WO2021253643A1 - 硫酸化多糖在抗新型冠状病毒中的应用 - Google Patents

硫酸化多糖在抗新型冠状病毒中的应用 Download PDF

Info

Publication number
WO2021253643A1
WO2021253643A1 PCT/CN2020/112597 CN2020112597W WO2021253643A1 WO 2021253643 A1 WO2021253643 A1 WO 2021253643A1 CN 2020112597 W CN2020112597 W CN 2020112597W WO 2021253643 A1 WO2021253643 A1 WO 2021253643A1
Authority
WO
WIPO (PCT)
Prior art keywords
fucoidan
sea cucumber
carrageenan
grams
parts
Prior art date
Application number
PCT/CN2020/112597
Other languages
English (en)
French (fr)
Inventor
朱蓓薇
宋爽
董秀萍
温成荣
艾春青
周大勇
启航
祁立波
李冬梅
杨静峰
Original Assignee
大连工业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202010560842.8A external-priority patent/CN111658664B/zh
Priority claimed from CN202010561815.2A external-priority patent/CN111686125A/zh
Priority claimed from CN202010561812.9A external-priority patent/CN111588732A/zh
Application filed by 大连工业大学 filed Critical 大连工业大学
Publication of WO2021253643A1 publication Critical patent/WO2021253643A1/zh
Priority to US17/567,945 priority Critical patent/US20220125827A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/731—Carrageenans
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41D—OUTERWEAR; PROTECTIVE GARMENTS; ACCESSORIES
    • A41D13/00—Professional, industrial or sporting protective garments, e.g. surgeons' gowns or garments protecting against blows or punches
    • A41D13/05—Professional, industrial or sporting protective garments, e.g. surgeons' gowns or garments protecting against blows or punches protecting only a particular body part
    • A41D13/11—Protective face masks, e.g. for surgical use, or for use in foul atmospheres
    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41D—OUTERWEAR; PROTECTIVE GARMENTS; ACCESSORIES
    • A41D31/00—Materials specially adapted for outerwear
    • A41D31/04—Materials specially adapted for outerwear characterised by special function or use
    • A41D31/30—Antimicrobial, e.g. antibacterial
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D46/00—Filters or filtering processes specially modified for separating dispersed particles from gases or vapours

Definitions

  • the invention relates to the application of sulfated polysaccharides in anti-new coronaviruses, especially the application of sea cucumber polysaccharides, fucoidan or carrageenan in anti-new coronaviruses, and belongs to the field of biomedical technology.
  • novel coronavirus (2019-nCoV), officially classified and named as severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), referred to as novel coronavirus (novel coronavirus), began to appear at the end of 2019. It has spread rapidly around the world and has infected millions of people. Common signs of people infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death.
  • the new coronavirus After the new coronavirus enters the body through the skin and mucous membranes, it binds to the angiotensin converting enzyme 2 (ACE2) receptor on the cell through the spike protein (S protein) on the surface of the virus, and then the cell membrane will adsorb and internalize the virus. After the virus enters the cytoplasm, it unshells and releases the genome. After the genome is released into the cytoplasm, the RNA of the virus will be combined with the ribosome of the host cell to translate two sets of proteins. Then, proteolytic enzymes will cut the two sets of proteins apart to generate enzymes and proteins for the virus to continue to assemble and replicate. .
  • ACE2 angiotensin converting enzyme 2
  • S protein spike protein
  • the newly generated enzymes include RNA-dependent RNA polymerase (RdRp), whose role is to replicate to form new viral RNA.
  • the new viral RNA will combine with the protein produced by the hydrolase to assemble into a new virus, release the original host cell, and continue to infect other cells.
  • the research and development of anti-new coronavirus drugs mainly starts from two aspects. One is to prevent the virus from binding to the host cell, and the target is S protein or ACE2; the other is to prevent the production of the new virus in the host cell.
  • the target is RdRp or proteolytic enzyme.
  • Sea cucumber polysaccharide is an important nutrient component of sea cucumber body wall, and its content can account for more than 6% of the total organic matter of dried ginseng.
  • Sea cucumber polysaccharide is composed of sea cucumber fucoidan sulfate and sea cucumber fucosylated chondroitin sulfate.
  • Sea cucumber fucoidan sulfate is a type of sulfated polysaccharide formed by polymerization of L-fucose; sea cucumber fucosylated chondroitin sulfate is a type of backbone structure consisting of glucuronic acid-acetylgalactosamine repeating units Structure, with fucosyl branched sulfated polysaccharides.
  • Sea cucumber polysaccharides have multiple functions such as enhancing immunity, lowering blood lipids, losing weight, and improving intestinal flora.
  • Fucoidan also known as fucoidan sulfate, fucoidan, and fucoidan sulfate, is mainly derived from brown algae. It is a type of polysaccharide containing fucose and sulfuric acid groups. In addition, it may also contain other monomers. Sugar (mannose, galactose, glucose, uronic acid, etc.). Fucoidan has multiple functions such as removing Helicobacter pylori, lowering blood lipids, losing weight, and improving intestinal flora.
  • Carrageenan (Carrageenan), also called carrageenan, carrageenan, Irish moss gum.
  • Carrageenan is a kind of galactan sulfate, the backbone structure is formed by alternately connecting 1,3- ⁇ -D-galactose and 1,4- ⁇ -D-galactose.
  • Carrageenan can be divided into seven types according to the position where the half ester sulfate group is connected to the galactose (that is, the difference in composition and structure): ⁇ -carrageenan, ⁇ -carrageenan, ⁇ -carrageenan, ⁇ -carrageenan Gum, ⁇ -carrageenan, ⁇ -carrageenan, ⁇ -carrageenan.
  • carrageenan Due to the functional properties of carrageenan, it is used as a thickening agent, gelling agent, suspending agent, emulsifier and stabilizer in the food, daily chemical and other industrial fields. In addition, studies have found that carrageenan has a variety of physiological activities, including anticoagulant, hypolipidemic, immune regulation, anti-gastric ulcer, anti-rheumatoid arthritis and other activities.
  • the technical problem to be solved by the present invention is the lack of substances that can effectively inhibit the new coronavirus.
  • the present invention provides the use of sea cucumber polysaccharide, fucoidan or carrageenan in anti-new coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2 or 2019-nCoV).
  • the anti-new coronavirus refers to the prevention or treatment of pneumonia caused by the new coronavirus, including binding to the S protein on the surface of the SARS-CoV-2 virus to prevent the SARS-CoV-2 virus from invading the cells of the body.
  • the use of sea cucumber polysaccharide, fucoidan or carrageenan can be used to prepare medicines, foods, and daily chemical products for preventing or treating coronavirus infections.
  • sea cucumber polysaccharides are derived from sea cucumbers, including sea cucumber species of Las Vegas, Lasticulaceae, Stichopus, taroceae, and Citrusaceae, and are composed of sea cucumber fucoidan sulfate and sea cucumber fucose. It is composed of chondroitin sulfate.
  • Sea cucumber fucoidan sulfate is a type of sulfated polysaccharide formed by polymerization of L-fucose; sea cucumber fucosylated chondroitin sulfate is a type of backbone structure consisting of glucuronic acid-acetylgalactosamine repeating units Structure, with fucosyl branched sulfated polysaccharides.
  • the fucoidan is derived from brown algae.
  • the sulfuric acid group content of the fucoidan is 20%-35%, the total sugar content is 45%-65%, the proportion of fucose in the monosaccharide composition is greater than 20% or the fucose content is greater than 15%.
  • the sulfate group content of the fucoidan is 20%-35%, the total sugar content is 45%-65%, the proportion of fucose in the monosaccharide composition is greater than 30% or the fucose content is greater than 20%. %.
  • the carrageenan is ⁇ -carrageenan.
  • the present invention provides the use of sea cucumber polysaccharide, fucoidan or carrageenan in the preparation of a medicine for preventing or treating coronavirus infections.
  • Said medicine uses sea cucumber polysaccharide, fucoidan or carrageenan as an inhibitor for the entry of coronavirus into body cells. Active ingredient.
  • the drug also includes pharmaceutically acceptable excipients, including: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavors, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers , Surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.
  • pharmaceutically acceptable excipients including: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regul
  • the dosage form of the medicine can be: spray, aerosol, powder mist, lotion, ointment, liniment, nasal spray, effervescent tablet, gargle, powder, emulsion, suspension, solution .
  • the invention provides the use of fucoidan and sea cucumber polysaccharide in the preparation of protective articles for preventing coronavirus infection.
  • the protective articles include hand lotion, hand sanitizer, and mouthwash.
  • the invention also provides the application of carrageenan in the preparation of hand sanitizers, mouthwashes, hand lotions, mask filters, air filter filters, frozen food ice coating preparations and edible films for preventing coronavirus infection.
  • the fucoidan is derived from brown algae.
  • the sulfuric acid group content of the fucoidan is 20%-35%, the total sugar content is 45%-65%, the proportion of fucose in the monosaccharide composition is greater than 20% or the fucose content is greater than 15%.
  • the sulfate group content of the fucoidan is 20%-35%, the total sugar content is 45%-65%, the proportion of fucose in the monosaccharide composition is greater than 30% or the fucose content is greater than 20%. %.
  • sea cucumber polysaccharides are derived from sea cucumbers, including sea cucumber species of Las Vegas, Lasticulaceae, Stichopus, taroceae, and Citrusaceae, and are composed of sea cucumber fucoidan sulfate and sea cucumber fucose. It is composed of chondroitin sulfate.
  • Sea cucumber fucoidan sulfate is a type of sulfated polysaccharide formed by polymerization of L-fucose; sea cucumber fucosylated chondroitin sulfate is a type of backbone structure consisting of glucuronic acid-acetylgalactosamine repeating units Structure, with fucosyl branched sulfated polysaccharides.
  • the carrageenan is ⁇ -carrageenan.
  • the invention provides a method for preparing protective articles against coronavirus infection by using sea cucumber polysaccharide, fucoidan or carrageenan, which is to add sea cucumber polysaccharide, fucoidan or carrageenan in the process of preparing the protective article.
  • the present invention provides a preparation method of an inhalant for the prevention of coronavirus infection.
  • 30-50 grams of sodium chloride and 70-120 grams of citron are respectively mixed with 8-50 times the amount of purified water.
  • Acid 70-120 grams of sodium citrate, 0.5-2 grams of benzalkonium chloride, stir and dissolve;
  • the purified water is diluted to 5-20 liters, filtered with 0.5 micron filter membrane, and canned.
  • the present invention provides a preparation method of oral liquid for preventing coronavirus infection. Take 1 to 2 kilograms of medlar, 1 to 2 kilograms of longans and add 12 to 20 liters of water to boil, keep it on a low heat for 0.5 to 3 hours, and filter to obtain the filtrate , Concentrated to 8-15 liters, add 40-100 grams of sea cucumber polysaccharide or fucoidan, 8-16 grams of pectin, 500-1000 grams of honey, stir evenly, let it cool naturally, and let it stand for 2-6 hours to make the precipitation complete; Filter the filtrate by pressing, add 500-1500 grams of sugar, 35-80 grams of citric acid, 3-10 grams of salt, 15-60 grams of vitamin C and mix well, filter with diatomaceous earth under pressure, and then pass the pore size of 5 microns and It is filtered by a 0.5 micron two-stage microporous membrane, and then the filtrate is sterilized (70-90 degrees Celsius, 20-50 minutes), and it can be canned.
  • the present invention provides a method for preparing a hand lotion for preventing coronavirus infection.
  • the formula of the hand lotion is as follows, in terms of mass ratio: oil phase: 3-6 parts stearic acid, 1-4 parts stearic acid Monoglyceride, 1 to 5 parts of isopropyl palmitate, 1 to 4 parts of petrolatum, 5 to 10 parts of white mineral oil, 1 to 4 parts of cetyl alcohol, 0.1 to 0.5 parts of flavor, 0.1 to 0.4 parts of paraben Ester, 0.1 to 0.4 parts of propyl paraben; water phase: 5 to 10 parts of glycerin, 0.1 to 0.5 parts of urea sac, 0.1 to 0.4 parts of dimethyl p-chlorophenol, 0.5 to 1 part of triethanolamine, 0.05 to 0.5 part Sea cucumber polysaccharide or fucoidan, 65 ⁇ 75 water;
  • Oil phase stearic acid, monoglyceride stearate, isopropyl palmitate, petrolatum, white mineral oil, cetyl alcohol, methyl paraben, propyl paraben
  • dissolve the sea cucumber polysaccharide Or fucoidan is dissolved in water, then add glycerol and triethanolamine, heat and stir to dissolve; then slowly pour the water phase into the oil phase under vigorous stirring; after homogenizer treatment, start heating and stirring, and cool to 45 ⁇ 55°C
  • Add allantoin, dimethyl p-chlorophenol and flavor at the same time continue to stir and cool to 25 ⁇ 35°C when the material is discharged; after aging for 2 ⁇ 3 days, there is no change, and the package will be qualified after inspection.
  • the present invention provides a method for preparing a mask filter element for preventing coronavirus infection, adding 1-3g of PVA, 8-12g of PET, 40-60mg of xylozoline hydrochloride, and 0.5-1.0mg of NaCl to In 100 mL of a carrageenan aqueous solution with a concentration of 0.005-0.2g/mL, stir in a water bath at 80-95°C for 20-40 minutes; use electrostatic spinning to spin the resulting viscous solution on a non-woven fabric to prepare a carrageenan film.
  • the working voltage of the electrostatic spinning method is 10-20kV
  • the working time of electrostatic spinning is 1.5-4h
  • the jetting distance is 10-30cm.
  • the present invention provides a method for preparing a disposable hand sanitizer for preventing coronavirus infection.
  • the formula of the disposable hand sanitizer is calculated according to the volume ratio: 75% ethanol 45-60%, glycerol 1-3%, and hydroxyethyl Cellulose 4-8%, sodium citrate 0.05-0.1%, lauric acid diethanolamide 1-2%, vitamin E 5-10%, citrus oil 2-4%, the balance is 0.005-0.2g/mL cara Glue solution.
  • the present invention proves through experiments that sea cucumber polysaccharide at a concentration of 3.9 ⁇ g/mL and above, fucoidan at a concentration of 15.6 ⁇ g/mL and above, and carrageenan at a concentration of 62.5 ⁇ g/mL and above, can significantly inhibit SARS-CoV-2 virus infection Body cells. Both can prevent the cell membrane of the body from adsorbing and internalizing the SARS-CoV-2 virus by binding to the S protein on the surface of the SARS-CoV-2 virus, thereby preventing the SARS-CoV-2 virus from infecting the body’s cells. This indicates that sea cucumber polysaccharides, Fucoidan has an anti-coronavirus effect.
  • sea cucumber polysaccharide or fucoidan External use of sea cucumber polysaccharide or fucoidan has a moisturizing effect, while oral administration of sea cucumber polysaccharide has the functions of enhancing immunity, lowering blood sugar, lowering blood lipid, weight loss, and improving intestinal flora. Eating fucoidan can eliminate Helicobacter pylori, lower blood lipids, and lose weight. , Improve intestinal flora, etc. It can be seen that sea cucumber polysaccharides and fucoidan are both beneficial and harmless to the human body for external use and oral administration.
  • sea cucumber polysaccharides and fucoidan can be used to prepare inhalants (nasal sprays), which enter the nasal cavity, trachea, and lungs to prevent the virus from infecting body tissues, and are used to prevent and treat the new coronavirus; use sea cucumber polysaccharides or fucoidan Adding to the hand lotion formula can increase the anti-new coronavirus effect of the hand lotion and prevent the new coronavirus infection; the oral liquid made of sea cucumber polysaccharide and fucoidan can inhibit the SARS-CoV-2 virus from invading the digestive system. It can prevent and assist the treatment of pneumonia caused by the new type of coronavirus, and has the functions of enhancing immunity, eliminating Helicobacter pylori, lowering blood sugar, lowering blood lipids, losing weight, and improving intestinal bacteria.
  • Carrageenan is a commonly used food additive. Both external use and oral administration are beneficial and harmless to the human body, and the price is cheap. Therefore, carrageenan can be prepared as an inhalant (nasal spray), which enters the nasal cavity, trachea, and lungs to prevent the virus from infecting the body tissues, and is used to prevent and treat the new coronavirus; adding carrageenan to the hand sanitizer formula can increase hand washing The anti-coronavirus effect of the liquid is to prevent the novel coronavirus infection; the mask filter element made of carrageenan can increase the effectiveness of the mask to protect against the novel coronavirus infection.
  • inhalant nasal spray
  • the mask filter element made of carrageenan can increase the effectiveness of the mask to protect against the novel coronavirus infection.
  • Sea cucumber polysaccharides and fucoidan are derived from food, are non-toxic, and have no toxic side effects when used to prevent and treat the new coronavirus.
  • FIG. 1 The inhibitory effect of sea cucumber polysaccharide (SCSP) on pseudoviruses with S protein.
  • Blank control (Blank) is a blank medium, and positive control (Positive control) is EK1 peptide. * p ⁇ 0.05, ** p ⁇ 0.01.
  • FIG. 1 Cellular immunofluorescence image of SARS-CoV-2 virus mixed with different concentrations of sea cucumber polysaccharides.
  • the final concentration of sea cucumber polysaccharide is 31.3, 15.6, 7.8, 3.9 ⁇ g/mL.
  • the negative control is a blank medium.
  • FIG. 3 The inhibitory effect of sea cucumber polysaccharide (SCSP) on SARS-CoV-2 virus.
  • the final concentration of sea cucumber polysaccharide is 31.3, 15.6, 7.8, 3.9 ⁇ g/mL.
  • Blank is a blank medium. * p ⁇ 0.05, ** p ⁇ 0.01, ** p ⁇ 0.001.
  • FIG. 4 The effect of sea cucumber polysaccharide (SCSP) on the survival rate of Vero E6 cells.
  • the final concentration of sea cucumber polysaccharide is 31.3, 15.6, 7.8, 3.9 ⁇ g/mL.
  • Blank is a blank medium. * p ⁇ 0.05, ** p ⁇ 0.01, ** p ⁇ 0.001.
  • Figure 6 Cellular immunofluorescence image of SARS-CoV-2 virus mixed with fucoidan of different concentrations.
  • concentration of fucoidan is 62.5, 31.3, 15.6, 7.8 ⁇ g/mL.
  • the negative control is a blank medium.
  • Figure 7 Fucoidan's inhibitory effect on SARS-CoV-2 virus.
  • the final concentration of fucoidan is 62.5, 31.3, 15.6, 7.8 ⁇ g/mL.
  • Blank is a blank medium. * p ⁇ 0.05, ** p ⁇ 0.01, ** p ⁇ 0.001.
  • Figure 8 The effect of Fucoidan on the viability of Vero E6 cells.
  • the final concentration of fucoidan is 62.5, 31.3, 15.6, 7.8 ⁇ g/mL.
  • Blank is a blank medium. * p ⁇ 0.05, ** p ⁇ 0.01, ** p ⁇ 0.001.
  • Figure 9 Cellular immunofluorescence image of SARS-CoV-2 virus mixed with different concentrations of carrageenan.
  • concentration of carrageenan is 500, 250, 125, 62.5, 31.3 ⁇ g/mL.
  • the negative control is a blank medium without ⁇ -carrageenan.
  • Figure 10 The inhibitory effect of carrageenan on SARS-CoV-2 virus.
  • the final concentration of sea cucumber polysaccharide is 500, 250, 125, 62.5, 31.3 ⁇ g/mL.
  • Blank is a blank medium. * p ⁇ 0.05, ** p ⁇ 0.01, ** p ⁇ 0.001.
  • Figure 11 The effect of carrageenan on the viability of Vero E6 cells.
  • the final concentration of sea cucumber polysaccharide is 500, 250, 125, 62.5, 31.3 ⁇ g/mL.
  • Blank is a blank medium. * p ⁇ 0.05, ** p ⁇ 0.01, ** p ⁇ 0.001.
  • SSP sea cucumber polysaccharide
  • the sea cucumber (Stichopus japonicus) is washed, boiled, drained, cut into small pieces, and then freeze-dried.
  • the lyophilized samples were soaked in acetone at 4°C for 24 hours and dried at room temperature. Take 1g freeze-dried sample as an example, add 30mL 0.1mol/L sodium acetate buffer solution (pH 6.0), 100mg papain (specific enzyme activity 2units/mg), 48mg ethylenediaminetetraacetic acid and 18mg cysteine, vortex Mix, shake and enzymatically hydrolyze in a 60°C water bath for 24 hours, centrifuge the reaction mixture (6000g, 15min, room temperature), and take the supernatant.
  • This embodiment may also include pretreatment steps such as solution preparation and ultrapure water preparation.
  • the content of uronic acid was determined by the meta-hydroxybiphenyl method.
  • sea cucumber polysaccharide contains fucoidan sulfate and fucosylated chondroitin sulfate.
  • the molecular weight of fucosylated chondroitin sulfate is >670kDa, and the molecular weight of fucosylated chondroitin sulfate is 179kDa; the sulfate content is 25.8 ⁇ 2.4%; the main monosaccharides are glucosamine (GlcN), galactosamine (GalN), glucose (Glc), galactose (Gal), fucose (Fuc), and the molar ratio is 1.0:1.7:1.1:1.8 :16.0; uronic acid content is 16.5 ⁇ 0.5%.
  • the full-length sequence of the gene encoding the HCoV-19 spike protein was cloned into the pCAGGS vector for pseudovirus production, and the constructed recombinant vector was called pCAGGS-HCoV-19-S.
  • the successful construction of pCAGGS-HCoV-19-S was confirmed by DNA sequencing.
  • the pCAGGS-HCoV-19-S and pNL4-3 plasmids were co-transfected into HEK 293T cells. After 48 hours of culture, the supernatant containing the SARS-CoV-2 pseudovirus was collected, and 50% of the pseudovirus was determined by infecting Huh7 cells The amount of tissue cell infection (TCID 50 ).
  • Example 3 The effect of sea cucumber polysaccharide on SARS-CoV-2 true virus
  • SARS-CoV-2 true virus from the Second Military Medical University
  • sea cucumber polysaccharide SCSP
  • DMEM medium containing 5% fetal bovine serum to make the final concentration of sea cucumber polysaccharide 31.3, 15.6, 7.8, 3.9 ⁇ g /mL, placed at 37 degrees Celsius for 1 hour, added to a 96-well plate inoculated with Vero E6 cells 12 hours in advance (the original cell culture medium was aspirated before adding), cultured for 24 hours, then immunofluorescence was used to detect viral proteins and DAPI stained the nucleus.
  • the inhibitory effect of sea cucumber polysaccharides with concentration gradients of 31.3, 15.6, 7.8, and 3.9 ⁇ g/mL on the virus was observed with an immunofluorescence microscope, and the results are shown in Figure 2.
  • the SPSS software Tukey HSD was used to count the quantified results of the virus (green fluorescence).
  • the SPSS software Tukey HSD was used to calculate the quantification results of the nuclei (blue fluorescence).
  • the sea cucumber polysaccharides did not appear to be cytotoxic at the concentrations of 31.3, 15.6, 7.8, and 3.9 ⁇ g/mL.
  • step S2 Soak the leaf algae powder A described in step S1 in absolute ethanol at 25°C for 4 hours, filter the precipitate A with gauze, stir the precipitate A in absolute ethanol at 25°C for 4 hours, filter the precipitate B with gauze , The precipitate B was soaked in absolute ethanol at 25° C. for 4 hours, and the precipitate C was filtered with gauze and dried at room temperature to remove lipids and lipid-soluble small molecules to obtain the leaf algae powder B; wherein, the leaf algae The weight-volume ratios of powder A, precipitation B and the absolute ethanol mentioned in this step are all 1:4g/mL;
  • the resulting mixture was centrifuged at 4500r/min for 15 minutes at room temperature, and the supernatant was taken; wherein, the vesicular algae powder B and the The weight-volume ratio of disodium hydrogen phosphate-citrate buffer is 1:30g/mL; the phytophyllum powder B, cellulase (specific enzyme activity 100units/mg), pectinase (specific enzyme activity 50units/mg) The weight ratio of) to papain (specific enzyme activity 2units/mg) is 12500:42:6:6;
  • step S4 Add excess CaCl 2 to the supernatant described in step S3 while stirring, centrifuge at 4500r/min for 15 minutes at room temperature, remove the alginate precipitate, and take the supernatant; use the supernatant and CaCl 2 of step S3
  • the volume-to-weight ratio is 20:1mL/g
  • step S5 Add cetyltrimethylammonium bromide (CTAB) to the supernatant of step S4 to precipitate fucoidan, and centrifuge the resulting mixture at 4500r/min for 15min at room temperature to collect the precipitate, and dissolve the precipitate in 3mol /L CaCl 2 solution, add absolute ethanol, place at 4°C for 24h to precipitate fucoidan, centrifuge at 4500r/min, 4°C for 15min, and collect the precipitate;
  • the volume-to-weight ratio of the supernatant and CTAB used in step S4 is 50:1mL/g;
  • the weight-volume ratio of the precipitate and the 3mol/L CaCl 2 solution is 1:3g/mL;
  • the volume ratio of the CaCl 2 solution and the absolute ethanol is 2:3;
  • step S6 Wash the precipitate described in step S5 with ethanol with a volume fraction of 80% for 3 times, then wash the precipitate with ethanol with a volume fraction of 95% for 3 times, dry at room temperature, and dissolve in ultrapure water, using tap water with a molecular weight of 3500Da in a dialysis bag Dialysis for 24 hours, and then use ultrapure water as the dialysate for 48 hours to remove the calcium chloride and other salt ions contained in the fucoidan.
  • the dialysate is changed every 2 hours at a vacuum of 1 pa and a temperature of -60°C Freeze-drying for 72 hours under the conditions of, to obtain the algae fucoidan (ANP);
  • the weight-volume ratio of the precipitate and the 80% ethanol solution is 1:3g/mL;
  • the precipitate and the volume fraction The weight-volume ratio of the 95% ethanol solution is 1:3 g/mL;
  • the weight-volume ratio of the precipitate and the ultrapure water is 1:150 g/mL.
  • This embodiment may also include pretreatment steps such as solution preparation and ultrapure water preparation.
  • the gelatin turbidimetric method was used to determine the sulfate group content of Fucoidan
  • the BCA method was used to determine the content of Fucoidan
  • the phenol-sulfuric acid method was used to determine the total sugar content of the algae fucoidan
  • the monosaccharide composition of algae polysaccharide is: fucose, mannose and galactose with a molar ratio of 6.5:1.1:1; functional groups include hydroxyl, carboxyl and sulfate groups.
  • the full-length sequence of the gene encoding the HCoV-19 spike protein was cloned into the pCAGGS vector for pseudovirus production, and the resulting recombinant vector was called pCAGGS-HCoV-19-S.
  • the successful construction of pCAGGS-HCoV-19-S was confirmed by DNA sequencing.
  • the plasmids of pCAGGS-HCoV-19-S and pNL4-3 were co-transfected into HEK 293T cells. After 48 hours of culture, the supernatant containing the SARS-CoV-2 pseudovirus model was collected, and the pseudovirus was determined by infection of Huh7 cells. 50% tissue cell infection (TCID 50 ).
  • Luciferase value at 48h Refer to Promega's Luciferase Assay System Protocol or Dual Luciferase Reporter Assay System Protocol. Specific operation: invert the 96-well plate, wash with PBS twice to ensure that the PBS is sucked dry, then add 30 ⁇ L of lysis buffer, lyse at room temperature for 30min, aspirate 10 ⁇ L on the white board, substrate 50 ⁇ L, and determine the luciferase value.
  • the polysaccharide from the algae fucoidum can effectively inhibit the SARS-CoV-2 virus infection of cells.
  • the same experimental method was used to detect the inhibitory effect of multiple concentrations of Alcophyllum algae fucoidan on the new coronavirus, and the IC50 value was calculated.
  • the results are shown in Fig. 5, the IC50 of Fucoidan against neocoronavirus is 0.327mg/mL.
  • the model used is a SARS-CoV-2 pseudovirus with only S protein, it can be deduced that the target of Alcohol Frophyllum fucoidan is S protein.
  • Example 6 Analyze the composition of purchased commercial fucoidan and verify its anti-coronavirus effect
  • Example 7 Analyze the composition of purchased commercial tau-carrageenan and verify its anti-coronavirus effect
  • the monosaccharide composition of ⁇ -carrageenan was determined by high performance liquid chromatography after acid hydrolysis and PMP derivatization, and it was found that the main monosaccharides in this carrageenan were glucose and galactose with a ratio of 1.0:5.2; gelatin was used for turbidity
  • the content of sulfuric acid was 10.4 ⁇ 0.5% by the method; the content of uronic acid was 0.9 ⁇ 0.1% by the m-hydroxybiphenyl method.
  • SARS-CoV-2 true virus SARS-CoV-2 true virus source: Second Military Medical University
  • tau-carrageenan in DMEM medium containing 5% fetal bovine serum, place at 37°C for 1 hour, add in advance Inoculate a 96-well plate of Vero E6 cells for 12 hours (add the original cell culture medium before aspirating), culture for 24 hours, then detect the virus protein by immunofluorescence, and stain the nucleus with DAPI.
  • the inhibitory effects of carrageenan at concentrations of 500, 250, 125, 62.5, and 31.3 ⁇ g/mL were observed with immunofluorescence microscope, and the results are shown in Figure 9.
  • the formula of hand lotion is as follows:
  • Element Proportion Oil phase To water box To Stearic acid 5 glycerin 6 Stearic acid monoglyceride 2 Allantoin 0.3 Isopropyl palmitate 3 Dimethyl p-chlorophenol 0.1 Vaseline 2 Triethanolamine 0.7
  • the oil phase (stearic acid, monoglyceride stearate, isopropyl palmitate, petrolatum, white mineral oil, cetyl alcohol, methyl paraben, propyl paraben) is heated and stirred to dissolve. Dissolve sea cucumber polysaccharides in water, add glycerin and triethanolamine, heat and stir to dissolve. Then slowly pour the water phase into the oil phase under vigorous stirring. After homogenizer treatment, start heating and stirring, add allantoin, dimethyl p-chlorophenol and flavor when cooled to 50°C, and continue to stir and cool to 30°C when the material is discharged. There is no change in Chen Fang for 2 to 3 days, and it will be packaged after passing the inspection.
  • the formula of hand lotion is as follows:
  • Element Proportion Oil phase To water box To Stearic acid 5 glycerin 6 Stearic acid monoglyceride 2 Allantoin 0.3 Isopropyl palmitate 3 Dimethyl p-chlorophenol 0.1
  • Vaseline 2 Triethanolamine 0.7 White mineral oil 8 Algae fucoidan 0.1 Cetyl Alcohol 2 water 70.4 essence 0.3 To To Methyl paraben 0.2. To To Propylparaben 0.1 To To To Propylparaben 0.1 To To
  • the oil phase (stearic acid, monoglyceride stearate, isopropyl palmitate, petrolatum, white mineral oil, cetyl alcohol, methyl paraben, propyl paraben) is heated and stirred to dissolve. Dissolve the algae fucoidan in water, add glycerin and triethanolamine, heat and stir to dissolve. Then slowly pour the water phase into the oil phase under vigorous stirring. After homogenizer treatment, start heating and stirring, add allantoin, dimethyl p-chlorophenol and flavor when cooled to 50°C, and continue to stir and cool to 30°C when the material is discharged. There is no change in Chen Fang for 2 to 3 days, and it will be packaged after passing the inspection.

Abstract

硫酸化多糖在抗新型冠状病毒中的应用。证实了海参多糖、岩藻多糖或卡拉胶能够通过与SARS-CoV-2病毒表面的S蛋白结合，防止机体细胞的细胞膜对SARS-CoV-2病毒进行吸附和内化，从而阻止SARS-CoV-2病毒感染机体细胞，这表明海参多糖、岩藻多糖或卡拉胶具有预防和治疗新型冠状病毒所致肺炎的效果。这些多糖可用于制备具有预防和治疗新型冠状病毒感染功效的防护用品，例如吸入剂、护手乳液、口服液、口罩等。

Description

硫酸化多糖在抗新型冠状病毒中的应用 技术领域
本发明涉及硫酸化多糖在抗新型冠状病毒中的应用，尤其是海参多糖、岩藻多糖或卡拉胶在抗新型冠状病毒中的应用，属于生物医学技术领域。
背景技术
新型冠状病毒(2019-nCoV)，正式分类命名为严重急性呼吸综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)，简称新冠病毒(novel coronavirus)，于2019年底开始出现，在全球迅速传播扩散，已造成上百万人感染。人感染了冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。
新冠病毒通过皮肤黏膜进入体内后，通过病毒表面的刺突蛋白(简称S蛋白)，与细胞上的血管紧张素转化酶酶2(简称ACE2)受体结合，然后细胞膜将对病毒进行吸附和内化，病毒进入细胞质后脱壳，释放基因组。基因组被释放入细胞质以后，病毒的RNA会与宿主细胞的核糖体结合，翻译出两组蛋白质，紧接着，蛋白质水解酶会把这两组蛋白质切开，生成病毒继续组装、复制的酶和蛋白质。新生成的酶包括RNA依赖性RNA聚合酶(RdRp)，它的作用就是复制形成新的病毒RNA。新的病毒RNA将和水解酶产生的蛋白质结合，组装成新的病毒，释放出原宿主细胞，继续感染其它细胞。
根据新冠病毒的染病机理，抗新冠病毒药物的研发主要从两方面入手，一是阻止病毒和宿主细胞结合，作用靶点为S蛋白或者ACE2；二是阻止新病毒在宿主细胞内的产生，作用靶点为RdRp或者蛋白质水解酶。
海参多糖是海参体壁的一类重要营养成分，其含量可占干参总有机物的6％以上。海参多糖由海参岩藻聚糖硫酸酯和海参岩藻糖基化硫酸软骨素组成。海参岩藻聚糖硫酸酯是一类由L-岩藻糖聚合而成的硫酸化多糖；海参岩藻糖基化硫酸软骨素是一类主链结构由葡萄糖醛酸-乙酰氨基半乳糖重复单元构成、带有岩藻糖基支链的硫酸化多糖。海参多糖具有增强免疫力、降血脂、减肥、改善肠道菌群等多种功效。
岩藻多糖又称为岩藻聚糖硫酸酯、褐藻糖胶、褐藻多糖硫酸酯等，主要来源于褐藻，是一类含有岩藻糖和硫酸基团的多糖，此外，还可能会含有其他单糖(甘露糖、半乳糖、葡萄糖、糖醛酸等)。岩藻多糖具有清除幽门螺杆菌、降血脂、减肥、改善肠道菌群等多种功效。
卡拉胶(Carrageenan)，也叫角叉菜胶、鹿角藻胶、爱尔兰苔菜胶。卡拉胶是一种硫酸半乳聚糖，骨架结构由1,3-β-D-半乳糖和1,4-α-D-半乳糖交替连接形成的。根据半酯式硫酸基在半乳糖上所连接的位置不同(即组成和结构的不同)卡拉胶可分为七种类型：κ-卡拉胶、τ-卡拉胶、λ-卡拉胶、μ-卡拉胶、ν-卡拉胶、θ-卡拉胶、ξ-卡拉胶。其中工业主要生产和使用的是前三种。由于卡拉胶的功能特性，它在食品、日化等工业领域中被用作增稠剂、胶凝剂、悬浮剂、乳化剂和稳定剂等。另外，还有研究发现卡拉胶具有多种生理活性包括抗凝血、降血脂、免疫调节、抗胃溃疡、抗类风湿性关节炎等活性。
发明内容
[技术问题]
本发明要解决的技术问题是缺乏有效抑制新型冠状病毒的物质。
[技术方案]
本发明提供了海参多糖、岩藻多糖或卡拉胶在抗新型冠状病毒(Severe acute respiratory syndrome coronavirus 2，SARS-CoV-2或2019-nCoV)中的用途。所述的抗新型冠状病毒是指预防或治疗新型冠状病毒所致肺炎，包括与SARS-CoV-2病毒表面的S蛋白结合、阻止SARS-CoV-2病毒侵入机体细胞。海参多糖、岩藻多糖或卡拉胶的这种用途可用于制备用于预防或治疗冠状病毒感染的药物、食品、日化产品。
所述海参多糖来源于海参，包括楯手目海参科、楯手目刺参科、芋参目尻参科和枝手目瓜参科的海参品种，由海参岩藻聚糖硫酸酯和海参岩藻糖基化硫酸软骨素组成。海参岩藻聚糖硫酸酯是一类由L-岩藻糖聚合而成的硫酸化多糖；海参岩藻糖基化硫酸软骨素是一类主链结构由葡萄糖醛酸-乙酰氨基半乳糖重复单元构成、带有岩藻糖基支链的硫酸化多糖。
所述岩藻多糖来源于褐藻。所述岩藻多糖的硫酸基含量为20％～35％，总糖为含量45％～65％，岩藻糖在单糖组成中的比例大于20％或岩藻糖含量大于15％。可选地，所述岩藻多糖的硫酸基含量为20％～35％，总糖为含量45％～65％，岩藻糖在单糖组成中的比例大于30％或岩藻糖含量大于20％。
所述卡拉胶为τ-卡拉胶。
本发明提供了海参多糖、岩藻多糖或卡拉胶在制备用于预防或治疗冠状病毒感染的药物中的用途，所述药物以海参多糖、岩藻多糖或卡拉胶为抑制冠状病毒进入机体细胞的活性成分。
所述药物还包括药学上可以接受的辅料，包括：溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、 矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、释放阻滞剂等。
所述药物的剂型可以是：喷雾剂、气雾剂、粉雾剂、洗剂、软膏剂、搽剂、喷鼻剂、泡腾片、含漱剂、散剂、乳剂、混悬剂、溶液剂。
本发明提供了岩藻多糖、海参多糖在制备用于防护冠状病毒感染的防护用品中的用途，所述的防护用品包括：护手乳液、洗手液、漱口水。
本发明还提供了卡拉胶在制备用于防护冠状病毒感染的洗手液、漱口水、护手乳液、口罩滤芯、空气过滤器滤芯、冷冻食品冰衣制剂和可食用膜中的应用。
所述岩藻多糖来源于褐藻。所述岩藻多糖的硫酸基含量为20％～35％，总糖为含量45％～65％，岩藻糖在单糖组成中的比例大于20％或岩藻糖含量大于15％。可选地，所述岩藻多糖的硫酸基含量为20％～35％，总糖为含量45％～65％，岩藻糖在单糖组成中的比例大于30％或岩藻糖含量大于20％。
所述海参多糖来源于海参，包括楯手目海参科、楯手目刺参科、芋参目尻参科和枝手目瓜参科的海参品种，由海参岩藻聚糖硫酸酯和海参岩藻糖基化硫酸软骨素组成。海参岩藻聚糖硫酸酯是一类由L-岩藻糖聚合而成的硫酸化多糖；海参岩藻糖基化硫酸软骨素是一类主链结构由葡萄糖醛酸-乙酰氨基半乳糖重复单元构成、带有岩藻糖基支链的硫酸化多糖。
所述卡拉胶为τ-卡拉胶。
本发明提供了利用海参多糖、岩藻多糖或卡拉胶制备冠状病毒感染防护用品的方法，是在制备防护用品的过程中加入海参多糖、岩藻多糖或卡拉胶。
本发明提供了一种用于预防冠状病毒感染的吸入剂的制备方法，在无菌环境中，分别用8～50倍量的纯净水将30～50克氯化钠、70～120克枸橼酸、70～120克枸橼酸钠、0.5～2克苯扎氯铵搅拌溶解；用500mL的纯净水将5～20克海参多糖、岩藻多糖或卡拉胶搅拌溶解；合并以上溶液后，用纯净水稀释至5～20升，用0.5微米滤膜过滤后，罐装。
本发明提供了一种用于预防冠状病毒感染的口服液的制备方法，取1～2公斤枸杞子、1～2公斤桂圆加12～20升水煮开，文火保持0.5～3小时，过滤取滤液，浓缩至8～15升，加入40～100克海参多糖或岩藻多糖，8～16克果胶，500～1000克蜂蜜，搅拌均匀，自然冷却，静置2～6小时，使沉淀完全；采用压滤取滤液，添加500～1500克白糖、35～80克柠檬酸、3～10克食盐、15～60克维生素C混匀，以硅藻土加压过滤，再经孔径为5微米和0.5微米的两级微孔滤膜过滤，再将滤液进行巴士杀菌(70～90摄氏度，20～50分钟)，即可罐装。
本发明提供了一种用于预防冠状病毒感染的护手乳液的制备方法，护手乳液的配方如下，以质量比计：油相：3～6份硬脂酸，1～4份硬脂酸单甘酯，1～5份棕榈酸异丙酯，1～4份凡士林，5～10份白矿油，1～4份十六醇，0.1～0.5份香精，0.1～0.4份尼泊金甲酯，0.1～0.4份尼泊金丙酯；水相：5～10份甘油，0.1～0.5份尿素囊，0.1～0.4份二甲基对氯苯酚，0.5～1份三乙醇胺，0.05～0.5份海参多糖或岩藻多糖，65～75水；
将油相(硬脂酸、硬脂酸单甘酯、棕榈酸异丙酯、凡士林、白矿油、十六醇、尼泊金甲酯、尼泊金丙酯)加热搅拌溶解；将海参多糖或岩藻多糖溶于水中，再加入甘油和三乙醇胺，加热搅拌溶解；然后在剧烈搅拌下将水相慢慢倒入油相；均质器处理后，开始加热搅拌，冷却至45～55℃时加入尿囊素、二甲基对氯苯酚和香精，继续搅拌冷却至25～35℃时出料；陈放2～3天没有变化，检验合格后包装。
本发明提供了一种用于预防冠状病毒感染的口罩滤芯的制备方法，将1-3g的PVA、8-12g的PET、40-60mg的盐酸赛洛唑啉、0.5-1.0mg的NaCl加入到100mL浓度为0.005-0.2g/mL的卡拉胶水溶液中，80-95℃水浴搅拌20-40min；采用静电纺丝法将所得的粘稠的溶液在无纺布上纺丝，制备卡拉胶膜，用做卡拉胶口罩滤芯；静电纺丝法的工作电压为10-20kV，静电纺丝工作时间为1.5-4h，喷射距离为10-30cm。
本发明提供了一种用于预防冠状病毒感染的免洗洗手液的制备方法，免洗洗手液的配方按照体积比计算是：75％乙醇45-60％，甘油1-3％，羟乙基纤维素4-8％，柠檬酸钠0.05-0.1％，月桂酸二乙醇酰胺1-2％，维生素E 5-10％，柑橘油2-4％，余量为0.005-0.2g/mL的卡拉胶水溶液。将羟乙基纤维素、柠檬酸钠溶于卡拉胶水溶液中，得到水溶部分；将甘油、月桂酸二乙醇酰胺、维生素E、柑橘油溶于75％乙醇中，得到油溶部分；将水溶部分和油溶部分混合搅拌至均匀，用1微米滤膜过滤后，罐装。
[有益效果]
本发明通过实验证实海参多糖在3.9μg/mL及以上浓度、岩藻多糖在15.6μg/mL及以上浓度，卡拉胶在62.5μg/mL及以上浓度，能够抑显著制SARS-CoV-2病毒感染机体细胞。均能够通过与SARS-CoV-2病毒表面的S蛋白结合防止机体细胞的细胞膜对SARS-CoV-2病毒进行吸附和内化，从而阻止SARS-CoV-2病毒感染机体细胞，这表明海参多糖、岩藻多糖具有抗新型冠状病毒的效果。
外用海参多糖或岩藻多糖有保湿作用，内服海参多糖则具有增强免疫力、降血糖、降血脂、减肥、改善肠道菌群等作用，食用岩藻多糖具有清除幽门螺杆菌、降血脂、减肥、改善肠道菌群等作用，可见，海参多糖、岩藻多糖外用和口服均对人体有益无害。因此，海参多 糖、岩藻多糖可用于制备吸入剂(喷鼻剂)，进入鼻腔、气管、肺部，防止病毒的感染机体组织，用于预防和治疗新型冠状病毒；将海参多糖或岩藻多糖加入到护手乳液配方中，可增加护手乳液抗新型冠状病毒的效果，预防新型冠状病毒感染；海参多糖、岩藻多糖制成的口服液可抑制SARS-CoV-2病毒从消化系统侵入，起到预防和辅助治疗新型冠状病毒所致肺炎的作用，并具有增强免疫力、清除幽门螺杆菌、降血糖、降血脂、减肥、改善肠道菌等作用。
卡拉胶是常用的食品添加剂，外用和口服均对人体有益无害，而且价格便宜。因此，卡拉胶可制备吸入剂(喷鼻剂)，进入鼻腔、气管、肺部，防止病毒的感染机体组织，用于预防和治疗新型冠状病毒；卡拉胶加入免洗洗手液配方，可增加洗手液抗新型冠状病毒的效果，预防新型冠状病毒感染；用卡拉胶制成的口罩滤芯，可增加口罩防护新型冠状病毒感染的功效。
海参多糖、岩藻多糖来源于食物、无毒性，用于防治新冠病毒时，无毒副作用。
附图说明
图1海参多糖(SCSP)对带有S蛋白的假病毒的抑制作用。空白对照(Blank)为空白培养基，阳性对照(Positive control)为EK1肽。 *p<0.05, **p<0.01。
图2将SARS-CoV-2病毒与不同浓度的海参多糖混合处理后的细胞免疫荧光图。海参多糖的终浓度为31.3、15.6、7.8、3.9μg/mL。阴性对照为空白培养基。
图3海参多糖(SCSP)对SARS-CoV-2病毒的抑制作用。海参多糖的终浓度为31.3、15.6、7.8、3.9μg/mL。Blank为空白培养基。 *p<0.05, **p<0.01, **p<0.001。
图4海参多糖(SCSP)对Vero E6细胞存活率的影响。海参多糖的终浓度为31.3、15.6、7.8、3.9μg/mL。Blank为空白培养基。 *p<0.05, **p<0.01, **p<0.001。
图5不同浓度的泡叶藻岩藻多糖对新冠病毒的抑制作用，横坐标：泡叶藻岩藻多糖的浓度，纵坐标：感染细胞率(％)＝进入细胞的病毒数量/总病毒数量×100。
图6将SARS-CoV-2病毒与不同浓度的岩藻多糖混合处理后的细胞免疫荧光图。岩藻多糖的浓度为62.5、31.3、15.6、7.8μg/mL。阴性对照为空白培养基。
图7岩藻多糖(Fucoidan)对SARS-CoV-2病毒的抑制作用。岩藻多糖的终浓度为62.5、31.3、15.6、7.8μg/mL。Blank为空白培养基。 *p<0.05, **p<0.01, **p<0.001。
图8岩藻多糖(Fucoidan)对Vero E6细胞存活率的影响。岩藻多糖的终浓度为62.5、31.3、15.6、7.8μg/mL。Blank为空白培养基。 *p<0.05, **p<0.01, **p<0.001。
图9将SARS-CoV-2病毒与不同浓度的卡拉胶混合处理后的细胞免疫荧光图。卡拉胶的浓度为500、250、125、62.5、31.3μg/mL。阴性对照为不含τ-卡拉胶的空白培养基。
图10卡拉胶(carrageenan)对SARS-CoV-2病毒的抑制作用。海参多糖的终浓度为500、250、125、62.5、31.3μg/mL。Blank为空白培养基。 *p<0.05, **p<0.01, **p<0.001。
图11卡拉胶(carrageenan)对Vero E6细胞存活率的影响。海参多糖的终浓度为500、250、125、62.5、31.3μg/mL。Blank为空白培养基。 *p<0.05, **p<0.01, **p<0.001。
具体实施方式
实施例1 制备海参多糖的方法
(1)海参多糖(SCSP)的制备方法如下：
将海参(刺参，Stichopus japonicus)洗净、水煮、沥干、剪成小块，然后冻干。将冻干样品置于4℃丙酮中浸泡24h，室温下晾干。以1g冻干样品为例，加入30mL 0.1mol/L乙酸钠缓冲溶液(pH 6.0)、100mg木瓜蛋白酶(比酶活2units/mg)、48mg乙二胺四乙酸和18mg半胱氨酸，涡旋混合，于60℃水浴振荡酶解24h，将反应混合物离心(6000g，15min，室温)，取上清液。向上清液中加入1.6mL 10％氯化十六烷基吡啶溶液，室温下放置24h后，离心(8000g，15min，室温)取沉淀。将沉淀溶解于15mL 3mol/LNaCl-乙醇(100：15v/v)溶液中，再加入30mL 95％乙醇溶液，4℃放置24h，离心(8000g，15min，室温)取沉淀。将沉淀用10mL 80％乙醇洗2至3次后，再用10mL 95％乙醇洗2至3次，室温晾干，蒸馏水溶解，用透析袋(3500Da)进行除盐，冻干，得海参多糖。
本实施例还可以包括溶液配制、超纯水的制备等前处理步骤。
(2)确定前述步骤制备的海参多糖的结构特性和组成
采用 1H NMR进行海参多糖结构特性和纯度检测；
采用凝胶渗透色谱法进行海参多糖分子量检测；
采用明胶比浊法进行海参多糖硫酸根含量检测；
采用高效液相色谱+PMP衍生化法进行海参多糖单糖组成检测；
采用傅里叶红外进行海参多糖官能团检测。
采用间羟基联苯法测定糖醛酸含量。
结果表明，海参多糖含有岩藻聚糖硫酸酯和岩藻糖基化硫酸软骨素，岩藻聚糖硫酸酯分子量＞670kDa，岩藻糖基化硫酸软骨素的分子量为179kDa；硫酸根含量为25.8±2.4％；主要组成单糖有氨基葡萄糖(GlcN)、氨基半乳糖(GalN)、葡萄糖(Glc)、半乳糖(Gal)、岩藻糖(Fuc)，摩尔比为1.0：1.7：1.1：1.8：16.0；糖醛酸含量为16.5±0.5％。
实施例2 使用假病毒模型评价海参多糖的抗新型冠状病毒的作用
将编码HCoV-19刺突蛋白的基因的全长序列克隆到pCAGGS载体用于假病毒的生产，构建得到的重组载体称为pCAGGS-HCoV-19-S。通过DNA测序确认pCAGGS-HCoV-19-S构建成功。将pCAGGS-HCoV-19-S和pNL4-3质粒共转染到HEK 293T细胞，培养48h后，收集含有SARS-CoV-2假病毒的上清液，并通过感染Huh7细胞确定假病毒的50％组织细胞感染量(TCID 50)。
使用该SARS-CoV-2假病毒模型评价海参多糖的抗新型冠状病毒的作用，具体步骤如下：
(1)选择生长状态良好的Huh7细胞，胰酶消化后，96孔铺板，培养过夜，至18-24h时细胞达到80-100％；
(2)每孔100TCID 50假病毒，与含有海参多糖的无血清培养基混合，混合后海参多糖的终浓度为0.01mg/mL、0.1mg/mL和1mg/mL，于37摄氏度孵育30min。EK1肽作为阳性对照，空白无血清培养基作为阴性对照。
(3)以PBS洗涤Huh7细胞去除血清后，用3倍倍比稀释病毒与海参多糖的混合物并感染Huh7细胞，每个孔100μL，每个样品设置三个平行孔，4-6h后，补加含有5％FBS血清的培养基100μL。
(4)48h测定Luciferase值。参考Promega公司Luciferase Assay System Protocol或Dual Luciferase Reporter Assay System Protocol。具体操作：倒扣96孔板，用PBS洗2遍，确保吸干PBS，然后加入30μL的裂解液，常温裂解30min，吸出10μL于白板上，底物50μL，测定luciferase值，结果如图1所示。
结果表明，海参多糖终浓度为100μg/mL和1000μg/mL时，都能够有效的抑制SARS-CoV-2病毒进入细胞。而且由于所使用的模型为具有S蛋白的SARS-CoV-2假病毒，可以推出海参多糖的作用靶点为S蛋白。
实施例3 海参多糖对SARS-CoV-2真病毒的作用
SARS-CoV-2真病毒(来源于第二军医大学)和海参多糖(SCSP)用含有5％胎牛血清的DMEM培养基混匀，使海参多糖的终浓度为31.3、15.6、7.8、3.9μg/mL，37摄氏度放置1小时，加入提前12小时接种Vero E6细胞的96孔板(加入以前吸除原细胞培养液)，培养24小时，然后用免疫荧光检测病毒蛋白，DAPI染色细胞核。用免疫荧光显微镜观测了31.3、15.6、7.8、3.9μg/mL浓度梯度的海参多糖对病毒的抑制作用，结果如图2所示。采用SPSS软件Tukey HSD对病毒(绿色荧光)的量化结果进行了统计，如图3所示，在大于3.9μg/mL浓度范围内，海参多糖均能够显著抑制新冠病毒对细胞的感染。同时，也采用SPSS软件Tukey  HSD对细胞核(蓝色荧光)的量化结果进行了统计，如图4所示，在31.3、15.6、7.8、3.9μg/mL浓度下海参多糖均未出现细胞毒。
实施例4 制备泡叶藻(褐藻)岩藻多糖
(1)泡叶藻岩藻多糖的制备方法如下：
S1、将泡叶藻(Ascophyllum nodosum)洗净、沥干、自然风干、粉碎并过80目筛，得泡叶藻粉A；
S2、将步骤S1所述泡叶藻粉A置于25℃无水乙醇中浸泡4h，纱布过滤取沉淀A，将所述沉淀A置于25℃无水乙醇中搅拌4h，纱布过滤取沉淀B，将所述沉淀B置于25℃无水乙醇中浸泡4h，纱布过滤取沉淀C室温晾干，从而除去脂类及脂溶性小分子，得泡叶藻粉B；其中，所述泡叶藻粉A、沉淀B和本步骤所述无水乙醇的重量体积比均为1:4g/mL；
S3、取步骤S2所述泡叶藻粉B，加入pH＝5的磷酸氢二钠-柠檬酸缓冲液、纤维素酶、果胶酶和木瓜蛋白酶，搅拌混匀，50℃水浴振荡酶解4h，使岩藻多糖解离，后加热至98℃，保持10分钟以钝化酶活，所得混合物于4500r/min室温离心15min，取上清液；其中，所述泡叶藻粉B和所述磷酸氢二钠-柠檬酸缓冲液的重量体积比为1:30g/mL；所述泡叶藻粉B、纤维素酶(比酶活100units/mg)、果胶酶(比酶活50units/mg)和木瓜蛋白酶(比酶活2units/mg)的重量比为12500:42:6:6；
S4、向步骤S3所述上清液中边搅拌边加入过量的CaCl 2，于4500r/min室温离心15min，将褐藻胶沉淀除去，取上清液；使用的步骤S3的上清液和CaCl 2体积重量比是20:1mL/g；
S5、向步骤S4所述上清液中加入十六烷基三甲基溴化铵(CTAB)，使岩藻多糖沉淀，将所得混合物在4500r/min室温离心15min收集沉淀，将沉淀溶解于3mol/L CaCl 2溶液中，再加无水乙醇，4℃放置24h，使岩藻多糖沉淀，4500r/min、4℃离心15min，收集沉淀；使用的步骤S4的上清液和CTAB体积重量比是50:1mL/g；所述沉淀和所述3mol/L CaCl 2溶液的重量体积比是1:3g/mL；所述CaCl 2溶液和所述无水乙醇的体积比为2:3；
S6、将步骤S5所述沉淀用体积分数为80％的乙醇洗涤3次，再使用体积分数为95％乙醇洗沉淀3次，室温晾干，超纯水溶解，使用分子量3500Da的透析袋自来水流水透析24小时，然后使用超纯水作为透析液透析48小时，除去岩藻多糖中含有的氯化钙及其它盐离子，其中每2小时换透析液，在真空度为1pa和温度为-60℃的条件下冻干72小时，得到泡叶藻岩藻多糖(ANP)；所述沉淀和所述体积分数为80％的乙醇溶液的重量体积比是1:3g/mL； 所述沉淀和所述体积分数为95％的乙醇溶液的重量体积比是1:3g/mL；所述沉淀和所述超纯水的重量体积比是1:150g/mL。
本实施例还可以包括溶液配制、超纯水的制备等前处理步骤。
(2)确定实施例1所制备的泡叶藻岩藻多糖的结构特性和组成
采用明胶比浊法进行泡叶藻岩藻多糖硫酸基含量的测定；
采用BCA法进行泡叶藻岩藻多糖蛋白含量的测定；
采用间羟基联苯法进行泡叶藻岩藻多糖糖醛酸含量的测定；
采用苯酚硫酸法进行泡叶藻岩藻多糖总糖含量的测定；
采用凝胶渗透色谱法进行泡叶藻岩藻多糖分子量的测定；
采用高效液相色谱+PMP衍生化法进行泡叶藻岩藻多糖单糖组成的测定；
采用傅里叶红外光谱法进行泡叶藻岩藻多糖官能团的测定。
结果表明，泡叶藻岩藻多糖的分子量为490kDa；糖醛酸含量为2.9～3.2％；蛋白含量为3.8～4.0％；硫酸基含量为28～30％；总糖含量为54％；所述褐藻岩藻多糖的单糖组成为：摩尔比为6.5:1.1:1的岩藻糖、甘露糖和半乳糖；官能团包括羟基、羧基和硫酸基等官能团。
实施例5 验证泡叶藻岩藻多糖阻止SARS-CoV-2病毒侵入机体细胞的用途
将编码HCoV-19刺突蛋白的基因的全长序列克隆到pCAGGS载体用于假病毒的生产，所得到的重组载体称为pCAGGS-HCoV-19-S。通过DNA测序确认pCAGGS-HCoV-19-S构建成功。将pCAGGS-HCoV-19-S和pNL4-3的质粒共转染到HEK 293T细胞，培养48h后，收集含有SARS-CoV-2假病毒模型的上清液，并通过感染Huh7细胞确定假病毒的50％组织细胞感染量(TCID 50)。
使用该SARS-CoV-2假病毒模型评价泡叶藻岩藻多糖的抗新型冠状病毒的作用，具体步骤如下：
(1)选择生长状态良好的Huh7细胞，胰酶消化后，96孔铺板，培养过夜，至18-24h时细胞达到80-100％；
(2)每孔100TCID 50假病毒，与含有泡叶藻岩藻多糖的无血清培养基混合，混合后泡叶藻岩藻多糖的终浓度为0.01mg/mL、0.1mg/mL和1mg/mL，37摄氏度孵育30min。EK1肽作为阳性对照，空白无血清培养基作为阴性对照。
(3)以PBS洗涤Huh7细胞去除血清后，用3倍倍比稀释病毒与泡叶藻岩藻多糖的混合物感染Huh7细胞，每个孔100μL，每个样品设置三个平行孔，4-6h后，补加含有5％FBS血清的培养基100μL。
(4)48h测定Luciferase值。参考Promega公司Luciferase Assay System Protocol或Dual Luciferase Reporter Assay System Protocol。具体操作：倒扣96孔板，用PBS洗2遍，确保吸干PBS，然后加入30μL的裂解液，常温裂解30min，吸出10μL于白板上，底物50μL，测定luciferase值。
如下表1所示，泡叶藻岩藻多糖在浓度为0.01mg/mL、0.1mg/mL和1mg/mL时，都能够有效的抑制SARS-CoV-2病毒感染细胞。
表1
Figure PCTCN2020112597-appb-000001
又进一步采用相同实验方法检测了多个浓度的泡叶藻岩藻多糖对新冠病毒的抑制作用，计算了IC50值。结果如图5所示，泡叶藻岩藻多糖抑制新冠病毒的IC50为0.327mg/mL。
而且，由于所使用的模型为仅具有S蛋白的SARS-CoV-2假病毒，可以推出泡叶藻岩藻多糖的作用靶点为S蛋白。
实施例6 分析购买的商品化岩藻多糖的组成并验证其抗新冠病毒的效果
参考中华人民共和国水产行业标准SC/T 3404-2012，测得青岛明月海藻集团有限公司的商品化岩藻多糖中硫酸基含量为22.8±0.7％，总糖含量为63.2±2.6％，岩藻糖含量为36.9±3.8％。采用间羟基联苯法测定糖醛酸含量为4.7±0.4％。
将SARS-CoV-2真病毒(来源于第二军医大学)和岩藻多糖(Fucoidan)用含有5％胎牛血清的DMEM培养基混匀，37摄氏度放置1小时，加入提前12小时接种Vero E6细胞的96孔板(加入以前吸除原细胞培养液)，培养24小时，然后用免疫荧光检测病毒蛋白，DAPI染色细胞核。采用免疫荧光显微镜观测了终浓度为62.5、31.3、15.6、7.8μg/mL的岩藻多糖对病毒的抑制作用，结果如图6所示。采用SPSS软件Tukey HSD统计，如图7所示，在浓 度大于15.6μg/mL时，岩藻多糖均能够显著抑制新冠病毒对细胞的感染。同时，也采用SPSS软件Tukey HSD对细胞核(蓝色荧光)的量化结果进行了统计，如图8所示，在62.5、31.3、15.6、7.8μg/mL浓度下岩藻多糖均未出现细胞毒。
实施例7 分析购买的商品化τ-卡拉胶的组成并验证其抗新冠病毒的作用
采用酸水解、PMP衍生化后高效液相色谱测定τ-卡拉胶的单糖组成，发现该卡拉胶(carrageenan)中主要组成单糖为葡萄糖和半乳糖，比例为1.0:5.2；采用明胶比浊法测定硫酸基含量为10.4±0.5％；采用间羟基联苯法测定糖醛酸含量为0.9±0.1％。
将SARS-CoV-2真病毒(SARS-CoV-2真病毒来源：第二军医大学)和τ-卡拉胶用含有5％胎牛血清的DMEM培养基混匀，37摄氏度放置1小时，加入提前12小时接种Vero E6细胞的96孔板(加入以前吸除原细胞培养液)，培养24小时，然后用免疫荧光检测病毒蛋白，DAPI染色细胞核。采用免疫荧光显微镜观测了浓度分别为500、250、125、62.5、31.3μg/mL的卡拉胶对病毒的抑制作用，结果如图9所示。用SPSS软件Tukey HSD统计，如图10所示，在62.5～500μg/mL浓度范围内，卡拉胶均能够显著抑制新冠病毒对细胞的感染。同时，也采用SPSS软件Tukey HSD对细胞核(蓝色荧光)的量化结果进行了统计，如图11所示，在62.5～500μg/mL浓度下卡拉胶均未出现细胞毒。
实施例8 海参多糖喷鼻剂的制备方法
在无菌环境中，分别用10倍量的纯净水将40克氯化钠、100克枸橼酸、100克枸橼酸钠、1克苯扎氯铵搅拌溶解。用500mL的纯净水将10克海参多糖搅拌溶解。合并以上溶液后，用纯净水稀释至10升，用0.5微米滤膜过滤后，罐装。
实施例9 海参多糖护手乳液的制备方法
护手乳液的配方如下表2：
表2
成分 比例 成分 比例
油相   水相  
硬脂酸 5 甘油 6
硬脂酸单甘酯 2 尿囊素 0.3
棕榈酸异丙酯 3 二甲基对氯苯酚 0.1
凡士林 2 三乙醇胺 0.7
白矿油 8 海参多糖 0.1
十六醇 2 水 70.4
香精 0.3    
尼泊金甲酯 0.2.    
尼泊金丙酯 0.1    
将油相(硬脂酸、硬脂酸单甘酯、棕榈酸异丙酯、凡士林、白矿油、十六醇、尼泊金甲酯、尼泊金丙酯)加热搅拌溶解。将海参多糖溶于水中，再加入甘油和三乙醇胺，加热搅拌溶解。然后在剧烈搅拌下将水相慢慢倒入油相。均质器处理后，开始加热搅拌，冷却至50℃时加入尿囊素、二甲基对氯苯酚和香精，继续搅拌冷却至30℃时出料。陈放2～3天没有变化，检验合格后包装。
实施例10 海参多糖口服液的制备方法
取1.6公斤枸杞子、1.6公斤桂圆加16升水煮开，文火保持1小时，过滤取滤液，浓缩至12升，加入60克海参多糖，12克果胶，840克蜂蜜，搅拌均匀，自然冷却，静置3小时，使沉淀完全。采用压滤取滤液，添加910克白糖、52克柠檬酸、6.5克食盐、26克维生素C混匀，以硅藻土加压过滤，再经孔径为5微米和0.5微米的两级微孔滤膜过滤，再将滤液进行巴士杀菌(80摄氏度，30分钟)，即可罐装。
实施例11 泡叶藻岩藻多糖喷鼻剂的制备方法
在无菌环境中，分别用10倍量的纯净水将40克氯化钠、100克枸橼酸、100克枸橼酸钠、1克苯扎氯铵搅拌溶解。用500mL的纯净水将10克泡叶藻岩藻多糖搅拌溶解。合并以上溶液后，用纯净水稀释至10升，用0.5微米滤膜过滤后，罐装。
实施例12 泡叶藻岩藻多糖护手乳液的制备方法
护手乳液的配方如下表3：
表3
成分 比例 成分 比例
油相   水相  
硬脂酸 5 甘油 6
硬脂酸单甘酯 2 尿囊素 0.3
棕榈酸异丙酯 3 二甲基对氯苯酚 0.1
凡士林 2 三乙醇胺 0.7
白矿油 8 泡叶藻岩藻多糖 0.1
十六醇 2 水 70.4
香精 0.3    
尼泊金甲酯 0.2.    
尼泊金丙酯 0.1    
将油相(硬脂酸、硬脂酸单甘酯、棕榈酸异丙酯、凡士林、白矿油、十六醇、尼泊金甲酯、尼泊金丙酯)加热搅拌溶解。将泡叶藻岩藻多糖溶于水中，再加入甘油和三乙醇胺，加热搅拌溶解。然后在剧烈搅拌下将水相慢慢倒入油相。均质器处理后，开始加热搅拌，冷却至50℃时加入尿囊素、二甲基对氯苯酚和香精，继续搅拌冷却至30℃时出料。陈放2～3天没有变化，检验合格后包装。
实施例13 泡叶藻岩藻多糖口服液的制备方法
取1.6公斤枸杞子、1.6公斤桂圆加16升水煮开，文火保持1小时，过滤取滤液，浓缩至12升，加入60克泡叶藻岩藻多糖，12克果胶，840克蜂蜜，搅拌均匀，自然冷却，静置3小时，使沉淀完全。采用压滤取滤液，添加910克白糖、52克柠檬酸、6.5克食盐、26克维生素C混匀，以硅藻土加压过滤，再经孔径为5微米和0.5微米的两级微孔滤膜过滤，再将滤液进行巴士杀菌(80摄氏度，30分钟)，即可罐装。
实施例14 卡拉胶喷鼻剂的制备方法
在无菌环境中，分别用10倍量的纯净水将40克氯化钠、100克枸橼酸、100克枸橼酸钠、1克苯扎氯铵搅拌溶解。用500mL的纯净水将10克卡拉胶搅拌溶解。合并以上溶液后，用纯净水稀释至10升，用0.5微米滤膜过滤后，罐装。
实施例15 卡拉胶口罩滤芯的制备方法
将2g的PVA、10g的PET、40mg的盐酸赛洛唑啉、0.5mg的NaCl加入到100mL浓度为0.01g/mL的卡拉胶水溶液中，90℃水浴搅拌30min。采用静电纺丝法将所得的粘稠的溶液在无纺布上纺丝，制备卡拉胶膜，用做卡拉胶口罩滤芯。工作电压为15kV，静电纺丝工作时间为3h，喷射距离为25cm，得到卡拉胶口罩滤芯。
实施例16 卡拉胶免洗洗手液的制备方法
配方：75％乙醇50％，甘油2％，羟乙基纤维素4％，柠檬酸钠0.05％，月桂酸二乙醇酰胺1％，维生素E 8％，柑橘油2％，余量为0.005g/mL的卡拉胶水溶液。将羟乙基纤维素、柠檬酸钠溶于卡拉胶水溶液中，得到水溶部分；将甘油、月桂酸二乙醇酰胺、维生素E、柑橘油溶于75％乙醇中，得到油溶部分；将水溶部分和油溶部分混合搅拌至均匀，用1微米滤膜过滤后，罐装。

Claims (16)

  1. 海参多糖、岩藻多糖或卡拉胶在制备用于治疗或预防冠状病毒感染的药物或日化用品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述冠状病毒包括新型冠状病毒SARS-CoV-2。
  3. 根据权利要求1所述的应用，其特征在于，所述治疗或预防冠状病毒感染，包括海参多糖、岩藻多糖或卡拉胶与SARS-CoV-2表面的S蛋白结合、阻止SARS-CoV-2病毒侵入机体细胞。
  4. 根据权利要求1-3任一项所述的应用，其特征在于，所述海参多糖来源于海参，包括楯手目海参科、楯手目刺参科、芋参目尻参科和枝手目瓜参科的海参品种，含有海参岩藻聚糖硫酸酯和海参岩藻糖基化硫酸软骨素。
  5. 根据权利要求1-3任一项所述的应用，其特征在于，所述岩藻多糖来源于褐藻。
  6. 根据权利要求1-3任一项所述的应用，其特征在于，所述卡拉胶为τ-卡拉胶。
  7. 根据权利要求1所述的应用，其特征在于，所述药物还包括药学上可以接受的辅料；所述药物的剂型包括：喷雾剂、气雾剂、粉雾剂、洗剂、软膏剂、搽剂、喷鼻剂、泡腾片、含漱剂、散剂、乳剂、混悬剂、溶液剂。
  8. 根据权利要求1所述的应用，其特征在于，所述的日化用品包括：护手乳液、洗手液、漱口水、口罩滤芯、空气过滤器滤芯、冷冻食品冰衣制剂、可食用膜。
  9. 用于治疗或预防冠状病毒感染的药物或吸入剂或喷鼻剂或护手乳液或洗手液或漱口水，其特征在于，以海参多糖、岩藻多糖或卡拉胶为抑制冠状病毒进入机体细胞的活性成分。
  10. 一种用于预防冠状病毒感染的吸入剂，其特征在于，在无菌环境中，分别用8～50倍量的纯净水将30～50克氯化钠、70～120克枸橼酸、70～120克枸橼酸钠、0.5～2克苯扎氯铵搅拌溶解；用500mL的纯净水将5～20克岩藻多糖或海参多糖或卡拉胶搅拌溶解；合并以上溶液后，用纯净水稀释至5～20升，微滤后，罐装。
  11. 一种用于预防冠状病毒感染的口服液，其特征在于，取1～2公斤枸杞子、1～2公斤桂圆加12～20升水煮开，文火保持0.5～3小时，过滤取滤液，浓缩至8～15升，加入40～100克岩藻多糖或海参多糖，8～16克果胶，500～1000克蜂蜜，搅拌均匀，自然冷却，静置2～6小时，使沉淀完全；采用压滤取滤液，添加500～1500克白糖、35～80克柠檬酸、3～10克食盐、15～60克维生素C混匀，以硅藻土加压过滤，再经两级微孔滤膜过滤，再将滤液进行巴士杀菌，即可罐装。
  12. 一种用于预防冠状病毒感染的护手乳液，其特征在于，护手乳液的配方如下，以质量比计：
    油相：3～6份硬脂酸，1～4份硬脂酸单甘酯，1～5份棕榈酸异丙酯，1～4份凡士林，5～10份白矿油，1～4份十六醇，0.1～0.5份香精，0.1～0.4份尼泊金甲酯，0.1～0.4份尼泊金丙酯；水相：5～10份甘油，0.1～0.5份尿素囊，0.1～0.4份二甲基对氯苯酚，0.5～1份三乙醇胺，0.05～0.5份岩藻多糖或海参多糖，65～75水。
  13. 一种用于预防冠状病毒感染的口罩滤芯，其特征在于，将1-3g的PVA、8-12g的PET、40-60mg的盐酸赛洛唑啉、0.5-1.0mg的NaCl加入到100mL浓度为0.005-0.2g/mL的卡拉胶水溶液中，80-95℃水浴搅拌20-40min；采用静电纺丝法将所得的粘稠的溶液在无纺布上纺丝，制备卡拉胶膜，用做卡拉胶口罩滤芯；静电纺丝法的工作电压为10-20kV，静电纺丝工作时间为1.5-4h，喷射距离为10-30cm。
  14. 一种用于预防冠状病毒感染的免洗洗手液，其特征在于，配方为：75％乙醇45-60％，甘油1-3％，羟乙基纤维素4-8％，柠檬酸钠0.05-0.1％，月桂酸二乙醇酰胺1-2％，维生素E5-10％，柑橘油2-4％，余量为0.005-0.2g/mL的卡拉胶水溶液。
  15. 海参多糖、岩藻多糖或卡拉胶在用于人或动物的治疗或预防冠状病毒感染中的用途。
  16. 海参多糖、岩藻多糖或卡拉胶中的两种的混合物或三种的混合物在制备用于治疗或预防冠状病毒感染的药物或日化用品中的应用。
PCT/CN2020/112597 2020-06-18 2020-08-31 硫酸化多糖在抗新型冠状病毒中的应用 WO2021253643A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/567,945 US20220125827A1 (en) 2020-06-18 2022-01-04 Application of Sulfated Polysaccharides in Resisting novel Coronavirus

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN202010561812.9 2020-06-18
CN202010560842.8 2020-06-18
CN202010560842.8A CN111658664B (zh) 2020-06-18 2020-06-18 海参多糖在抗新型冠状病毒中的应用
CN202010561815.2A CN111686125A (zh) 2020-06-18 2020-06-18 卡拉胶在抗新型冠状病毒中的应用
CN202010561815.2 2020-06-18
CN202010561812.9A CN111588732A (zh) 2020-06-18 2020-06-18 岩藻多糖在抗新型冠状病毒中的应用

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/567,945 Continuation US20220125827A1 (en) 2020-06-18 2022-01-04 Application of Sulfated Polysaccharides in Resisting novel Coronavirus

Publications (1)

Publication Number Publication Date
WO2021253643A1 true WO2021253643A1 (zh) 2021-12-23

Family

ID=79269114

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/112597 WO2021253643A1 (zh) 2020-06-18 2020-08-31 硫酸化多糖在抗新型冠状病毒中的应用

Country Status (2)

Country Link
US (1) US20220125827A1 (zh)
WO (1) WO2021253643A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114573727A (zh) * 2022-03-24 2022-06-03 自然资源部第一海洋研究所 海参岩藻多糖及其制备方法和在制备防治由幽门螺杆菌引起的疾病的药物和保健品中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101784272A (zh) * 2007-08-24 2010-07-21 玛丽诺姆德生物技术公司 包含硫酸多糖的抗病毒组合物
CN107847430A (zh) * 2015-07-14 2018-03-27 玛丽诺姆德生物技术股份公司 一种具有抗病毒活性的鼻塞疏通组合物
CN111471088A (zh) * 2020-04-21 2020-07-31 北京中科微盾生物科技有限责任公司 一种抑制sars-cov-2感染的多肽、组合物及其用途
CN111588732A (zh) * 2020-06-18 2020-08-28 大连工业大学 岩藻多糖在抗新型冠状病毒中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101784272A (zh) * 2007-08-24 2010-07-21 玛丽诺姆德生物技术公司 包含硫酸多糖的抗病毒组合物
CN107847430A (zh) * 2015-07-14 2018-03-27 玛丽诺姆德生物技术股份公司 一种具有抗病毒活性的鼻塞疏通组合物
CN111471088A (zh) * 2020-04-21 2020-07-31 北京中科微盾生物科技有限责任公司 一种抑制sars-cov-2感染的多肽、组合物及其用途
CN111588732A (zh) * 2020-06-18 2020-08-28 大连工业大学 岩藻多糖在抗新型冠状病毒中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PEREIRA LEONEL , CRITCHLEY ALAN T: "The COVID 19 Novel Coronavirus Pandemic 2020: Seaweeds to the Rescue? Why Does Substantial, Supporting Research about the Antiviral Properties of Seaweed Polysaccharides Seemto go Unrecognized by the Pharmaceutical Community in These Desperate Times", JOURNAL OF APPLIED PHYCOLOGY, vol. 32, no. 3, 1 June 2020 (2020-06-01), pages 1875 - 1877, XP037165769, ISSN: 0921-8971, DOI: 10.1007/s10811-020-02143-y *
RITESH TANDON; SHARP JOSHUA S; ZHANG FUMING; POMIN VITOR H; ASHPOLE NICOLE M; MITRA DIPANWITA; JIN WEIHUA; LIU HAO; SHARMA POONAM;: "Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives", 8 June 2020 (2020-06-08), pages 1 - 20, XP055754631, Retrieved from the Internet <URL:https://www.researchgate.net/publication/342044922_Effective_Inhibition_of_SARS-CoV-2_Entry_by_Heparin_and_Enoxaparin_Derivatives/fulltext/5edf81cb92851cf1386c47d2/Effective-Inhibition-of-SARS-CoV-2-Entry-by-Heparin-and-Enoxaparin-Derivatives.pdf> *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114573727A (zh) * 2022-03-24 2022-06-03 自然资源部第一海洋研究所 海参岩藻多糖及其制备方法和在制备防治由幽门螺杆菌引起的疾病的药物和保健品中的应用
CN114573727B (zh) * 2022-03-24 2023-03-21 自然资源部第一海洋研究所 海参岩藻多糖及其制备方法和在制备防治由幽门螺杆菌引起的疾病的药物和保健品中的应用

Also Published As

Publication number Publication date
US20220125827A1 (en) 2022-04-28

Similar Documents

Publication Publication Date Title
CN111658664B (zh) 海参多糖在抗新型冠状病毒中的应用
CN111588732A (zh) 岩藻多糖在抗新型冠状病毒中的应用
KR101571571B1 (ko) 황화 폴리사카라이드를 포함하는 항바이러스 조성물
US20210052710A1 (en) Compositions and methods for protecting against airborne pathogens and irritants
CA2992352C (en) Stuffy nose deblocking composition having antiviral activity
MXPA06006650A (es) Composiciones farmaceuticas antivirales.
CN111686125A (zh) 卡拉胶在抗新型冠状病毒中的应用
CN101616687A (zh) 非特异性免疫刺激剂
CN112220799A (zh) 一种抑制病毒的产品及用途
WO2021253643A1 (zh) 硫酸化多糖在抗新型冠状病毒中的应用
US20230270802A1 (en) Antiviral pharmaceutical composition
EP2040716B1 (en) Cellulose sulfate for the treatment of rhinovirus infection
CN111773182B (zh) 一种预防病毒传染的复方制剂及其配制/使用方法与应用
DE60204967T2 (de) Verwendung von übersulfatierten polysacchariden als hiv-hemmer
WO2021236626A1 (en) Mucoretentive antiviral technologies
CN114732827B (zh) 不同海洋生物来源硫酸多糖的应用及其药物组合物
AU2021235223A1 (en) Composition for the treatment of lesions of the respiratory system
CN112516156A (zh) 一种抑制病毒的产品、制备方法及用途
CN104721221B (zh) 琼枝麒麟菜多糖用于制备抗呼吸道病毒药物中的用途
CN114316081B (zh) 一种具有抑制SARS-CoV-2活性的长茎葡萄蕨藻硫酸化多糖及其制备方法和应用
CN114404446B (zh) 一种抗病毒感染的复合物及其制备方法和用途
US20230241098A1 (en) Antiviral pharmaceutical composition
CN101544690B (zh) 从墨草(P.octandra)所提取的一类植物蛋白、其制备方法及用途
US20240058385A1 (en) Anti-viral compositions and methods
JPH01199915A (ja) ウイルス性疾患処置剤

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20941213

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20941213

Country of ref document: EP

Kind code of ref document: A1