WO2022041634A1 - 一种预防病毒传染的复方制剂及其配制/使用方法与应用 - Google Patents

一种预防病毒传染的复方制剂及其配制/使用方法与应用 Download PDF

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WO2022041634A1
WO2022041634A1 PCT/CN2021/071507 CN2021071507W WO2022041634A1 WO 2022041634 A1 WO2022041634 A1 WO 2022041634A1 CN 2021071507 W CN2021071507 W CN 2021071507W WO 2022041634 A1 WO2022041634 A1 WO 2022041634A1
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preparation
fucoidan
formulation
viral infection
preventing viral
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French (fr)
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宋淑亮
吉爱国
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山东大学
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    • 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
    • A61K9/0043—Nose
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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
    • 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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the invention belongs to the field of medicine and biology, and particularly relates to a compound preparation for preventing virus infection.
  • the mucous membrane is a layer of tissue structure covering the inner wall of the digestive, respiratory, urinary, reproductive and other organs in the human body, and is composed of epithelial tissue and loose connective tissue. According to different parts, there are different names such as oral mucosa, gastrointestinal mucosa, nasal mucosa, tracheal mucosa, vaginal mucosa, and eyelid mucosa. Their structures are also different due to their different functions and parts.
  • the nasal mucosa referred to as the nasal mucosa, covers the surface of the nasal cavity. The nasal mucosa can make the nose play an important role in filtering, regulating temperature and moisturizing, so that the air inhaled into the lungs can maintain the appropriate temperature and humidity.
  • Airborne transmission of the virus is the main transmission mode of respiratory infectious diseases, including droplet transmission, droplet nuclear transmission and dust transmission.
  • the virus When the virus is transmitted through the air, the virus enters the human nasal cavity through the human body's breathing. Due to the warmth and humidity in the nasal cavity, the virus is easily attached to the mucous membrane of the nasal cavity, thereby invading the cells of the human body and infecting people with the virus. Therefore, if the virus on the nasal mucosa can be blocked from invading human cells, the spread of the virus can be blocked.
  • Fucoidan is a sulfated polysaccharide containing high proportions of L-fucose and organic sulfates, mainly derived from brown algae. Current studies have shown that fucoidan sulfate has anti-inflammatory, anti-tumor, antioxidant, anti-viral and hypolipidemic effects. The latest research shows that fucoidan has the effect of protecting cells from viruses such as influenza virus and COVID-19 virus.
  • the inventors found that the antiviral effect of the sulfated polysaccharides such as fucoidan is not easily attached to the nasal mucosa.
  • the present invention provides a compound preparation for preventing virus infection.
  • the preparation is a preparation capable of combining fucoidan with the nasal mucosa with high efficiency.
  • a first aspect of the present invention provides a compound preparation for preventing viral infection, comprising: preparation A and preparation B;
  • the preparation A includes: chitosan, phenoxyethanol, buffer;
  • the formulation B includes: fucoidan, phenoxyethanol, buffer;
  • the deacetylation degree of chitosan in this formula is more than 90%, and the molecular weight is less than 10000Da.
  • the molecular weight of fucoidan in this formula is greater than 10000Da, and the molecular weight is less than 100000Da.
  • the compound preparation for preventing virus infection of the present invention can remarkably improve the binding ability of fucoidan and mucin according to the order of first administering chitosan and then administering fucoidan, so that fucoidan can be efficiently combined with the preparation of nasal mucosa .
  • a second aspect of the present invention provides a method for preparing any of the above-mentioned compound preparations for preventing viral infection, including:
  • the preparation method of the invention is simple, convenient to use, strong practicability and easy to popularize.
  • a third aspect of the present invention provides a method for using any of the above-mentioned compound preparations for preventing viral infection, including:
  • the use method of the invention is simple, solves the problem that currently sulfated polysaccharides such as fucoidan are not easily attached to the nasal mucosa, and enhances the antiviral effect.
  • the present invention has better antiviral effect and binding ability with mucin, it is expected to be widely used in the preparation of antiviral drugs.
  • the compound preparation for preventing viral infection of the present invention can significantly improve the binding ability of fucoidan and mucin according to the sequence of administering chitosan first and then administering fucoidan.
  • the compound preparation for preventing viral infection of the present invention solves the problem that currently sulfated polysaccharides such as fucoidan are not easily attached to the nasal mucosa, thereby enhancing the antiviral effect.
  • the method of the present invention is simple, has good antiviral effect, strong practicability, and is easy to popularize.
  • a compound preparation for preventing viral infection comprising: preparation A and preparation B;
  • the preparation A includes: chitosan, phenoxyethanol, buffer;
  • the formulation B includes: fucoidan, phenoxyethanol, buffer;
  • the compound preparation for preventing viral infection of the present invention solves the problem that currently sulfated polysaccharides such as fucoidan are not easy to adhere to the nasal mucosa, thereby enhancing the antiviral effect.
  • the structure of chitosan is not particularly limited in this application.
  • the degree of deacetylation of chitosan is greater than 90%, and the molecular weight is less than 10000 Da, so as to better improve fucoidan and mucin binding ability.
  • the structure of fucoidan is not particularly limited in this application.
  • the molecular weight of the fucoidan is greater than 10,000 Da, and the molecular weight is less than 100,000 Da, so as to obtain better antiviral effects.
  • the buffer is a phosphate buffer to stabilize the pH of the combination formulation.
  • the pH of the formulation A or the formulation B is about 6.5, so as to obtain a better drug absorption effect under the condition of ensuring the safety and stability of the formulation.
  • the mass ratio of chitosan, phenoxyethanol, and fucoidan is 1-3:1-3:3-5, so that chitosan and fucoidan are dispersed in the solvent Evenly, improve the dosing effect.
  • This compound preparation consists of preparation A and preparation B.
  • the deacetylation degree of chitosan in this formula is more than 90%, and the molecular weight is less than 10000Da.
  • Formulation B was formulated as follows:
  • the molecular weight of fucoidan in this formula is greater than 10000Da, and the molecular weight is less than 100000Da.
  • FITC-tyramine-fucosan solutions were obtained, respectively.
  • the obtained FITC-tyramine-fucosan solution was centrifuged at 4000 rpm to take the supernatant, which was subjected to ultrafiltration (MW3000) through a tangential flow membrane filtration system. Each time, 10 ml of the solution was diluted 10 times the volume, and the ultrafiltration was carried out for 3 times. liquid 65ml, and the small molecular substances were removed by Superdex30 column chromatography, the first peak was collected, and lyophilized to obtain pure FITC-tyramine-fucosan.
  • Negative control group firstly add 100 ⁇ L of 100 ⁇ g/mL chitosan solution with a concentration of 100 ⁇ g/mL and incubate for 1 h, wash three times with pH 6.5 phosphate buffer after 1 h, and then add 100 ⁇ L of pH 6.5 with a concentration of 100 ⁇ g/mL The FITC-tyramine-fucosan solution was incubated for 1 h, washed four times with phosphate buffer at the corresponding pH for 1 h, and the phosphate buffer was retained for the last time.
  • Experimental group 1 firstly add 100 ⁇ L of 100 ⁇ g/mL chitosan solution with a concentration of 100 ⁇ g/mL and incubate for 1 h, wash three times with pH 6.5 phosphate buffer after 1 h, and then add 100 ⁇ L of pH 6.5 with a concentration of 100 ⁇ g/mL The FITC-tyramine-fucosan solution was incubated for 1 h, washed four times with phosphate buffer at the corresponding pH for 1 h, and the phosphate buffer was retained for the last time.
  • Experiment group 2 First add 100 ⁇ L of FITC-tyramine-fucosan solution with a concentration of 100 ⁇ g/mL pH 6.5 for 1 h, wash three times with pH 6.5 phosphate buffer after 1 h, and then add 100 ⁇ L of pH 6.5 The chitosan solution with a concentration of 100 ⁇ g/mL was incubated for 1 h, and then washed four times with the corresponding pH phosphate buffer for 1 h, and the phosphate buffer was retained for the last time.
  • Experimental group 3 First mix 100 ⁇ L of FITC-tyramine-fucosan solution with a concentration of 100 ⁇ g/mL pH 6.5 and 100 ⁇ L of chitosan solution with a concentration of 100 ⁇ g/mL, then add it to the 96-well plate and incubate for 2 hours. After 2 h, the cells were washed four times with phosphate buffer at the corresponding pH, and the phosphate buffer was retained for the last time.
  • Experimental group 4 Add 100 ⁇ L of FITC-tyramine-fucosan solution with a concentration of 100 ⁇ g/mL pH 6.5 for 2 h, then wash with phosphate buffer of corresponding pH for 4 times for 2 h, and retain the phosphate buffer for the last time.
  • Negative control group firstly add 100 ⁇ L of 100 ⁇ g/mL chitosan solution with a concentration of 100 ⁇ g/mL and incubate for 1 h, wash three times with pH 6.5 phosphate buffer after 1 h, and then add 100 ⁇ L of pH 6.5 with a concentration of 100 ⁇ g/mL FITC-tyramine-fucosan solution was incubated for 1 h.
  • Experimental group 1 firstly add 100 ⁇ L of 100 ⁇ g/mL chitosan solution with a concentration of 100 ⁇ g/mL and incubate for 1 h, wash three times with pH 6.5 phosphate buffer after 1 h, and then add 100 ⁇ L of pH 6.5 with a concentration of 100 ⁇ g/mL FITC-tyramine-fucosan solution was incubated for 1 h.
  • Experiment group 2 First add 100 ⁇ L of FITC-tyramine-fucosan solution with a concentration of 100 ⁇ g/mL pH 6.5 for 1 h, wash three times with pH 6.5 phosphate buffer after 1 h, and then add 100 ⁇ L of pH 6.5 Chitosan solution with a concentration of 100 ⁇ g/mL was incubated for 1 h.
  • Experimental group 3 100 ⁇ L of FITC-tyramine-fucosan solution with a concentration of 100 ⁇ g/mL pH 6.5 and 100 ⁇ L of chitosan solution with a concentration of 100 ⁇ g/mL were mixed evenly, and then added to the 96-well plate and incubated for 2 h.
  • Experimental group 4 Add 100 ⁇ L of FITC-tyramine-fucosan solution with a concentration of 100 ⁇ g/mL pH6.5 and incubate for 2 h.
  • mice were randomly divided into 4 groups according to body weight, namely normal control group, fucoidan control group, chitosan control group and compound preparation group, with 10 mice in each group.
  • the normal control group, the fucoidan control group, the chitosan control group, and the compound preparation group were given the corresponding nasal spray 3 times respectively, and the normal control group was given normal saline as the control.
  • After 15 minutes of spraying all mice were exposed to the air of influenza A H1N1 virus liquid for 15 minutes. On day 5 after infection, the number of infected animals was counted.
  • the compound preparation can significantly reduce the number of mice infected with the virus, and has the effect of preventing the airborne transmission of the virus.

Abstract

一种预防病毒传染的复方制剂及其配制/使用方法与应用。该复方制剂包括制剂A和制剂B，其中制剂A包括：壳聚糖、苯氧乙醇、缓冲液；制剂B包括：岩藻聚糖、苯氧乙醇、缓冲液。该复方制剂使用方法为：先给予包括壳聚糖的制剂A再给予包括岩藻聚糖的制剂B。

Description

一种预防病毒传染的复方制剂及其配制/使用方法与应用 技术领域
本发明属于医药生物领域，具体涉及一种预防病毒传染的复方制剂。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
黏膜是覆盖在人体内消化、呼吸、泌尿、生殖等器官管腔内壁的一层组织结构，由上皮组织和疏松结缔组织构成。根据部位不同，有口腔黏膜、胃肠黏膜、鼻腔黏膜、气管黏膜、阴道黏膜、眼睑黏膜等不同名称。它们的结构也因功能、部位不一而不尽相同。鼻腔黏膜，简称鼻黏膜，覆盖于鼻腔表面。鼻黏膜能使鼻子起到过滤、调温及湿润的重要作用，使吸到肺部的空气能维持合适的温度及湿度。当鼻腔受到致病源侵袭时，这些致病菌就会刺激局部鼻黏膜，人体出于自身保护作用，鼻黏膜局部的毛细血管就会扩张，充血，水肿，渗出，分泌出大量的组织液以杀灭入侵的病原微生物。
病毒的传染途径中经空气传播是呼吸系统传染病的主要传播方式，包括飞沫传播、飞沫核传播和尘埃传播等三种传播途径。病毒经空气传播时，病毒经过人体的呼吸进入人的鼻腔中，由于鼻腔内温暖潮湿使病毒极易附着在鼻腔的黏膜上，进而侵害人体的细胞，使人感染病毒。因此如果能够阻断鼻腔黏膜上的病毒侵害人体细胞，就可以阻断病毒的传播。
岩藻聚糖是一种含有高比例L-岩藻糖和有机硫酸酯的硫酸多糖，主要来源 于褐藻。目前的研究表明，岩藻聚糖硫酸酯具有抗炎、抗肿瘤、抗氧化、抗病毒和降血脂等作用。最新研究表明岩藻聚糖具有保护细胞不受流感病毒、COVID-19病毒等病毒感染的作用。
但发明人发现：由于岩藻聚糖等硫酸化多糖不易附着鼻腔黏膜，使其抗病毒效果受到一定影响。
发明内容
为了克服上述问题，本发明提供了一种预防病毒传染的复方制剂。该制剂是一种可使岩藻聚糖高效率结合于鼻黏膜的制剂。
为实现上述技术目的，本发明采用如下技术方案：
本发明的第一个方面，提供了一种预防病毒传染的复方制剂，包括：制剂A和制剂B；
所述制剂A包括：壳聚糖、苯氧乙醇、缓冲液；
所述制剂B包括：岩藻聚糖、苯氧乙醇、缓冲液；
本配方中壳聚糖脱乙酰度大于90％，分子量低于10000Da。本配方中岩藻聚糖分子量大于10000Da，分子量低于100000Da。
本发明的预防病毒传染的复方制剂按照先给予壳聚糖再给予岩藻聚糖的顺序能显著提高岩藻聚糖与黏蛋白的结合能力，使岩藻聚糖高效率结合于鼻黏膜的制剂。
本发明的第二个方面，提供了任一上述的预防病毒传染的复方制剂的配制方法，包括：
取壳聚糖、苯氧乙醇、NaH 2PO 4、NaH 2PO 4，加水混合均匀后，定容，得制剂A；
取岩藻聚糖、苯氧乙醇、NaH 2PO 4、NaH 2PO 4，加水混合均匀后，定容，得制剂B。
本发明的配制方法简单、使用方便，实用性强，易于推广。
本发明的第三个方面，提供了任一上述的预防病毒传染的复方制剂的使用方法，包括：
先将制剂A喷于鼻黏膜上2-3下，2-3min后再将制剂B喷于鼻黏膜上2-3下即可。
本发明的使用方法简单，解决了目前岩藻聚糖等硫酸化多糖不易附着鼻腔黏膜的问题，使其抗病毒效果得到增强。
由于本发明具有较好的抗病毒效果和与黏蛋白的结合能力，因此，有望在制备抗病毒药物中得到广泛的应用。
本发明的有益效果在于：
(1)本发明的预防病毒传染的复方制剂按照先给予壳聚糖再给予岩藻聚糖的顺序能显著提高岩藻聚糖与黏蛋白的结合能力。
(2)本发明的预防病毒传染的复方制剂解决了目前岩藻聚糖等硫酸化多糖不易附着鼻腔黏膜的问题，使其抗病毒效果得到增强。
(3)本发明的方法简单、抗病毒效果好、实用性强，易于推广。
具体实施方式
应该指出，以下详细说明都是示例性的，旨在对本发明提供进一步的说明。除非另有指明，本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限 制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
一种预防病毒传染的复方制剂，包括：制剂A和制剂B；
所述制剂A包括：壳聚糖、苯氧乙醇、缓冲液；
所述制剂B包括：岩藻聚糖、苯氧乙醇、缓冲液；
本发明的预防病毒传染的复方制剂解决了目前岩藻聚糖等硫酸化多糖不易附着鼻腔黏膜的问题，使其抗病毒效果得到增强。
本申请中对壳聚糖的结构并不作特殊的限定，在一些实施例中，所述壳聚糖脱乙酰度大于90％，分子量低于10000Da，以更好地改善岩藻聚糖与黏蛋白的结合能力。
本申请中对岩藻聚糖的结构并不作特殊的限定，在一些实施例中，所述岩藻聚糖分子量大于10000Da，分子量低于100000Da，以获得更优的抗病毒效果。
在一些实施例中，所述缓冲液为磷酸盐缓冲液，以稳定复方制剂的pH。
研究发现：pH对本发明的复方制剂的稳定性、溶解度和药效等都有一定影响。因此，在一些实施例中，所述制剂A或制剂B的pH为6.5左右，以在保证制剂安全、稳定的情况下，获得更优的药物吸收效果。
在一些实施例中，所述壳聚糖、苯氧乙醇、岩藻聚糖的质量比为1～3:1～3:3～5，以使壳聚糖、岩藻聚糖在溶剂中分散均匀，提高给药效果。
下面结合具体的实施例，对本发明做进一步的详细说明，应该指出，所述具体实施例是对本发明的解释而不是限定。
实施例1
预防病毒传染的复方制剂的制备：
本复方制剂由制剂A和制剂B组成。
其中制剂A的配方如下：
Figure PCTCN2021071507-appb-000001
本配方中壳聚糖脱乙酰度大于90％，分子量低于10000Da。取上述配方中相应质量或者体积的壳聚糖、苯氧乙醇、0.2M NaH 2PO 4、0.2M NaH 2PO 4，加适量纯净水搅拌均匀后，定容于1000mL即得。
制剂B的配方如下：
Figure PCTCN2021071507-appb-000002
本配方中岩藻聚糖分子量大于10000Da，分子量低于100000Da。取上述配方中相应质量或者体积的岩藻聚糖、苯氧乙醇、0.2M NaH 2PO 4、0.2M NaH 2PO 4，加适量纯净水搅拌均匀后，定容于1000mL即得。
复方制剂的使用方法：
使用时，先将A溶液喷于鼻黏膜上2-3下，2-3min后再将B溶液喷于鼻黏膜上2-3下即可。
岩藻聚糖的荧光标记：
取岩藻聚糖(75000Da)1g，溶解于100ml pH为7.4的磷酸盐缓冲液中，4℃下充分溶解12h，加入2g酪胺Tyr，充分混合溶解。保鲜膜封口，置于150r/min的摇床中，37℃条件下反应24h；加入1g氰基硼氢化钠，保鲜膜封口，置于150r/min的摇床中，反应96h。反应液于4000rpm离心，取上清液，酪胺-岩藻聚糖溶液。
加入25mg异硫氰酸荧光素FITC于上述酪胺-岩藻聚糖溶液中，保鲜膜封口，置于150r/min的摇床中，37℃条件下反应36h。分别得FITC-酪胺-岩藻聚糖溶液。
将得到FITC-酪胺-岩藻聚糖溶液4000rpm离心取上清，经切向流膜过滤系统进行超滤(MW3000)，每次取10ml溶液稀释10倍体积，连续超滤3次，收集浓缩液65ml，经Superdex30柱层析脱除小分子物质，收集第一峰，冻干，得纯品FITC-酪胺-岩藻聚糖。
复方pH的选择：
将壳聚糖(脱乙酰度95％)、FITC-酪胺-岩藻聚糖各0.01g分别溶解于100mL pH为6.0、6.5、7.0的0.02M的磷酸缓冲液中。
取高亲和ELISA 96孔板，每孔先用100μL浓度为10μg/mL的黏蛋白MUC5AC在4℃条件下孵育24h(阴性对照组不加MUC5AC)，再用100μL浓度为100μg/mL的脱脂牛奶封闭4h，然后用pH7.2的磷酸缓冲液清洗三次，先分别加入100μL浓度为100μg/mL的不同pH的壳聚糖溶液孵育1h，1h后用相应 pH的磷酸缓冲液清洗三次，再加入相应pH的100μL浓度为100μg/mL的FITC-酪胺-岩藻聚糖溶液孵育1h，1h再用相应pH的磷酸缓冲液清洗四次，最后一次保留磷酸缓冲液，采用多功能酶标仪Cytation5检测荧光强度，并计算FITC-酪胺-岩藻聚糖的结合率。
表1 不同pH下岩藻聚糖与黏蛋白结合能力实验结果(n＝3)
Figure PCTCN2021071507-appb-000003
**与阴性对照组相比P＜0.01。
上述实验结果表明，pH6.5条件下岩藻聚糖与黏蛋白的结合能力最强，能显著提高岩藻聚糖与黏蛋白的结合率。pH降低或者升高会降低两者的结合力，这是发明人经多次实验得到实验结果。
复方使用顺序的选择1：
取高亲和ELISA 96孔板，每孔先用100μL浓度为10μg/mL的黏蛋白MUC5AC在4℃条件下孵育24h(阴性对照组不加MUC5AC)，再用100μL浓度为100μg/mL的脱脂牛奶封闭4h，然后用pH6.5的磷酸缓冲液清洗三次，按以下分组进行孵育：
阴性对照组：先加入100μL浓度为100μg/mL的pH6.5的壳聚糖溶液孵育1h，1h后用pH6.5的磷酸缓冲液清洗三次，再加入pH6.5的100μL浓度为100μg/mL的FITC-酪胺-岩藻聚糖溶液孵育1h，1h再用相应pH的磷酸缓冲液清 洗四次，最后一次保留磷酸缓冲液。
实验组一：先加入100μL浓度为100μg/mL的pH6.5的壳聚糖溶液孵育1h，1h后用pH6.5的磷酸缓冲液清洗三次，再加入pH6.5的100μL浓度为100μg/mL的FITC-酪胺-岩藻聚糖溶液孵育1h，1h再用相应pH的磷酸缓冲液清洗四次，最后一次保留磷酸缓冲液。
实验组二：先加入100μL浓度为100μg/mL的pH6.5的FITC-酪胺-岩藻聚糖溶液孵育1h，1h后用pH6.5的磷酸缓冲液清洗三次，再加入pH6.5的100μL浓度为100μg/mL的壳聚糖溶液孵育1h，1h再用相应pH的磷酸缓冲液清洗四次，最后一次保留磷酸缓冲液。
实验组三：先将100μL浓度为100μg/mL的pH6.5的FITC-酪胺-岩藻聚糖溶液和100μL浓度为100μg/mL的壳聚糖溶液混合均匀后再加入96孔板孵育2h，2h再用相应pH的磷酸缓冲液清洗四次，最后一次保留磷酸缓冲液。
实验组四：加入pH6.5的100μL浓度为100μg/mL的FITC-酪胺-岩藻聚糖溶液孵育2h，2h再用相应pH的磷酸缓冲液清洗四次，最后一次保留磷酸缓冲液。
最后统一采用多功能酶标仪Cytation5检测荧光强度，并计算FITC-酪胺-岩藻聚糖的结合率。
表2 不同添加顺序岩藻聚糖与黏蛋白结合能力实验结果(n＝3)
Figure PCTCN2021071507-appb-000004
Figure PCTCN2021071507-appb-000005
**与阴性对照组相比P＜0.01。
从上述结果可以看出，不同的给药顺序对于岩藻聚糖和黏蛋白的结合能力影响极大，只有按照先给予壳聚糖再给予岩藻聚糖的顺序才能显著提高岩藻聚糖与黏蛋白的结合能力。
复方使用顺序的选择2：
取高亲和ELISA 96孔板，每孔先用100μL浓度为10μg/mL的黏蛋白MUC5AC在4℃条件下孵育24h(阴性对照组不加MUC5AC)，再用100μL浓度为100μg/mL的脱脂牛奶封闭4h，然后用pH6.5的磷酸缓冲液清洗三次，按以下分组进行孵育：
阴性对照组：先加入100μL浓度为100μg/mL的pH6.5的壳聚糖溶液孵育1h，1h后用pH6.5的磷酸缓冲液清洗三次，再加入pH6.5的100μL浓度为100μg/mL的FITC-酪胺-岩藻聚糖溶液孵育1h。
实验组一：先加入100μL浓度为100μg/mL的pH6.5的壳聚糖溶液孵育1h，1h后用pH6.5的磷酸缓冲液清洗三次，再加入pH6.5的100μL浓度为100μg/mL的FITC-酪胺-岩藻聚糖溶液孵育1h。
实验组二：先加入100μL浓度为100μg/mL的pH6.5的FITC-酪胺-岩藻聚糖溶液孵育1h，1h后用pH6.5的磷酸缓冲液清洗三次，再加入pH6.5的100μL浓度为100μg/mL的壳聚糖溶液孵育1h。
实验组三：先将100μL浓度为100μg/mL的pH6.5的FITC-酪胺-岩藻聚糖溶液和100μL浓度为100μg/mL的壳聚糖溶液混合均匀后再加入96孔板孵育2h。
实验组四：加入pH6.5的100μL浓度为100μg/mL的FITC-酪胺-岩藻聚糖溶液孵育2h。
所有组孵育完成后，立即取适量上清液采用半胱氨酸盐酸盐法检测各孔上清中岩藻聚糖，并计算吸附率。。
表3 不同添加顺序岩藻聚糖与黏蛋白结合能力实验结果(n＝3)
Figure PCTCN2021071507-appb-000006
**与阴性对照组相比P＜0.01。
从上述结果可以看出，不同检测方法结果基本一致，不同的给药顺序对于岩藻聚糖和黏蛋白的结合能力影响极大，只有按照先给予壳聚糖再给予岩藻聚糖的顺序才能显著提高岩藻聚糖与黏蛋白的结合能力。
复方制剂预防病毒感染活性研究：
小鼠按体重随机分为4组，分别为正常对照组、岩藻聚糖对照组、壳聚糖对照组、复方制剂组，每组10只。正常对照组、岩藻聚糖对照组、壳聚糖对照组、复方制剂组分别给予相应鼻喷雾3次，正常对照组使用生理盐水作为对照。喷雾15min后，将所有小鼠暴露于甲型H1N1流感病毒液空气中15min。感染后第5天，计算感染动物数。
表4 不同鼻喷雾剂预防病毒感染活性结果
Figure PCTCN2021071507-appb-000007
从上表中可以看出复方制剂可以明显减低小鼠感染病毒的数量，具有预防病毒空气传播的作用。
最后应该说明的是，以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。上述虽然对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，所属领域技术人员应该明白，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。

Claims (10)

  1. 一种预防病毒传染的复方制剂，其特征在于，包括：制剂A和制剂B；
    所述制剂A包括：壳聚糖、苯氧乙醇、缓冲液；
    所述制剂B包括：岩藻聚糖、苯氧乙醇、缓冲液。
  2. 如权利要求1所述的预防病毒传染的复方制剂，其特征在于，所述壳聚糖脱乙酰度大于90％，分子量低于10000Da。
  3. 如权利要求1所述的预防病毒传染的复方制剂，其特征在于，所述岩藻聚糖分子量大于10000Da，分子量低于100000Da。
  4. 如权利要求1所述的预防病毒传染的复方制剂，其特征在于，所述缓冲液为磷酸盐缓冲液。
  5. 如权利要求1所述的预防病毒传染的复方制剂，其特征在于，所述壳聚糖、苯氧乙醇、岩藻聚糖的质量比为1～3:1～3:3～5。
  6. 如权利要求1所述的预防病毒传染的复方制剂，其特征在于，所述制剂A和/或制剂B的pH为6.3～6.7。
  7. 如权利要求6所述的预防病毒传染的复方制剂，其特征在于，所述制剂A和/或制剂B的pH为6.5。
  8. 权利要求1-7任一项所述的预防病毒传染的复方制剂的配制方法，其特征在于，包括：
    取壳聚糖、苯氧乙醇、NaH 2PO 4、NaH 2PO 4，加水混合均匀后，定容，得制剂A；
    取岩藻聚糖、苯氧乙醇、NaH 2PO 4、NaH 2PO 4，加水混合均匀后，定容，得制剂B。
  9. 权利要求1-7任一项所述的预防病毒传染的复方制剂的使用方法，其特征 在于，包括：
    先将制剂A喷于鼻黏膜上2-3下，2-3min后再将制剂B喷于鼻黏膜上2-3下即可。
  10. 岩藻聚糖在制备抗炎、抗肿瘤、抗氧化、抗病毒或降血脂药物中的应用。
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