Oral care composition, mouth wash and preparation method and application of mouth wash

Abstract

The invention relates to an oral care composition which is characterized by comprising the following components: a traditional Chinese medicine extract and virus blocking protein. The oral care composition and the mouthwash do not contain ethanol, so that the use scene and crowd of the mouthwash are expanded, and the oral care composition and the mouthwash can be widely applied. Wherein the key component, namely the virus blocking protein, is creatively added, the virus blocking agent has the characteristics of high affinity and multi-angle capture and adhesion of coronavirus, so that the virus loses the function and way of infecting an organism, finally, the transmission and infection of the virus, especially the coronavirus, are blocked, and the virus blocking protein is non-toxic to a human body, and has no toxic or side effect. By adding the compound into the oral care composition, specific disinfection on viruses can be effectively achieved.

Description

Technical Field

[0001] The present application belongs to the technical field of daily necessities, and relates to an oral care composition, a mouthwash, and a preparation method and application thereof, and in particular to an oral care composition, a mouthwash, and a preparation method and application thereof with the function of blocking coronavirus. Background Art

[0002] Oral diseases are common and frequently occurring diseases caused by various pathogenic bacteria in the oral cavity, which directly affect people's physical health. At the same time, the new coronavirus can also be transmitted and infected through the oral cavity. For a long time, people have mainly used various toothpastes and mouthwashes to clean their oral cavity to prevent the occurrence of oral diseases. However, the number of times toothpaste is used and the scenarios in which it is used are limited. In some scenarios, mouthwash can replace toothpaste to clean the oral cavity and prevent various diseases caused by the oral cavity. In order to improve the anti-inflammatory and antibacterial effects of oral care products, people have developed various anti-inflammatory mouthwashes. The existing anti-inflammatory effects are mainly achieved by adding disinfectants such as alcohol. However, the addition of alcohol will limit the applicable population and applicable occasions, such as use by minors and drivers. At the same time, the existing mouthwash products on the market do not contain active protein components that have a blocking effect on the new coronavirus (SARS-CoV-2). Summary of the invention

[0003] The technical problem to be solved by the present invention is: to solve the shortcomings of the mouthwash in the above-mentioned technology, thereby providing an oral care composition and mouthwash that are non-toxic to the human body and can effectively kill viruses. Since the oral care composition and mouthwash do not contain alcohol, they are theoretically suitable for all people.

[0004] The first aspect of the present invention provides an oral care composition, characterized in that it comprises the following ingredients: a traditional Chinese medicine extract and a virus blocking protein.

[0005] Furthermore, the Chinese medicine extract is selected from one or more of honeysuckle extract, green tea extract, grapefruit peel extract, orange peel extract, violet extract, scutellaria baicalensis extract, stevia extract, wild chrysanthemum extract or gallnut extract.

[0006] Furthermore, the Chinese herbal medicine extract is preferably honeysuckle extract and green tea extract.

[0007] Furthermore, the virus blocking protein is selected from coronavirus blocking protein.

[0008] Furthermore, the virus blocking protein is a spike protein (RBD) blocking protein; preferably, the virus blocking protein is preferably a SARS-CoV-2 spike protein (RBD) blocking protein.

[0009] Furthermore, the virus blocking protein includes the following domains: a spike protein binding domain (SBD), a rigid domain, an oligomer site and a membrane exclusion tail.

[0010] Furthermore, the different domains of the virus blocking protein are connected by covalently linked linking groups, preferably the linking groups are selected from polypeptides or disulfide bonds, and the connection order between the domains is not particularly limited. For example, in a preferred embodiment, the spike protein binding domain (SBD), rigid domain, oligomer site and membrane repulsion tail of the blocker are sequentially connected from segment N to segment C. The spike protein binding domain is a region that specifically binds to the spike protein. The present invention designs spike binding domains of various lengths (10-20 residues) based on the binding site of ACE2.

[0011] Furthermore, the spike binding domain comprises a polypeptide as shown in any one of the amino acid sequences of SEQ ID NO.1-3:

[0012] SEQ ID NO.1:

[0013] QVQLVESGGGLMQAGGSLRLSCAVSGRTFSTAAMGWFRQAPGKEREFVAAI RWSGGSA YYADSVKGRFTISRDKAKNTVYLQMNSLKYEDTAVYYCAQTHY VSYLLSDYATWPYDYWGQGTQVTVSSK;

[0014] SEQ ID NO.2:

[0015] DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEE VER;

[0016] SEQ ID NO.3:

[0017] SAEIDLGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSRA

[0018] SEAAKRIAKAIRKAADAIAEAAKIAARAAKDGDAARNAENAARKAKEFAEEQAKLADMYAELAKNGDKSSVLEQLKTFADK AFHEMEDRFYQAALAVFEAAAEAAAG

[0019] Further, the spike binding domain comprises a polypeptide having at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% homology with the polypeptide shown in any one of the amino acid sequences of SEQ ID NO.1-3.

[0020] Furthermore, the spike binding domain comprises a sequence that has one or several amino acid substitutions, deletions or additions compared to a polypeptide as shown in any of the amino acid sequences of SEQ ID NO.1-3, and the number of the substituted, deleted or added amino acids may be 1-20, preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0021] Preferably, in order to increase the binding affinity of the blocker to the spike protein, the blocker comprises at least one spike binding domain. Most preferably, the viral blocker has two spike binding domains, preferably comprising more than two complementary determining regions, such as 1-10, preferably 2-8, and most preferably 4-8.

[0022] The rigid domain can maintain the three-dimensional structure and improve the biological activity of the blocker. The rigid domain comprises eGFP (SEQ ID NO. 4), or rigid proteins improved on the basis of eGFP, such as mTagBFP2, moxCerulean3, AmCyanl, MiCy, ZsGreen, Clover, mVenus, ZsYellow1, mKO2, TurboRFP, tdTomato, eqFP611, mKate1.3, mNeptune2, miRFP670, mAme-trine, PAmCherry 2, mEos3.2, etc.

[0023] Furthermore, the rigid domain comprises a polypeptide having at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% homology to the polypeptide shown in the amino acid sequence of SEQ ID NO.4.

[0024] SEQ ID NO.4:

[0025] VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGK

[0026] LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG

[0027] TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQ

[0028] KNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0029] The membrane repelling tail further causes the blocker complex to repel the host cell membrane, thereby hindering the virus from entering the host cell. In combination, the modularly assembled blockers are expected to weaken the cell attachment ability of SARS-CoV-2 variants. The membrane repelling tail is a short-chain polymer of amino acids with negative charge, and the number of amino acids in the short-chain polymer is 0-50, preferably 2-40, more preferably 3-30, and most preferably 4-20. The negatively charged amino acids are aspartic acid and / or glutamic acid.

[0030] Furthermore, the oligomer site promotes oligomerization of the blocker and aggregation of the virus-blocker complex. The oligomer site is a single-chain protein or polypeptide with a polymerization function, and the polymerization function means that the single-chain protein has the function of naturally polymerizing into a polymer. Furthermore, the polymer includes but is not limited to oligomers with a monomer number of 2-10, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers and mixtures thereof, oligomers with a monomer number of 11-20 and polymers with a monomer number of more than 20. In a feasible embodiment, the oligomer site has

[0031] The polymerizable single-chain proteins are derived from natural polymerizable proteins and their functional variants.

[0032] Preferably, the single-chain protein with polymerization function has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% homology with a single peptide chain of streptavidin protein (SEQ ID NO.5).

[0033] Furthermore, the virus blocker is preferably the ConstantinTM blocking protein or a protein having at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% homology with its amino acid sequence.

[0034] SEQ ID NO.5:

[0035] SDVQSSLTGTWYNELNSKMELTANKDGTLTGKYLSKVGDVYVPYPLSGRYNLQPPAGQGVALGWAVSWENSKIHSATTWSGQFFSESSPVILTQWLLSSSTARGDVWESTLVGNDSFTKTAPTEQQIAHAQLHCRAPRLK.

[0036] Further preferably, the virus blocking protein is the Constin protein produced by Kangma (Shanghai) Biotechnology Co., Ltd., or a protein having at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% homology with its amino acid sequence.

[0037] The second aspect of the present invention provides a mouthwash comprising the oral care composition provided by the first aspect of the present invention.

[0038] Furthermore, the mouthwash further comprises a wetting agent, an emulsifier, a flavor enhancer, a cooling agent, an antibacterial agent, a pH regulator, a color enhancer and water.

[0039] Furthermore, the wetting agent is selected from one or more of propylene glycol, glycerol, sorbitol, ethylene glycol, polyethylene glycol or butylene glycol, and more preferably propylene glycol or glycerol.

[0040] Further, the emulsifier is selected from one or more of polysorbate, polysorbate, lecithin, poloxamer, hydroxypropyl-β-cyclodextrin, arginine, glycine, aspartic acid and glutamine. Further preferably, the emulsifier is selected from polysorbate.

[0041] Furthermore, the flavor enhancer is selected from one or more of xylitol, honey, cyclamate, sorbitol, mannitol, sucralose, trichlorogalactose, saccharin sodium, steviol glycoside, hydrogenated starch hydrolysate or aspartame.

[0042] Further, the cooling agent is selected from one or more of menthol, menthyl lactate, menthyl acetate, menthone glycerol ketal or eucalyptus oil, and more preferably menthol or menthyl amide.

[0043] Furthermore, the antibacterial agent is selected from one or more of cetylpyridinium chloride, potassium sorbate, domiphene, sodium benzoate, and parabens and their salts, and more preferably cetylpyridinium chloride or potassium sorbate.

[0044] Furthermore, the pH regulator is selected from one or more of citric acid, sodium citrate, boric acid, malic acid and sorbic acid, and more preferably citric acid or sodium citrate.

[0045] Furthermore, the color enhancer is selected from one or more of brilliant blue, sunset yellow, lemon yellow, carmine, allure red, food green and apple green, and more preferably brilliant blue.

[0046] Furthermore, the mouthwash comprises: a virus blocking protein having a molar concentration of 1 to 100 nmol / L; the following components recorded in weight percentage: 0.01% to 2% of a Chinese herbal medicine extract, 0.5% to 5% of a wetting agent, 0.01% to 1% of an emulsifier, 0.1% to 2% of a flavor enhancer, 0.01% to 1% of a cooling agent, 0.01% to 1% of an antibacterial agent, 0.01% to 1% of a pH adjuster, 0.0001% to 0.1% of a color enhancer; and the remainder of water.

[0047] Furthermore, the concentration of the virus blocking protein is 1 to 100 nmol / L, preferably 1 to 80 nmol / L, and more preferably 5 to 50 nmol / L.

[0048] Furthermore, in terms of weight percentage, the content of the Chinese herbal medicine extract is preferably 0.05% to 2%, more preferably 0.1% to 1.5%, and even more preferably 0.2% to 1%.

[0049] Furthermore, in terms of weight percentage, the wetting agent content is preferably 0.7% to 5%, more preferably 1% to 5%.

[0050] Furthermore, in terms of weight percentage, the emulsifier content is preferably 0.01% to 0.8%, more preferably 0.05% to 0.5%.

[0051] Furthermore, in terms of weight percentage, the content of the flavor enhancer is preferably 0.1% to 1.5%, more preferably 0.2% to 1%, and even more preferably 0.5% to 1%.

[0052] Furthermore, in terms of weight percentage, the content of the cooling agent is preferably 0.01% to 0.8%, more preferably 0.03% to 0.5%, and even more preferably 0.05% to 0.5%.

[0053] Furthermore, in terms of weight percentage, the content of the antibacterial agent is preferably 0.02% to 0.8%, more preferably 0.03% to 0.5%, and even more preferably 0.05% to 0.5%.

[0054] Furthermore, in terms of weight percentage, the content of the pH adjuster is preferably 0.05% to 1%, more preferably 0.05% to 0.8%, and even more preferably 0.1% to 0.5%.

[0055] Furthermore, in terms of weight percentage, the content of the color enhancer is preferably 0.0001% to 0.005%, more preferably 0.0001% to 0.003%, and even more preferably 0.0001% to 0.001%.

[0056] A third aspect of the present invention provides a method for preparing a mouthwash, characterized in that it comprises the following steps:

[0057] (1) mixing a wetting agent and a cooling agent, heating them, and then cooling them; (2) sequentially adding a Chinese herbal extract, an emulsifier, a flavor enhancer, a cooling agent, an antibacterial agent, a pH adjuster, a color enhancer, and the remaining water to the system of step (1), and mixing them evenly; (3) finally adding a virus blocking protein to the system, and stirring them evenly.

[0058] The fourth invention of the present invention provides the use of the oral care composition provided in the first aspect of the present invention and the mouthwash provided in the second aspect in the preparation of drugs for preventing viral infections, preventing or treating oral diseases and daily oral care.

[0059] Furthermore, the virus is a coronavirus.

[0060] The beneficial effects of the present invention are:

[0061] (1) This oral care composition and mouthwash do not contain ethanol, which expands the use scenarios and population of mouthwash and can be widely used. The key ingredient-virus blocking protein is creatively added. The virus blocking agent has the characteristics of high affinity and multi-angle capture and adhesion of coronavirus, thereby making the virus lose the function and path of infecting the body, and finally blocking the spread and infection of viruses, especially coronaviruses. The virus blocking protein is non-toxic to the human body. By adding it to the oral care composition, it can effectively achieve specific disinfection of viruses.

[0062] (2) The oral care composition and mouthwash contain Chinese medicinal extracts such as honeysuckle extract and green tea extract, which can have antibacterial and anti-inflammatory effects and prevent oral inflammation to a certain extent; the addition of cooling agents such as menthol and menthyl amine can effectively freshen the breath and remove bad breath; the addition of antibacterial agents such as cetylpyridinium chloride and potassium sorbate can effectively reduce the growth of oral bacteria. DETAILED DESCRIPTION

[0063] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

[0064] The present invention is further described below in conjunction with specific embodiments and examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably followed or referred to the conditions indicated in the specific embodiments described above, and then can be followed according to conventional conditions or according to the conditions recommended by the manufacturer.

[0065] Unless otherwise specified, the percentages and parts mentioned in the present invention are percentages and parts by weight.

[0066] Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

[0067] Unless otherwise specified, the temperature units in this application are all degrees Celsius (°C).

[0068] Nouns and terms

[0069] The following is an explanation or description of the meaning of some relevant "nouns" and "terms" used in the present invention, so as to better understand the present invention. The corresponding explanation or description applies to the full text of the present invention, both below and above. When the present invention involves references, the definitions of relevant terms, nouns, and phrases in the references are also quoted, but when they conflict with the definitions in the present invention, the definitions in the present invention shall prevail. When the definitions in the references conflict with the definitions in the present invention, it does not affect the cited components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc., which shall be subject to the contents determined in the references.

[0070] In the present invention, preferred embodiments such as “preferred”, “better”, “more preferred”, “better”, “most preferred” and “further preferred” do not constitute any limitation on the scope of coverage and protection scope of the invention, and are not used to limit the scope and embodiments of the present invention, but are only used to provide some embodiments as examples.

[0071] In the description of the present invention, preferred modes such as "one of the preferred embodiments", "one of the preferred modes", "one of the preferred embodiments", "one of the preferred examples", "preferred examples", "in a preferred embodiment", "some preferred examples", "some preferred modes", "preferably", "preferably", "preferably", "more preferably", "more preferably", "further preferably", "most preferably", and illustrative enumeration modes such as "one of the embodiments", "one of the modes", "example", "specific example", "for example", "as an example", "for example", "such as", "such as", etc., do not constitute any limitation on the scope of coverage and protection scope of the invention, and the specific features described in each mode are included in at least one specific embodiment of the present invention. In the present invention, the specific features described in each mode may be combined in a suitable manner in any one or more specific embodiments. In the present invention, the technical features or technical solutions corresponding to each preferred mode may also be combined in any suitable manner.

[0072] In the present invention, "any combination thereof" means "greater than 1" in terms of quantity, and means a group consisting of the following situations in terms of coverage: "select any one of them, or select a group consisting of at least two of them".

[0073] In the present invention, the descriptions of "one or more", "one or more" and "one or more" have the same meaning as "at least one", "at least one", "a combination thereof", "or a combination thereof", "and combinations thereof", "or any combination thereof", "and any combination thereof", etc. and can be used interchangeably to indicate that the quantity is equal to "1" or "greater than 1".

[0074] In the present invention, "or / and" and "and / or" are used to represent "optionally one of them or optionally a combination of them", and also represent at least one of them.

[0075] The prior art means described in the present invention in such ways as “usually”, “conventional”, “general”, “frequently” and “often” are also cited as references to the contents of the present invention. Unless otherwise specified, they can be regarded as one of the preferred ways of some technical features of the present invention. It should be noted that they do not constitute any limitation on the scope of coverage and protection scope of the invention.

[0076] All documents mentioned in the present invention and documents directly or indirectly cited by these documents are cited in this application as references, just as if each document was cited individually as a reference.

[0077] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (including but not limited to the embodiments) can be combined with each other to form a new or preferred technical solution, as long as it can be used to implement the present invention. Due to space limitations, they will not be described one by one.

[0078] the term

[0079] Blocking

[0080] In the context of the present invention, blocking generally means reducing, preventing or inhibiting viral entry and / or reducing, preventing or inhibiting biological pathways mediated by viral envelope proteins and / or their viral receptors, as measured using appropriate in vitro, cellular or in vivo assays (such as those mentioned herein).

[0081] In particular, blocking may mean reducing, preventing or inhibiting viral entry and / or reducing, preventing or inhibiting biological pathways mediated by viral envelope proteins and / or their viral receptors, as measured using appropriate in vitro, cellular or in vivo assays (such as those mentioned herein), compared to normal (i.e., naturally occurring) viral entry and / or normal biological pathways mediated by viral envelope proteins and / or their viral receptors in the same assay under the same conditions but in the absence of the blocking agent of the invention, reducing, preventing or inhibiting by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more.

[0082] Specific binding

[0083] The term "specific binding" or similar terms refers to the formation of a relatively stable complex between two molecules under certain conditions. Specific binding is characterized by high affinity and low to medium capacity. Nonspecific binding is usually characterized by low affinity and medium to high capacity. Generally, when the affinity constant K is above 10 6 M -1 Or preferably higher than 10 8 M -1 When the binding conditions are changed, non-specific binding is considered to be specific. If necessary, non-specific binding can be reduced without substantially affecting specific binding by changing the binding conditions. The above conditions are well known in the art, and those skilled in the art can select appropriate conditions using conventional techniques. The conditions are usually defined by antibody concentration, solution ionic strength, temperature, time for binding, concentration of non-related molecules (e.g., serum albumin, casein), etc.

[0084] In vitro cell-free protein synthesis system

[0085] In the present invention, the expressions "in vitro cell-free protein synthesis system", "in vitro expression system", "in vitro protein synthesis system", "in vitro protein synthesis reaction system", "cell-free protein synthesis system" and the like have the same meaning. Description methods such as protein in vitro synthesis system, in vitro protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system) and the like. Including in vitro translation system, in vitro transcription translation system (IVTT system) and the like. In the present invention, the IVTT system is preferred. We also call the in vitro protein synthesis system a "protein synthesis factory" (Protein Factory). In vitro protein synthesis reaction refers to the reaction of synthesizing protein in an in vitro cell-free synthesis system, which at least includes the translation process. Including but not limited to IVTT reaction (in vitro transcription translation reaction). In the present invention, IVTT reaction is preferred. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein in vitro. Therefore, we also call this type of in vitro protein synthesis system the D2P system, D-to-P system, D_to_P system, and DNAto-Protein system; the corresponding in vitro protein synthesis method is also called the D2P method, D-to-P method, D_to_P method, and DNA-to-Protein method.

[0086] Coronavirus

[0087] The term "coronavirus" in the present invention refers to coronaviruses that belong to the genus Coronavirus of the family Coronaviridae of the order Nidovirales in systematic classification and use ACE2 as a binding receptor, including but not limited to SARS-CoV, MERS-CoV, SARS-CoV-2, etc.

[0088] spike protein

[0089] The "spike protein" in the present invention refers to a protein on the surface of the coronavirus that can directly (e.g., by interacting with a viral receptor (e.g., ACE2)) or indirectly (e.g., by mediating the interaction of one or more other proteins or molecules with a viral receptor such as ACE2) mediate viral attachment to a target host cell.

[0090] The virus blocking protein described in the present invention is produced by protein engineering technology. After coding optimization, the gene of the blocking protein is cloned into an expression vector and then transferred into a host cell or an in vitro cell-free reaction system. After incubation and purification, the virus blocking protein is obtained.

[0091] The host cell host cells are well known in the art, including but not limited to E. coli, CHO cells, Chinese hamster ovary, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, HEK-293 cells and many other cell lines. Spodoptera frugiperda or Trichoplusiani), amphibian cells, bacterial cells, plant cells and fungal cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataeaminuta, Pichialindneri), Pichia opuntiae, Pichia athermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0092] Furthermore, the in vitro cell-free protein synthesis system includes, but is not limited to, an in vitro protein synthesis system of Escherichia coli, an in vitro protein synthesis system of bacteria, an in vitro protein synthesis system of mammalian cells (such as HF9, Hela, CHO, HEK293), an in vitro protein synthesis system of plant cells, an in vitro protein synthesis system of yeast cells, and an in vitro protein synthesis system of insect cells. Preferably, it is an in vitro protein synthesis system of yeast, more preferably, an in vitro protein synthesis system of Kluyveromyces, and most preferably, an in vitro protein synthesis system of Kluyveromyces lactis.

[0093] The in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, various detection methods and other technical elements of the present invention can also be independently selected from the following documents. Suitable implementation methods or implementation methods include but are not limited to CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293 A, CN109971783 A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, CN110964736A, etc. Unless they conflict with the purpose of the present invention, these documents and their citations are cited in their entirety and for all purposes.

[0094] Further, the virus blocking protein is preferably produced by the D2P technology of Kangma (Shanghai) Biotech Co., Ltd., such as the D2P system, and the D2P technology is produced, such as comprising the following steps: the gene of the virus blocking protein is optimized and cloned into the pD2P vector. The plasmid is amplified using the Ampi system and then added to the protein factory rapid reaction system (Kangma (Shanghai) Biotech Co., Ltd.) at a volume ratio of 1:30. This incubated the reaction mixture at 30°C for 4 hours and then collected and purified by centrifugation. The supernatant of the cell-free mixture was rotated at 4°C for 1 hour with magnetic His Monster Beads (Kangma Healthcode (Shanghai) Biotech). Wash with buffer (50mM Tris-HCl, pH8.0, 500mM NaCl, 10mM imidazole) and elution buffer (50mM Tris-HCl, pH 8.0, 500mM NaCl, 250mM imidazole).

[0095] Further preferably, the virus blocking protein is selected from the Constin protein produced by D2P technology of Kangma (Shanghai) Biotechnology Co., Ltd., or a protein having at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% homology with its amino acid sequence.

[0096] Embodiment 1:

[0097] Add 0.03% menthol, 0.01% menthamide, and 0.7% propylene glycol by weight, stir and heat to completely dissolve the solid matter, then cool, add 0.4% glycerol, 0.06% polysorbate 80, stir to make the solution uniform, add 0.5% xylitol, 0.05% potassium sorbate, 0.1% sodium citrate, 0.009% anhydrous citric acid, 0.02% honeysuckle extract, 0.02% green tea extract, 0.02% cetylpyridinium chloride, 0.0002% brilliant blue and the rest of water, stir to dissolve evenly, finally add 20nmol / L of the new crown blocker protein produced by our company, Constantin protein, and stir evenly to obtain a clean and refreshing mouthwash that can prevent new coronavirus infection.

[0098] Embodiment 2:

[0099] The content of each component by weight percentage is: 0.03% menthol, 0.01% menthamide, 0.7% propylene glycol, 0.4% glycerol, 0.06% polysorbate 80, 0.5% xylitol, 0.05% potassium sorbate, 0.1% sodium citrate, 0.009% anhydrous citric acid, 0.2% honeysuckle extract, 0.2% green tea extract, 0.02% cetylpyridinium chloride, 0.0002% brilliant blue, the remainder is water and Constantin protein 20nmol / L.

[0100] The preparation method is the same as that of Example 1.

[0101] Embodiment 3:

[0102] The content of each component by weight percentage is: 0.03% menthol, 0.01% menthamide, 0.7% propylene glycol, 0.4% glycerol, 0.06% polysorbate 80, 0.5% xylitol, 0.05% potassium sorbate, 0.1% sodium citrate, 0.009% anhydrous citric acid, 0.4% sodium chloride, 0.02% honeysuckle extract, 0.02% green tea extract, 0.02% cetylpyridinium chloride, 0.0002% brilliant blue, the remainder is water and 20nmol / L of Constantin protein.

[0103] The preparation method is the same as that of Example 1.

[0104] Embodiment 4:

[0105] The content of each component by weight percentage is: 0.03% menthol, 0.01% menthamide, 0.7% propylene glycol, 0.4% glycerol, 0.06% polysorbate 80, 0.5% xylitol, 0.05% potassium sorbate, 0.1% sodium citrate, 0.009% anhydrous citric acid, 0.02% honeysuckle extract, 0.02% green tea extract, 0.02% cetylpyridinium chloride, 0.0002% brilliant blue, the remainder is water and Constantin protein 50nmol / L.

[0106] The preparation method is the same as that of Example 1.

[0107] Embodiment 5:

[0108] The content of each component by weight percentage: 0.03% menthol, 0.01% menthyl amine, 0.7% propylene glycol, 0.4% glycerol, 0.06% polysorbate 80, 0.5% xylitol, 0.05% potassium sorbate, 0.1% sodium citrate,

[0109] 0.009% anhydrous citric acid, 0.02% honeysuckle extract, 0.02% green tea extract, 0.2% cetylpyridinium chloride, 0.0002% brilliant blue, the remainder water and Constantin protein 20nmol / L.

[0110] The preparation method is the same as that of Example 1.

[0111] Embodiment 6:

[0112] The content of each component in mass percentage is: 0.03% menthol, 0.01% menthamide, 0.7% propylene glycol, 0.4% glycerol, 0.06% polysorbate 80, 0.5% xylitol, 0.05% potassium sorbate, 0.1% sodium citrate, 0.009% anhydrous citric acid, 0.02% honeysuckle extract, 0.02% green tea extract, 0.02% cetylpyridinium chloride, 0.002% brilliant blue, the remainder is water and 20nmol / L of Constantin protein.

[0113] The preparation method is the same as that of Example 1.

[0114] Embodiment 7:

[0115] The content of each component by weight percentage is: 0.05% menthol, 0.01% menthamide, 0.7% propylene glycol, 0.4% glycerol, 0.06% polysorbate 80, 0.5% xylitol, 0.05% potassium sorbate, 0.1% sodium citrate, 0.009% anhydrous citric acid, 0.02% honeysuckle extract, 0.02% green tea extract, 0.02% cetylpyridinium chloride, 0.0002% brilliant blue, the remainder is water and Constantin protein 20nmol / L.

[0116] The preparation method is the same as that of Example 1.

[0117] Example 8: Testing the blocking activity of different concentrations of Constine protein against the new coronavirus

[0118] Using PBS as the positive control, equal volumes of PBS, 100nM Constine protein (PBS solution), and 1000nM Constine protein (PBS solution) were mixed with a certain volume of the SARS-CoV-2 simulated virus, allowed to stand for 15 minutes, and the virus was cultured with cultured cells to observe the effects of the three on the pseudovirus.

[0119] 1) Take out the simulated virus from the -80°C freezer and dissolve it in a room temperature water bath.

[0120] 2) Dilute the simulated virus with PBS solution to 10 ml, the dilution factor is 1000 times, and mark it.

[0121] 3) Take 3 centrifuge tubes, add 1 ml of PBS solution, 1 ml of mouthwash solution, and 1 ml of artificial saliva and mouthwash solution (1:1 equal volume mixture) into the three centrifuge tubes respectively, shake after mixing, and let stand for 15 minutes to obtain three samples.

[0122] 4) Mix the virus dilution with complete medium at a ratio of 1:1, take 220ul of three samples and add 220ul of complete medium to mix, take out the 48-well plate with HEK293T-ACE2 cells in advance from the incubator, confirm the cell density and state, and remove the upper medium. Add 200uL of pseudovirus infection solution to each well along the well wall to avoid flushing the cells (each sample is added to two wells). Place the 48-well plate in the incubator and culture for 6 hours.

[0123] 5) After 6 hours, carefully aspirate the pseudovirus infection solution and replace it with 300 μL of fresh complete culture medium. Continue to culture in the incubator for 48 hours.

[0124] 6) After 48 hours, dilute the 5x cell lysate to 1x with water.

[0125] 7) Total volume of cell lysate = 65 μL / well × total number of wells × 1.1.

[0126] 8) Aspirate the culture medium and carefully add 200 μL 1× PBS to each well to rinse the cells, avoiding washing the cells. Carefully aspirate the PBS and add 65 μL 1× cell lysis buffer to each well and incubate at room temperature for 15-20 minutes.

[0127] 9) After 15-20 minutes, store at -20℃.

[0128] Take out the frozen cell well plate, thaw on ice, mix 5 μL of cell lysate with 5 μL of luciferase substrate (Promega E1501 luciferase detection system), add the mixture to a white 384-well plate, immediately detect the activity of luciferase on a PerkinElmer EnVision 2102 multifunctional microplate reader, and determine the neutralization efficiency of the virus blocker protein (as shown in Table 1 below). Among them, Well1~Well2 and the average value represent the detected virus fluorescence value. Among them, neutralization efficiency = (positive fluorescence value-sample fluorescence value) / positive fluorescence value.

[0129] Table 1

[0130]

[0131] From the results obtained in Table 1, it can be seen that Constin protein has a good blocking effect on the new coronavirus simulated virus, with a neutralization efficiency of up to 99.98%, and different concentrations of Constin protein have little effect on its blocking efficiency, all of which can reach more than 99%.

[0132] Embodiment 9:

[0133] The mouthwash in Example 1 was used as a verification object for verification.

[0134] Equal volumes of mouthwash, a mixture of artificial saliva and mouthwash were mixed with a certain volume of the new coronavirus simulated virus, left to stand for 15 minutes, and the virus was cultured using cultured cells to observe the effects of the three on the pseudovirus.

[0135] Steps 1)-9) are the same as in Example 8

[0136] Take out the frozen cell well plate, thaw on ice, mix 5 μL of cell lysate with 5 μL of luciferase substrate (Promega E1501 luciferase detection system), add the mixture to a white 384-well plate, immediately detect the activity of luciferase on a PerkinElmer EnVision 2102 multifunctional microplate reader, and determine the neutralization efficiency of the virus blocking protein (as shown in Table 2 below). Among them, Well1~Well3 and the average value represent the detected virus fluorescence value. Among them, neutralization efficiency = (positive fluorescence value-sample fluorescence value) / positive fluorescence value.

[0137] Table 2

[0138]

[0139] From the results in Table 2, it can be seen that the new coronavirus simulation virus can infect cells. After adding mouthwash, the virus infection level to cells is reduced, and the virus activity is significantly reduced. The neutralization efficiency can reach 99.94%. This mouthwash for preventing new coronavirus infection can adhere to the new coronavirus with high affinity, making the new coronavirus lose its function and pathway to infect the body, and ultimately achieve the effect of blocking the spread and infection of the new coronavirus, thereby preventing the oral infection of the new coronavirus.

[0140] Example 10: Antibacterial Experiment

[0141] The mouthwash solution prepared in Example 1 was used as the test solution. The test temperature was 20°C and the humidity was 46% RH. The suspension quantitative antibacterial test was performed in accordance with WS / T 650-2019 "Antibacterial and Antibacterial Effect Evaluation Method" 5.1.1. Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were used for the suspension quantitative antibacterial test. The test solution was used for 0.5 min, 1 min, and 1.5 min, and the test was repeated 3 times. See Tables 1-3 for details.

[0142] Table 1 Results of quantitative antibacterial test of Escherichia coli suspension

[0143]

[0144] Note: Negative control group: PBS. No bacteria grow in the culture medium

[0145] Table 2 Results of quantitative antibacterial test of suspension of Staphylococcus aureus

[0146]

[0147] Note: Negative control group: No bacteria growth in PBS and culture medium

[0148] Table 3 Results of quantitative antibacterial test of suspension of Pseudomonas aeruginosa

[0149]

[0150] Note: Negative control group: No bacteria growth in PBS and culture medium

[0151] After three repeated tests, the mouthwash prescription solution had a strong antibacterial effect on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa at ≥ 90% after being applied for 0.5min, 1min, and 1.5min, respectively, and met the requirements of 5.1.1.5 of WS / T 650-2019 "Methods for Evaluation of Antibacterial and Antibacterial Effects".

[0152] It can be proved from Examples 9 and 10 that this mouthwash for preventing viral infection can adhere to the new coronavirus with high affinity, causing the new coronavirus to lose its function and pathway to infect the body, and ultimately achieve the effect of blocking the spread and infection of the new coronavirus, thereby achieving the effect of preventing viral oral infection. At the same time, the addition of some antibacterial agents and cooling agents can effectively reduce the growth of oral bacteria and refresh the breath.

[0153] Based on the above ideal embodiments of this application, the relevant staff can make various changes and modifications without departing from the technical concept of this application through the above description. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. An oral care composition, characterized in that Contains the following ingredients: Chinese herbal extract and virus blocking protein.

2. The oral care composition according to claim 1, wherein the Chinese herbal extract is selected from the group consisting of: honeysuckle extract, green tea extract, grapefruit peel extract, orange peel extract, violet extract, scutellaria baicalensis extract, stevia extract, wild chrysanthemum extract or gallnut extract.

3. The oral care composition according to claim 1 or 2, wherein the virus blocking protein is selected from coronavirus blocking protein.

4. According to the oral care composition according to any one of claims 1 or 3, the virus blocking protein comprises the following domains: a spike protein binding domain (SBD), a rigid domain, an oligomer site and a membrane exclusion tail; further preferably, the spike protein binding domain (SBD), the rigid domain, the oligomer site and the membrane exclusion tail are connected in sequence from segment N to segment C.

5. A mouthwash, characterized in that: The oral care composition comprises the oral care composition according to any one of claims 1 to 4; preferably, the mouthwash further comprises one or more of the following components: a wetting agent, an emulsifier, a flavor enhancer, a cooling agent, an antibacterial agent, a pH adjuster, a color enhancer and water.

6. The mouthwash according to claim 5, characterized in that: The wetting agent is selected from one or more of propylene glycol, glycerol, sorbitol, ethylene glycol, polyethylene glycol or butylene glycol.

7. The mouthwash according to claim 5 or 6, wherein the emulsifier is selected from one or more of polysorbate, polysorbate, lecithin, poloxamer, hydroxypropyl-β-cyclodextrin, arginine, glycine, aspartic acid and glutamine.

8. The mouthwash according to any one of claims 5 to 7, wherein the flavor enhancer is selected from one or more of xylitol, honey, sodium cyclamate, sorbitol, mannitol, sucralose, trichlorogalactose, saccharin sodium, steviol glycoside, hydrogenated starch hydrolysate or aspartame.

9. The mouthwash according to any one of claims 5 to 8, wherein the cooling agent is selected from one or more of menthol, menthyl lactate, menthyl acetate, menthone glycerol ketal or eucalyptus oil.

10. The mouthwash according to any one of claims 5 to 9, wherein the antibacterial agent is selected from one or more of cetylpyridinium chloride, potassium sorbate, domiphene, sodium benzoate, and parabens and their salts.

11. The mouthwash according to any one of claims 5 to 10, wherein the pH regulator is selected from one or more of citric acid, sodium citrate, boric acid, malic acid and sorbic acid.

12. The mouthwash according to any one of claims 5 to 11, wherein the color enhancer is selected from one or more of brilliant blue, sunset yellow, lemon yellow, carmine, allura red, food green and apple green.

13. The mouthwash according to any one of claims 5 to 12, characterized in that include: The following components, calculated by weight percentage: 0.01% to 2% of Chinese herbal medicine extract, 0.5% to 5% of wetting agent, 0.01% to 1% of emulsifier, 0.1% to 2% of flavor enhancer, 0.01% to 1% of cooling agent, 0.01% to 1% of antibacterial agent, 0.01% to 1% of pH adjuster, 0.0001% to 0.1% of color enhancer; and the balance of water; The concentration of the virus blocking protein is preferably 1 to 100 nmol / L.

14. The method for preparing a mouthwash according to any one of claims 5 to 13, characterized in that: The following steps are involved: (1) Mix a wetting agent and a cooling agent, heat them, and then cool them to obtain system 1; (2) Add a Chinese herbal medicine extract, an emulsifier, a flavor enhancer, a cooling agent, an antibacterial agent, a pH adjuster, a color enhancer, and the remaining water to system 1 in sequence, and mix them evenly to obtain system 2; (3) Add a virus blocking protein to system 2 and stir them evenly.

15. Use of the oral care composition according to any one of claims 1 to 4 and the mouthwash according to any one of claims 5 to 14 in the preparation of drugs for preventing viral infections, preventing or treating oral diseases, and in daily oral care.

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