invention, the composition may be a composition for desorption, neutralization or both of the virus.
본 발명의 일 예로, 상기 조성물은, 체액, 검체, 세포(생체 외 및/또는 생체 내), 검체 채취용 기재, 인체 조직 등에 부착된 바이러스의 표면을 둘러싸서 포획하여 바이러스를 탈리 및/또는 중화시킬 수 있다. 예를 들어, 구강 표면, 코 표면, 타액, 콧물 및 소변 중 적어도 하나 이상에서 포함되거나 부착된 바이러스를 탈리 및/또는 중화시키고, 이를 수집하여 바이러스 검체를 획득할 수 있다. 예를 들어, 인체 구강 점막에 부착된 바이러스를 탈리하기 위해 가글로 세척하고, 구강 점막 외에도 인체 점막 등에 부착된 바이러스를 탈리하기 위해 세척할 수 있다.As an example of the present invention, the composition surrounds and captures the surface of a virus attached to a bodily fluid, specimen, cell (ex vivo and/or in vivo), substrate for specimen collection, human tissue, etc. to desorb and/or neutralize the virus. can make it For example, a virus sample may be obtained by detaching and/or neutralizing viruses contained or attached to at least one of the oral surface, nasal surface, saliva, nasal mucus, and urine, and collecting them. For example, it can be washed with a gargle to remove viruses attached to the oral mucosa of the human body, and washed to remove viruses attached to the human oral mucosa and the like in addition to the oral mucosa.
본 발명의 일 예로, 상기 체액은, 생체(또는, 인체)로부터 배출되는 액체이며, 상기 체액은, 타액, 콧물 및 소변 중 적어도 이상일 수 있다. 상기 체액은, 생체 조직 및/또는 세포 내 또는 표면에 존재할 경우에 상기 조성물의 적용 시 탈리 및/또는 중화된 세포와 함께 검체로 수득될 수 있다.As an example of the present invention, the bodily fluid is a liquid discharged from a living body (or human body), and the bodily fluid may be at least one of saliva, nasal mucus, and urine. The bodily fluid, when present in or on the surface of biological tissues and/or cells, may be obtained as a specimen together with detached and/or neutralized cells upon application of the composition.
본 발명의 일 예로, 상기 획득한 검체는, 바이러스 진단 키트 또는 PCR에 도입하여 바이러스 검사에 이용될 수 있다.As an example of the present invention, the obtained sample may be introduced into a virus diagnostic kit or PCR and used for virus testing.
본 발명의 일 실시예에 따라, 상기 조성물은, 생체 무독성이며, 예를 들어, T세포 분열을 억제 또는 프리(free)하므로 생체 독성의 유발을 방지할 수 있다.According to one embodiment of the present invention, the composition is non-toxic to the body, and, for example, suppresses or frees T cell division, so that the induction of toxicity to the body can be prevented.
본 발명의 일 실시예에 따라, 상기 바이러스는, 외피 보유 바이러스, 외피 미보유 바이러스 또는 이 둘이고, 예를 들어, 호흡기 바이러스일 수 있다. 상기 호흡기 바이러스는, 인플루엔자 바이러스(예를 들어, 인플루엔자바이러스 A (Influenza virus A : IVA) 및 인플루엔자바이러스 B(Influenza virus B : IVB)), 코로나바이러스, 세포융합 바이러스(RSV, 예를 들어, 호흡기세포융합바이러스 A (respiratory syncytial virus A : RSVA) 및 호흡기세포융합바이러스 B (respiratorysyncytial virus B : RSVB)), 아데노바이러스 (adenovirus), 인간 보카바이러스(human bocavirus), 리노바이러스(rhinovirus), 인간 메타뉴모바이러스(human metapneumovirus, hMPV), 중동호흡기 바이러스 (MERS-CoV), 사스 연관 코로나바이러스 (SARS-associated coronavirus: SARS-coV), 인간 파라인플루엔자바이러스 1(human parainfluenza virus 1: HPIV 1), 인간 파라인플루엔자바이러스 2(human parainfluenza virus 2: HPIV 2) 및 인간 파라인플루엔자바이러스 3(human parainfluenza virus 3: HPIV 3)로 이루어진 군에서 선택된 적어도 하나 이상을 포함할 수 있다.According to one embodiment of the present invention, the virus is an enveloped virus, a non-enveloped virus, or both, and may be, for example, a respiratory virus. The respiratory virus includes influenza virus (eg, influenza virus A (IVA) and influenza virus B (IVB)), coronavirus, syncytial virus (RSV, eg, respiratory cell Syncytial virus A (RSVA) and respiratory syncytial virus B (RSVB), adenovirus, human bocavirus, rhinovirus, human metapneumovirus (human metapneumovirus, hMPV), Middle East respiratory virus (MERS-CoV), SARS-associated coronavirus (SARS-coV), human parainfluenza virus 1 (HPIV 1), human parainfluenza virus 2 (human parainfluenza virus 2: HPIV 2) and human parainfluenza virus 3 (human parainfluenza virus 3: HPIV 3).
본 발명의 일 실시예에 따라, 상기 말단-절단된 카나발린은, 바이러스와 수용체의 결합 저해, 탈리 및/또는 중화를 제공하기 위해서, 상기 조성물 중 1 ppm 내지 1000; 10 ppm 내지 1000 ppm; 10 ppm 내지 500 ppm; 또는 10 ppm 내지 200 ppm일 수 있다.According to one embodiment of the present invention, the truncated canavalin is present in the composition in an amount of 1 ppm to 1000; 10 ppm to 1000 ppm; 10 ppm to 500 ppm; or 10 ppm to 200 ppm.
본 발명의 일 실시예에 따라, 상기 조성물은, 고상, 액상, 또는 이 둘의 혼합된 형태일 수 있으며, 예를 들어, 분말, 용액, 젤, 페이스트, 에멀젼, 콜로이드성 분산액, 서스펜션 또는 에어로졸 형태일 수 있다.According to one embodiment of the present invention, the composition may be in the form of a solid, a liquid, or a mixture of the two, for example, a powder, solution, gel, paste, emulsion, colloidal dispersion, suspension or aerosol form. can be
본 발명의 일 실시예에 따라, 상기 조성물은, 경구용 또는 비강용 조성물일 수 있으며, 이는 용도에 따라 의료용 또는 비의료용으로 구분될 수 있다.According to one embodiment of the present invention, the composition may be an oral or nasal composition, which may be classified into medical or non-medical use depending on the purpose.
예를 들어, 상기 조성물은, 가글, 치약 등의 경구 세척액, 비강 스프레이 등의 비강 세척액 등일 수 있으며, 비강 및/또는 경구 세척을 통해 바이러스 감염 예방, 치료 등을 위한 항바이러스 기능을 제공할 수 있다. 즉, 구강을 가글링으로 세척하여 바이러스에 대한 예방 및/또는 치료 효과를 제공하고, 구강을 가글링한 이후 가글액 내에 존재하는 탈리 및 중화된 바이러스를 수집하여 바이러스 검체를 수득할 수 있다. 또한, 비강 내에 분말, 액 등을 흡입, 분사 또는 튜브 등의 기구를 이용한 주입 방식 등으로 투여하고, 세척하여 바이러스에 대한 예방 및/또는 치료 효과를 제공하거나 탈리 및/또는 중화된 바이러스를 수집할 수 있다.For example, the composition may be a gargle, an oral wash such as toothpaste, a nasal wash such as a nasal spray, and the like, and may provide an antiviral function for preventing and treating viral infections through nasal and/or oral washing. . That is, it is possible to obtain a virus sample by washing the oral cavity with gargling to provide a preventive and/or therapeutic effect against viruses, and by collecting desorbed and neutralized viruses present in the gargle solution after gargling the mouth. In addition, powder, liquid, etc. may be administered in the nasal cavity by inhalation, spraying, or injection using a device such as a tube, and washed to provide a preventive and / or therapeutic effect against viruses or to collect desorbed and / or neutralized viruses. can
예를 들어, 상기 조성물은, 투여 경로에 따라 경구 투여용 또는 비강 투여용으로 구분되어 바이러스 감염 예방, 치료 등과 같은 항바이러스 기능을 제공할 수 있다.For example, the composition may be divided into oral administration or nasal administration according to the route of administration to provide antiviral functions such as prevention and treatment of viral infection.
예를 들어, 상기 조성물은, 경구 내 또는 비강 내 바이러스 검체 획득을 위한 체액 전처리 조성물일 수 있으며, 예를 들어, 가글을 통해 구강을 헹구고 뱉어내는 방식으로 부착된 바이러스를 탈리 및/또는 중화시켜 바이러스 검체를 수득할 수 있다. 또는 생체 외에 세포, 기재 등에 부착된 바이러스를 탈리 및/또는 중화시키고, 바이러스 검사, 진단 등 같이 다양한 목적에 따라 바이러스 검체를 수집할 수 있다.For example, the composition may be a bodily fluid pretreatment composition for obtaining a virus sample in the oral cavity or nasal cavity, and, for example, by rinsing the mouth through gargling and spitting it out, the virus is detached and/or neutralized. specimens can be obtained. Alternatively, viruses attached to cells, substrates, etc. may be desorbed and/or neutralized outside the body, and virus samples may be collected for various purposes such as virus testing and diagnosis.
본 발명의 일 실시예에 따라, 상기 조성물은, 본 발명의 목적을 벗어나지 않는다면, 제형에 따라 본 발명의 기술분야에서 적용되는 담체 성분, 베이서 성분, 향료, 첨가제, 용매 등을 포함할 수 있으며, 본 명세서에는 구체적으로 언급하지 않는다.According to one embodiment of the present invention, the composition may include a carrier component, a base component, a fragrance, an additive, a solvent, etc. applied in the technical field of the present invention according to the formulation, if it does not deviate from the object of the present invention, , which is not specifically mentioned in this specification.
본 발명은, 본 발명에 의한 조성물의 제조방법에 관한 것으로, 본 발명의 일 실시예에 따라, 상기 조성물의 제조방법은, 콩, 콩껍질 또는 이 둘을 발효시키는 단계; 및 발효물에서 말단-절단된 카나발린을 분리하는 단계;를 포함할 수 있다. 상기 제조방법은, 콩, 예를 들어, 작두콩에서 독성을 갖는 ConA 독성단백질을 발효 공정으로 제거하고, 항바이러스 유용단백질을 확보할 수 있는 발효 조건을 제공하고, 상기 유용단백질을 포함하는 조성물을 제공하는 것이다.The present invention relates to a method for producing a composition according to the present invention, and according to an embodiment of the present invention, the method for producing the composition includes fermenting beans, bean hulls or both; and separating the end-truncated canavalin from the fermented product. The manufacturing method removes ConA toxic protein, which is toxic in soybeans, for example, soybeans, through a fermentation process, provides fermentation conditions capable of securing useful antiviral proteins, and provides a composition containing the useful protein. is to do
본 발명의 일 실시예에 따라, 상기 발효시키는 단계 중 단백질 가수분해에 의해서 유용단백질인 말단-절단된 카나발린이 형성될 수 있다. 즉, 상기 발효시키는 단계는, 용매 내에 콩, 콩껍질 또는 이 둘을 혼합하고, 바실러스 서브틸러스를 접종한 이후 10℃ 내지 45 ℃; 20 ℃ 내지 40℃; 실온 내지 40 ℃또는 30 ℃내지 40 ℃의 온도; 30 % 내지 50 %의 용존 산소 분위기; 또는 이 둘의 조건에서 10 시간 이상; 1일 이상; 또는 1일 내지 5일; 또는 1일 내지 4일 동안; 발효 공정을 진행할 수 있다. 예를 들어, 상기 콩은, 작두콩이며, 작두콩 외에 다른 콩이 적용될 수 있다.According to one embodiment of the present invention, during the fermentation step, proteolysis can form useful protein, terminal-truncated canavalin. That is, in the step of fermenting, after mixing soybeans, soybean hulls, or both in a solvent, and inoculating Bacillus subtilus, the temperature is 10 ° C to 45 ° C; 20° C. to 40° C.; a temperature between room temperature and 40° C. or between 30° C. and 40° C.; 30% to 50% dissolved oxygen atmosphere; or at least 10 hours in both conditions; more than 1 day; or 1 to 5 days; or for 1 to 4 days; The fermentation process may proceed. For example, the beans are small beans, and other beans other than small beans may be applied.
본 발명의 일 예로, 상기 발효시키는 단계는, pH 5 내지 8; pH 6 내지 7.5 또는 바람직하게는 중성 영역에서 이루어질 수 있다.As an example of the present invention, the step of fermenting is pH 5 to 8; pH 6 to 7.5 or preferably in the neutral region.
본 발명의 일 실시예에 따라, 상기 용매는, 물, 유기용매 또는 이 둘을 포함하고, 예를 들어, 상기 유기용매는, 탄소수 1 내지 6의 알코올, 부틸렌글리콜 및 프로필렌글리콜 중 적어도 하나 이상을 포함할 수 있다.According to one embodiment of the present invention, the solvent includes water, an organic solvent, or both, and for example, the organic solvent includes at least one of alcohol having 1 to 6 carbon atoms, butylene glycol, and propylene glycol. can include
본 발명의 일 실시예에 따라, 상기 발효물에서 말단-절단된 카나발린을 분리하는 단계는, 상기 발효물에서 독성 물질을 제거하고 유용단백질 서열을 분리하여 말단-절단된 카나발린을 획득할 수 있다. 예를 들어, 상기 발효물을 크기 배제 크로마토그래피를 이용하여 말단-절단된 카나발린을 분리할 수 있다.According to one embodiment of the present invention, the step of separating the end-truncated canavalin from the fermented product may remove toxic substances from the fermented product and isolate useful protein sequences to obtain the end-truncated canavalin. there is. For example, the fermentation can be used to isolate end-truncated canavalin using size exclusion chromatography.
이하, 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.
(1) TCan(truncated canavalin)의 제조 및 정제(1) Preparation and purification of TCan (truncated canavalin)
용존산소(20-40% 포화도) 및 pH 7.0 조건 및 33 ℃에서 (또는, 정제수(96.059 % ~ 100 % 미만) 내에서) 4일 동안 작두콩 (sword bean, Canavalia gladiata)을 바실러스 서브틸러스(Bacillus subtilis)에 의해 단백질 가수분해(proteolysed)를 진행하였다. 단백질 가수분해 후, 생성물을 4 ℃에서 15분 동안 원심분리(12,000Хg)하고, 상청액을 수집한 이후 동결건조하였다(이하, BE (콩 발효 추출물)). 최종 정제는 PBS 완충액 (140mM NaCl, 2.7mM KCl, 6.5mM Na2H2PO, 1.5mM KH2PO, pH7.4)으로 평형화된 WTC-050S5 컬럼(Wyatt Technology, Santa Barbara, CA)를 사용하여 크기 배제 크로마토그래피(size-exclusion chromatography (SEC))로 수행하였다. 순수 단백질은 -80 ℃에서 보관된 LN2에서 동결되고, TCan의 SEC 프로필은 도 1에 나타내었다.Sword bean (Canavalia gladiata) was cultured under dissolved oxygen (20-40% saturation) and pH 7.0 conditions and at 33 ° C (or in purified water (96.059% to less than 100%)) for 4 days. subtilis) was proteolysed. After protein hydrolysis, the product was centrifuged (12,000 Хg) at 4° C. for 15 minutes, and the supernatant was collected and lyophilized (hereinafter referred to as BE (fermented soybean extract)). Final purification was performed using a WTC-050S5 column (Wyatt Technology, Santa Barbara, CA) equilibrated with PBS buffer (140 mM NaCl, 2.7 mM KCl, 6.5 mM Na 2 H 2 PO, 1.5 mM KH 2 PO, pH7.4). Size-exclusion chromatography (SEC) was performed. Pure protein was frozen in LN2 stored at -80 °C, and the SEC profile of TCan is shown in Figure 1.
(2) UPLC-ESI-QTOF 질량분석기를 이용하여 단백질 전체 질량값 분석(top-down법)(2) Total protein mass analysis using UPLC-ESI-QTOF mass spectrometer (top-down method)
온라인 하향식 MS(On-line top-down MS)는 Synapt G2-S QTOF 질량 분석기(Waters Corp., USA)에 연결된 waters ACQUITY UPLC I-Class 시스템에서 수행되었다. 정제된 TCan 2μl를 2.1 Х 50 mm ACQUITY UPLC BEH300 C4 컬럼(입자 크기 1.7um)에 주입하였다. 농도구배는 5 % B(아세토니트릴 중 0.1% 포름산)에서 시작하여 400 μl로 전달되었고, 6.7분에 95 % B로 상승하고, 9.0 분에 5 % B로 상승하였다.On-line top-down MS was performed on a waters ACQUITY UPLC I-Class system connected to a Synapt G2-S QTOF mass spectrometer (Waters Corp., USA). 2 μl of purified TCan was injected onto a 2.1 Х 50 mm ACQUITY UPLC BEH300 C4 column (particle size 1.7 μm). The gradient started at 5% B (0.1% formic acid in acetonitrile) and was delivered in 400 μl, rising to 95% B in 6.7 min and 5% B in 9.0 min.
질량 분석기는 ESI-포지티브 MSE 연속체 데이터 수집 모드(ESI-Positive MSE continuum data acquisition mode), 모세관 전압 3.0 kV, 콘 전압 50 V, 탈용매 가스 800 L/hr, 소스 온도 120 ℃, 탈용매화 온도 300 ℃에서 작동되었다. 데이터는 500-4,000 m/z에서 1 스펙트럼/초(spectra/second)로 수집되었다. 매스 디콘볼루션(mass deconvolution)에는 최대 엔트로피 알고리즘(MaxEnt1)이 사용되었다. 디콘볼루션은 MaxEnt1을 100회 반복하여 예상 질량, 목표 분해능 0.5Da를 중심으로 1,500-2,600Da 범위에서 수행되었다. TCan의 전자분무 스펙트럼은 도 2에 나타내었다.The mass spectrometer is ESI-Positive MSE continuum data acquisition mode, capillary voltage 3.0 kV, cone voltage 50 V, desolvation gas 800 L/hr, source temperature 120 °C, desolvation temperature 300 °C worked in Data were collected at 1 spectra/second from 500-4,000 m/z. A maximum entropy algorithm (MaxEnt1) was used for mass deconvolution. Deconvolution was performed in the range of 1,500-2,600 Da centered on the expected mass and target resolution of 0.5 Da by repeating MaxEnt1 100 times. The electrospray spectrum of TCan is shown in FIG. 2 .
(3) Tcan의 N-말단 시퀀싱(N-Terminal Sequencing)(3) N-Terminal Sequencing of Tcan
단백질 샘플을 SDS-PAGE 겔에서 PVDF 막으로 옮기고 LC 492 단백질 시퀀싱 시스템(LC 492 Protein Sequencing System, Applied Biosystems Instruments, USA)을 사용하여 막에서 전달된 단백질 밴드를 분석했습니다. 그 결과는 도 3에 나타내었다.Protein samples were transferred from an SDS-PAGE gel to a PVDF membrane and the protein bands transferred on the membrane were analyzed using the LC 492 Protein Sequencing System (Applied Biosystems Instruments, USA). The results are shown in Figure 3.
실시예 1: TCan 조성물의 hACE2 수용체와 RBD의 결합 저해능 평가Example 1: Evaluation of binding inhibitory ability of TCan composition between hACE2 receptor and RBD
hACE2 수용체와 SARS-CoV-2 spike receptor binding domain (RBD) 결합 저해능은 COVID-19 Neutralizing Antibody ELISA kit (Cat.# KA6111, abnova)를 사용하여 평가하였다. hACE2 수용체와 RBD는 kit에 동봉된 것을 사용하였으며, 제조사에서 제공하는 설명서에 따라 평가를 수행하였다. TCan 조성물이 RBD를 중화하여 hACE2 결합을 저해하는 효과 (Neutralization effect)는 다음과 같은 방법으로 실시하였다. RBD와 TCan 조성물을 37 ℃에서 2시간 동안 반응하였다. 이후 hACE2 수용체가 코팅된 플레이트에 첨가하여 RBD와 결합을 2시간 동안 유도하였다.The ability to inhibit hACE2 receptor and SARS-CoV-2 spike receptor binding domain (RBD) binding was evaluated using the COVID-19 Neutralizing Antibody ELISA kit (Cat.# KA6111, abnova). The hACE2 receptor and RBD included in the kit were used, and evaluation was performed according to the instructions provided by the manufacturer. The effect of the TCan composition neutralizing RBD to inhibit hACE2 binding (Neutralization effect) was performed by the following method. The RBD and TCan compositions were reacted at 37 °C for 2 hours. Then, the hACE2 receptor was added to the coated plate to induce binding with RBD for 2 hours.
TCan 조성물이 RBD와 hACE2간의 선(先)결합을 탈리시키는 효과 (Detachment effect)는 다음과 같은 방법으로 실시하였다. hACE2 수용체가 코팅된 플레이트에 RBD를 첨가하여 이들의 결합을 37 ℃에서 2시간 동안 유도하였다. 그런 다음, TCan 조성물로 이들 결합을 1분간 2회 세척하였다.The effect of the TCan composition to detach the prebond between RBD and hACE2 (Detachment effect) was performed in the following way. RBD was added to the hACE2 receptor-coated plate to induce their binding at 37 °C for 2 hours. These bonds were then washed twice for 1 minute with the TCan composition.
TCan 조성물이 hACE2와의 반응하여 RBD가 hACE2 결합을 예방하는 효과 (Prevention effect)는 다음과 같은 방법으로 실시하였다. hACE2 수용체가 코팅된 플레이트에 TCan 조성물을 첨가하고 37 ℃에서 1시간 동안 반응시켰다. 세척용 완충용액으로 세척한 후, RBD를 첨가하여 hACE2와 결합을 37 ℃에서 2시간 동안 유도하였다.The effect of the TCan composition reacting with hACE2 to prevent the binding of RBD to hACE2 was evaluated by the following method. The TCan composition was added to the hACE2 receptor-coated plate and reacted at 37° C. for 1 hour. After washing with a washing buffer, RBD was added to induce binding with hACE2 at 37 °C for 2 hours.
RBD 반응이 종료된 후, 모든 실험구는 세척용 완충용액으로 비결합된 RBD를 세척하고 hACE2-RBD 결합체를 검출할 수 있도록 kit에 동봉된 HRP가 결합된 항체를 첨가하여 37℃에서 1시간 동안 반응하였다. 완충용액으로 세척한 후, tetramethylbezidine (TMB) 용액으로 5분간 반응시킨 다음 1N HCl 용액을 첨가하여 TMB 반응을 정지하여 450 nm 파장에서 흡광도를 측정하였다. RBD-hACE2 결합율은 아래 계산식에 의해 산정하였다. 
는 TCan 무처리구 흡광도 값을 의미하며, 
는 농도별 TCan의 흡광도 값을 의미한다. After the completion of the RBD reaction, all experimental groups were washed with a washing buffer to unbound RBD, and the HRP-conjugated antibody included in the kit was added to detect the hACE2-RBD conjugate, followed by reaction at 37°C for 1 hour. did After washing with a buffer solution, the mixture was reacted with a tetramethylbezidine (TMB) solution for 5 minutes, and then 1N HCl solution was added to stop the TMB reaction, and absorbance was measured at a wavelength of 450 nm. RBD-hACE2 binding rate was calculated by the following formula. Means the absorbance value of the TCan untreated group, Means the absorbance value of TCan for each concentration.
도 4에 나타낸 바와 같이, TCan 조성물은 RBD와 hACE2 결합을 농도 의존적으로 저해하는 효과를 보였다. RBD와 hACE2 수용체와의 결합 정도를 50 % 억제시키는 TCan 조성물의 농도 (IC50)는 예방(prevention) 방법은 4.569 ppm, 중화(neutralization) 방법은 8.935 ppm, 탈리(detachment) 방법은 >35.89 ppm으로 확인되었다. 따라서, RBD와 hACE2 수용체와의 결합을 저해하는 TCan 조성물을 제공할 수 있다.As shown in Figure 4, the TCan composition showed an inhibitory effect on the binding between RBD and hACE2 in a concentration-dependent manner. The concentration (IC 50 ) of the TCan composition that inhibits the degree of binding between RBD and the hACE2 receptor by 50% was 4.569 ppm for the prevention method, 8.935 ppm for the neutralization method, and >35.89 ppm for the detachment method. Confirmed. Accordingly, it is possible to provide a TCan composition that inhibits the binding between RBD and the hACE2 receptor.
실시예 2: 생체 독성 실험Example 2: Biotoxicity test
(1) 쥐의 비장세포에서 Con A 및 BE (콩 발효 추출물)에 의한 유사분열 반응(Mitogenic response)(1) Mitogenic response by Con A and BE (fermented soybean extract) in mouse splenocytes
Con A는 면역 체계를 활성화하고 림프구를 모집하며 사이토카인 생성을 유도하는 비장 세포에서 유사분열 반응을 유도하는 것으로 잘 알려져 있다. Con A와 BE에 의해 유도된 유사분열 효과를 비교하기 위해서, 2·5 μg/mL의 농도에서 Con A 또는 BE로 비장세포를 처리하여 생산된 T 헬퍼(Th) 사이토카인((IFN-g for Th1, IL-17 for Th17, and IL-4, IL-5, and IL-13 for Th2 cells)을 조사하였다. 그 결과는 도 5a, 도 5b, 도 5c, 도 5d 및 도 5e에 나타내었다. 도 5a, 도 5b, 도 5c, 도 5d 및 도 5e에서 BE와 비교하여, Con A가 모든 Th 사이토카인의 훨씬 더 높은 생산을 유도하는 것을 보여준다. IFN-g, IL-17, IL-5 및 IL-13의 생산은 실험 기간 동안 BE 처리된 비장세포에서 증가했지만, 생산은 Con A 처리군보다 유의하게 낮다.Con A is well known to induce a mitotic response in spleen cells that activates the immune system, recruits lymphocytes, and induces cytokine production. To compare the mitotic effects induced by Con A and BE, T helper (Th) cytokine ((IFN-g for Th1, IL-17 for Th17, and IL-4, IL-5, and IL-13 for Th2 cells) were investigated The results are shown in Figures 5a, 5b, 5c, 5d and 5e. Figures 5A, 5B, 5C, 5D and 5E show that Con A induces significantly higher production of all Th cytokines compared to BE, IFN-g, IL-17, IL-5 and The production of IL-13 increased in BE-treated splenocytes during the experimental period, but the production was significantly lower than that of the Con A-treated group.
(2) Con A와 BE가 쥐의 생존율에 미치는 영향(2) Effects of Con A and BE on the survival rate of mice
생 시스템(iving system)에서 Con A와 BE의 독성 효과를 조사하고 비교하기 위해 C57BL/6 마우스에서 기관 내 점적 후 사망률에 대한 Con A와 BE의 영향을 조사하였다. 그 결과는, 도 6a 및 도 6b에 나타내었다. 도 6a 및 도 6b에서 생존율은 C57BL/6 마우스에서 7개의 그룹으로 나누어 매시간 모니터링하였다: 비히클 대조군(VC); 40, 80 및 160mg/kg 체중(BW) 농도에서 Con A의 처리된 그룹(Con A 40, Con A 80, Con A 160 그룹) 및 75, 160 및 200 mg/kg BW의 농도에서 BW 처리된 그룹(BE 75, BE 160, BE 200 groups)(처리 시간: 24 h.). VC 및 BE-처리군은 100 %의 생존율을 나타낸 반면, Con A 처리군은 생존율 감소를 나타낸다. Con A 처리군의 생쥐의 생존율은 처리 17시간째부터 감소하기 시작하고, Con A 160 처리군의 생쥐의 생존율은 처리 24시간째까지 관찰되지 않는다. 한편, Con A 40 및 Con A 80 그룹의 마우스에서는 처리 24시간 후 60 %의 생존이 관찰된다. 이러한 결과는 BE가 마우스에 독성이 없음을 나타내며, 이는 시험관내 세포독성 결과와 일치한다.To investigate and compare the toxic effects of Con A and BE in the iving system, the effects of Con A and BE on mortality after intratracheal instillation in C57BL/6 mice were investigated. The results are shown in FIGS. 6A and 6B. In Figures 6a and 6b, survival rate was monitored hourly in C57BL/6 mice divided into 7 groups: vehicle control (VC); Con A treated groups at concentrations of 40, 80 and 160 mg/kg body weight (BW) (Con A 40, Con A 80, and Con A 160 groups) and BW treated groups at concentrations of 75, 160 and 200 mg/kg BW (BE 75, BE 160, BE 200 groups) (treatment time: 24 h.). The VC and BE-treated groups showed a survival rate of 100%, whereas the Con A-treated group showed a reduced survival rate. The survival rate of mice in the Con A treatment group starts to decrease from 17 hours after treatment, and the survival rate of mice in the Con A 160 treatment group is not observed until 24 hours after treatment. On the other hand, 60% survival was observed in the mice of the Con A 40 and Con A 80 groups after 24 hours of treatment. These results indicate that BE is not toxic to mice, which is consistent with the in vitro cytotoxicity results.
(3) 쥐의 간 독성에 대한 Con A 및 BE의 효과 (3) Effects of Con A and BE on liver toxicity in rats
Con A는 T 세포를 활성화하여 간 손상을 유발하는 사이토카인을 분비한다. 간 손상에 대한 Con A와 BE의 영향을 비교하기 위해 간 기능의 바이오마커인 혈청 AST, ALT, TBIL 및 GGT의 수치는, 마우스에 Con A 및 BE 투여 후 24시간 후에 평가하였다. 그 결과는, 도 7a, 도 7b, 도 7c 및 도 7d에 나타내었다. 도 7a, 도 7b, 도 7c 및 도 7d에서 투여 방식이 간 바이오마커 수준에 영향을 미친다는 것을 보여준다. Con A 및 BE 처리 마우스 모두 기관 내 점적 투여는 간 바이오마커의 수준에 유의한 영향을 미치지 않았지만, 정맥내 투여된 고농도의 Con A(15 mg/kg BW)는 모듬 바이오 마커(p 값 < 0.05)의 발현을 현저하게 증가시키는 것을 알 수 있다.Con A activates T cells to secrete cytokines that cause liver damage. To compare the effects of Con A and BE on liver damage, levels of serum AST, ALT, TBIL, and GGT, which are biomarkers of liver function, were evaluated 24 hours after administration of Con A and BE to mice. The results are shown in Figs. 7a, 7b, 7c and 7d. Figures 7a, 7b, 7c and 7d show that the mode of administration affects liver biomarker levels. Intratracheal instillation of both Con A and BE-treated mice did not significantly affect the levels of liver biomarkers, but high concentrations of Con A (15 mg/kg BW) administered intravenously did not significantly affect all biomarkers (p-value < 0.05). It can be seen that the expression of
본 발명은, 작두콩 발효과정을 통하여 독성을 제거하고 항바이러스 특성을 가지는 유용단백질 서열을 정제하였다. 또한, 정확한 서열분석을 위해 질량분석기와 N-terminal sequencing 방법을 이용하여 intact 질량값을 확보하고 N-terminal sequencing을 통하여 말단 절단된 카나발린(Tcan, Truncated Canavalin Tcan)을 확인하였다. 또한, 정제된 단백질의 바이러스에 대한 특성을 확인하기 위하여 ELISA 방법으로 분석하였다. 즉, 작두콩 발효 추출물 중의 항바이러스 유용효과가 있는 Tcan을 확보하였고, Tcan은 hACE2에 강한 결합을 보여 RBD 가 결합하지 못하도록 하는 결과를 얻었다. 이는 SARS-CoV-2 바이러스의 spike 단백질 중 RBD 영역이 수용체에 결합하지 못하는 것으로 향후 바이러스 감염을 예방할 수 있는 가글 및 비강 세척제 등의 의약품 및 의약외품에 활용할 수 있다. 또한, hACE2에 결합된 RBD를 TCan이 떼어내는 결과로 보아 이미 구강 및 비강 내에 붙어 있는 바이러스를 효과적으로 씻어 낼 수 있는 가글 및 비강 세척제, 검체 수집을 위한 체액 전처리 용액 등의 의약품 및 의약외품에 활용할 수 있다.In the present invention, useful protein sequences having antiviral properties and removing toxicity through the fermentation process of soybean were purified. In addition, for accurate sequencing, mass spectrometry and N-terminal sequencing were used to secure intact mass values, and truncated canavalin (Tcan, Truncated Canavalin Tcan) was confirmed through N-terminal sequencing. In addition, in order to confirm the characteristics of the purified protein against the virus, it was analyzed by ELISA method. That is, Tcan, which has an antiviral useful effect in the fermented soybean extract, was secured, and Tcan showed strong binding to hACE2, preventing RBD from binding. This is because the RBD region of the spike protein of the SARS-CoV-2 virus cannot bind to the receptor, so it can be used for pharmaceuticals and quasi-drugs such as gargles and nasal washes that can prevent viral infection in the future. In addition, as a result of TCan detaching RBD bound to hACE2, it can be used for pharmaceuticals and quasi-drugs such as gargles and nasal washes that can effectively wash out viruses already attached to the oral and nasal cavities, and body fluid pretreatment solutions for sample collection. .
이상과 같이 실시예들이 비록 한정된 실시예와 도면에 의해 설명되었으나, 해당 기술분야에서 통상의 지식을 가진 자라면 상기의 기재로부터 다양한 수정 및 변형이 가능하다. 예를 들어, 설명된 기술들이 설명된 방법과 다른 순서로 수행되거나, 및/또는 설명된 구성요소들이 설명된 방법과 다른 형태로 결합 또는 조합되거나, 다른 구성요소 또는 균등물에 의하여 대치되거나 치환되더라도 적절한 결과가 달성될 수 있다. 그러므로, 다른 구현들, 다른 실시예들 및 특허청구범위와 균등한 것들도 후술하는 특허청구범위의 범위에 속한다.As described above, although the embodiments have been described with limited examples and drawings, those skilled in the art can make various modifications and variations from the above description. For example, even if the described techniques are performed in a different order from the described method, and/or the described components are combined or combined in a different form than the described method, or substituted or replaced by other components or equivalents. Appropriate results can be achieved. Therefore, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.
Claims (21)
- 콩 유래 말단-절단된 카나발린을 유효성분으로 포함하는, Containing soybean-derived end-truncated canavalin as an active ingredient,바이러스의 탈리, 중화 또는 이 둘을 위한, 조성물.A composition for desorption, neutralization or both of the virus.
- 제1항에 있어서,According to claim 1,상기 조성물은,The composition,바이러스 검사를 위한 검체 수집에 이용되는 것인, Which is used for sample collection for virus testing,조성물. composition.
- 제1항에 있어서,According to claim 1,상기 조성물은, The composition,바이러스가 부착된 부위에서 바이러스를 탈리, 중화 또는 이 둘에 의해서 바이러스 검체를 수집하는 것인, To collect virus samples by detaching, neutralizing, or both of the virus at the site where the virus is attached,조성물.composition.
- 제1항에 있어서,According to claim 1,상기 바이러스가 부착된 부위는, The site where the virus is attached is생체 세포, 생체 조직, 체액 및 기재 중 적어도 하나 이상인 것인,At least one or more of biological cells, biological tissues, body fluids, and substrates,조성물.composition.
- 콩 유래 말단-절단된 카나발린을 유효성분으로 포함하는, Containing soybean-derived end-truncated canavalin as an active ingredient,바이러스의 감염 예방 또는 치료를 위한, 조성물.A composition for preventing or treating viral infection.
- 제5항에 있어서,According to claim 5,상기 조성물은, The composition,바이러스가 부착된 부위에서 바이러스를 탈리, 중화 또는 이 둘에 의해서 바이러스에 의한 감염을 예방 또는 치료하는 것인, To prevent or treat infection by a virus by detaching, neutralizing, or both of the virus at the site where the virus is attached,조성물.composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 말단-절단된 카나발린은,The end-truncated canavalin,C-말단 절단된 카나발린인 것인, C-terminal truncated canavaline,조성물.composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 말단-절단된 카나발린은,The end-truncated canavalin,카나발린 아미노산 서열 중 C-말단 절단된 서열을 포함하는 것인, Including the C-terminal truncated sequence of the canavalin amino acid sequence,조성물.composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 말단-절단된 카나발린은,The end-truncated canavalin,바이러스 스파이크 수용체 결합 도메인과 수용체의 결합 저해 기능을 갖는 것인, Having a binding inhibitory function of the virus spike receptor binding domain and the receptor,조성물.composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 말단-절단된 카나발린은, The end-truncated canavalin,바이러스 스파이크 수용체 결합 도메인과 수용체의 결합을 예방하거나 또는 결합을 탈리시키는 것인, To prevent binding of the virus spike receptor binding domain and the receptor or to detach the binding,조성물.composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 조성물은,The composition,분말, 용액, 젤, 페이스트, 서스펜션 또는 에어로졸인 것인, which is a powder, solution, gel, paste, suspension or aerosol;조성물.composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 조성물은, 경구용 또는 비강용 조성물인 것인,The composition is a composition for oral or nasal use,조성물.composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 조성물은, The composition,경구 세척액 또는 비강 세척액인 것인, Oral lavage or nasal lavage,조성물.composition.
- 제13항에 있어서,According to claim 13,상기 경구 세척액은, 가글 또는 치약이고,The oral washing liquid is gargle or toothpaste,상기 비강 세척액은, 비강 스프레이인 것인, The nasal wash solution is a nasal spray,조성물.composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 바이러스는, 호흡기 바이러스인 것인,The virus is a respiratory virus,조성물.composition.
- 제15항에 있어서,According to claim 15,상기 호흡기 바이러스는,The respiratory virus,인플루엔자바이러스, 코로나바이러스, 세포융합 바이러스, 아데노바이러스 (adenovirus), 인간 보카바이러스(human bocavirus) 및 리노바이러스(rhinovirus)로 이루어진 군에서 선택된 적어도 하나 이상을 포함하는 것인,Including at least one selected from the group consisting of influenza virus, coronavirus, syncytial virus, adenovirus, human bocavirus and rhinovirus,조성물. composition.
- 제1항 또는 제5항에 있어서,According to claim 1 or 5,상기 말단-절단된 카나발린은, 작두콩 또는 콩 발효물에서 유래되고,The end-truncated canavalin is derived from soy beans or fermented soybeans,상기 말단-절단된 카나발린은, 생체 독성-프리(free)인 것인, Wherein the end-truncated canavaline is biotoxic-free,조성물.composition.
- 콩, 콩껍질 또는 이 둘을 발효시키는 단계; 및 fermenting soybeans, soybean hulls or both; and발효물에서 말단-절단된 카나발린을 분리하는 단계;Separating the end-truncated canavalin from the fermented product;를 포함하는, including,조성물의 제조방법. Method for preparing the composition.
- 제18항에 있어서,According to claim 18,상기 발효시키는 단계 중 단백질 가수분해에 의해서 유용단백질인 말단-절단된 카나발린이 생성되는 것인, During the fermentation step, proteolysis produces useful protein, terminal-truncated canavalin,조성물의 제조방법. Method for preparing the composition.
- 제18항에 있어서,According to claim 18,상기 발효시키는 단계는,The fermentation step is바실러스 서브틸러스를 접종한 이후에 30 ℃내지 40 ℃에서 발효시키는 것인, Fermented at 30 ° C to 40 ° C after inoculation with Bacillus subtilus,조성물의 제조방법. Method for preparing the composition.
- 제18항에 있어서,According to claim 18,상기 발효시키는 단계는,The fermentation step is30 % 내지 50 %의 용존 산소 분위기에서 1 일 이상 동안 발효시키는 것인, Fermented for at least one day in a dissolved oxygen atmosphere of 30% to 50%,조성물의 제조방법.Method for preparing the composition.
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| NAKATSUKA Y., T. NAGASAWA, Y. YUMOTO, F. NAKAZAWA, Y. FURUICHI: "Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection", JOURNAL OF PERIODONTAL RESEARCH, vol. 49, no. 6, 3 February 2014 (2014-02-03), pages 801 - 809, XP093029178, DOI: 10.1111/jre.12166 * |
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|---|---|
| KR20230018886A (en) | 2023-02-07 |
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