Use of kelp polysaccharide in antiviral and sterilization

Abstract

The present invention relates to the field of pharmaceutical biotechnology, and specifically to the use of laminarin for antiviral and auxiliary sterilization; a preparation method of laminarin and its application in preparing antiviral sterilization liquid are disclosed, and the preparation method is: take dried kelp, crush it, add it into water, add hydrolase to fully enzymolyze it, inactivate the enzyme after enzymolysis, centrifuge and take the supernatant for freeze-drying to obtain laminarin. Through experimental comparison, it is found that the laminarin prepared by papain is particularly effective in resisting influenza virus. The laminarin antiviral sterilization liquid of the present invention has laminarin as an active ingredient, has significant anti-influenza virus effect, especially influenza A virus, and has certain antibacterial effect, and can be used as a disinfection product or medicine with significant inhibition of influenza A virus and auxiliary inhibition of bacteria and fungi.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and in particular to the use of laminarin polysaccharide in antiviral and sterilization. Background Art

[0002] Kelp, also known as kelp, is a nutritious edible brown algae that contains a variety of active substances beneficial to the human body, including active polysaccharides, trace minerals, vitamins, etc. Kelp has extremely high economic and nutritional value and contains more than 60 nutrients, including iodine, fucose, mannitol, alginic acid, dietary fiber, multiple vitamins and multiple trace elements, etc. These ingredients can be used as the main source of many functional foods or pharmaceutical products. Kelp is low in calories, moderate in protein, and rich in minerals. It is an ideal natural marine food and also has the effects of anti-radiation, prevention and treatment of goiter, skin care and hair care.

[0003] Kelp is rich in laminarin, and studies have shown that it has anti-tumor, lipid-lowering, blood pressure-lowering and blood sugar-lowering functions. So far, three types of polysaccharides have been found: alginate, fucoidan, and brown algae starch. It also contains acidic polysaccharides, fucogalactopolysaccharide sulfate, zosteramin, galacturonic acid, laminin, taurine, bifidus factor and other active ingredients.

[0004] As the main algae product in my country, kelp is mainly distributed in Shandong Peninsula and Dalian coastal areas. It has high economic value in food processing and production. The development of products using kelp as raw material is conducive to promoting the cultivation of kelp in my country, and then promoting the high-value utilization of kelp, achieving simultaneous improvement of economic, ecological and social effects. However, in recent years, the products developed using kelp as raw material are limited to the form of snack foods and condiments, and the development of kelp functional products is still relatively lacking.

[0005] Influenza virus is a representative species of the Orthomyxoviridae family, also known as influenza virus. It includes human influenza virus and animal influenza virus. Human influenza virus mainly includes three types: A, B, and C. It is the pathogen of influenza (flu) and is mainly transmitted through droplets or indirect contact. Among them, influenza A virus (IAV) is prone to antigenic mutation and has caused global pandemics many times.

[0006] In a broad sense, pathogenic bacteria and viruses are both microorganisms, but in a narrow sense, they are quite different. Viruses are non-cellular organisms composed of proteins, nucleic acids, etc., while pathogenic bacteria (bacteria, fungi) are organisms with a cellular structure. The existence of viruses and pathogenic bacteria leads to the occurrence of many diseases and infectious diseases, such as pneumonia, norovirus-induced diarrhea, influenza, bacterial acute otitis media, and oral inflammation caused by fungi, etc., which seriously affect people's daily lives.

[0007] In recent years, with the continuous mutation of influenza A virus, it has also developed a certain degree of drug resistance to current anti-influenza drugs. This urgently requires the development of new substances with anti-influenza activity to provide ideas for the development of current anti-influenza drugs. Based on the traditional idea that medicine and food have the same origin, transforming food and medicine into products is also the basis of food and drug management. Nasal spray is currently a more popular product form, which can block viruses and pathogens from invading the human body from the source, avoiding the shortcomings of difficulty in absorption, unchanged dosage, and difficult to control dosage. It does not require digestion and absorption, reducing irritation to the gastrointestinal tract. It can be used as both a health product and a disinfectant product. Summary of the invention

[0008] The purpose of the present invention is to provide a method for using kelp polysaccharide for antiviral and sterilization in view of the above problems.

[0009] In order to achieve its purpose, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention provides a method for preparing laminarin, comprising the following steps: taking kelp (preferably dried kelp), crushing it and adding it into water, adding hydrolase to perform sufficient enzymolysis, inactivating the enzyme after enzymolysis, centrifuging and taking the supernatant for freeze-drying to obtain laminarin; preferably, the hydrolase is selected from cellulase, pectinase, papain and a composite enzyme mixed in any proportion.

[0011] Preferably, the hydrolase is papain, and the enzymolysis method is: adding the hydrolase in an amount of 1-2% of the mass of the kelp powder, adding water in a solid-liquid ratio of 1:30-100, enzymolysis in a water bath at 50-55°C for 3.0-4.0h, and inactivating the kelp enzymolysis solution after the enzymolysis time is over;

[0012] Preferably, the freeze drying means that the supernatant is first pre-frozen at -55°C to -45°C in a vacuum freeze dryer for 8 to 10 hours, and then maintained in a vacuum state before freeze drying is completed. Under the vacuum state, the temperature is first increased to -10°C at a gradient of 10°C / h, and then maintained at -10°C for 4.5 hours before being increased to -5°C, and then maintained for 5.5 hours before the temperature is increased to 0°C, and then maintained at 0°C and 5°C for 6 hours respectively, and then maintained at 10°C and 15°C for 4.5 hours respectively, and further maintained at 20°C for 6 hours before reaching 25°C, dried at 25°C for 12 hours, and then heated to 35°C, and finally dried at 35°C for 1 hour to complete freeze drying;

[0013] The preferred hydrolase is papain, the addition amount of which is 1.2-1.8% or 1.4-1.6% of the mass of the kelp powder, and the enzymolysis temperature and time are 50° C. and 3.5 hours.

[0014] The second aspect of the present invention provides a laminarin antiviral and bactericidal liquid, comprising an effective amount of laminarin prepared by the above-mentioned preparation method and one or more pharmaceutically acceptable excipients;

[0015] Preferably, the auxiliary materials include aromatics, sweeteners, and antioxidants; the aromatics include flavors and spices; and the antioxidants include vitamin C.

[0016] Preferably, the above-mentioned laminarin polysaccharide antiviral and bactericidal liquid comprises the following components in parts by weight: 0.4-0.6 parts of laminarin polysaccharide, 0-1.25 parts of vitamin C, 0-1.2 parts of aromatics, 0-10 parts of sweeteners, and 87-99.6 parts of purified water.

[0017] Preferably, the above-mentioned laminarin polysaccharide antiviral sterilization liquid comprises the following components in parts by weight: 0.4-0.6 parts of laminarin polysaccharide, 0.25-1.25 parts of vitamin C, and 87-99.6 parts of purified water;

[0018] It is further preferred that the laminarin polysaccharide antiviral sterilization solution further comprises 6 to 10 parts of sweetener,

[0019] It is further preferred that the kelp polysaccharide antiviral disinfectant solution further comprises 0.1 to 1.0 part or 0.4 to 1.0 part of an aromatic agent, wherein the aromatic agent is selected from mint flavor, strawberry flavor, passion fruit flavor, and lavender flavor;

[0020] It is further preferred that the aroma comprises 0.1 to 0.5 parts of mint essence and 0.3 to 0.7 parts of strawberry essence.

[0021] Preferably, the above-mentioned kelp polysaccharide antiviral sterilization liquid comprises the following components in parts by weight: 0.4-0.6 parts of kelp polysaccharide, 0.5-1.0 parts of vitamin C, 0.2-0.4 parts of mint flavor, 6-10 parts of sweetener, 0.4-0.6 parts of strawberry flavor, and 87.5-92.5 parts of purified water;

[0022] Preferably, the mass percentages of kelp polysaccharide, vitamin C, sweetener, mint flavor, strawberry flavor and purified water are 0.5%, 0.5%, 10.0%, 0.2%, 0.5% and 88.3% respectively;

[0023] The sweetener is selected from xylitol, glucose, erythritol and steviol glycoside.

[0024] The third aspect of the present invention provides a method for preparing the laminarin antiviral bactericidal liquid described in any one of the above, comprising the following steps:

[0025] (1) Weigh each raw material component and add purified water to fully dissolve;

[0026] (2) Aseptic canning;

[0027] (3) sterilization to obtain the kelp polysaccharide antiviral sterilization liquid.

[0028] In the technical scheme of the above preparation method, the raw materials are dissolved by ultrasound, the ultrasonic frequency is 45khz, and the ultrasonic time is 20 to 30 minutes; after mixed dissolution, they can be filtered through a 200-mesh sieve; ultra-high temperature instantaneous sterilization is adopted, and the conditions are sterilization at a temperature of 136 to 150°C for 5 to 10 seconds.

[0029] The fourth aspect of the present invention provides the use of laminarin prepared by any of the preparation methods described above or the laminarin antiviral and bactericidal liquid described above in the preparation of antiviral and / or auxiliary antibacterial products, wherein the virus includes influenza virus, and the bacteria are pathogenic fungi or pathogenic bacteria.

[0030] Preferably, in the above application technology scheme, the virus is influenza A virus, including H1N1, PR8, NWS and H3N2; the pathogenic fungi include Candida albicans, and the pathogenic bacteria include Escherichia coli and Staphylococcus aureus; the product includes a medicine, a disinfectant or a health product; the dosage form is a spray or a solution, preferably a nasal spray or an oral spray.

[0031] The beneficial effects of the present invention are:

[0032] The anti-influenza virus extract used in the product of the present invention is mainly laminarin polysaccharide extracted from kelp and its processing waste by enzymatic hydrolysis, which is suitable for most people in my country and has obvious kelp flavor. The laminarin polysaccharide has a broad-spectrum inhibitory effect on influenza A virus, has good color, and has obvious kelp flavor. The present invention has found through research that low-temperature freeze drying can better retain the quality and activity of the extracted laminarin polysaccharide.

[0033] The kelp polysaccharide antiviral sterilization liquid of the present invention has kelp polysaccharide as an active ingredient, has significant efficacy in inhibiting bacteria, fungi and influenza viruses, and can be used as a disinfection product or medicine with significant efficacy in inhibiting bacteria, fungi and influenza viruses. The present invention processes kelp in an enzymatic manner, obtains kelp polysaccharide by freeze drying technology, and then prepares it into a spray, enriches the product form of kelp, adds auxiliary materials such as antioxidants (vitamin C), sweeteners, and flavors, and adds vitamin C on the one hand to prevent the active ingredient polysaccharide therein from being decomposed and destroyed, prolong the shelf life of the product, and on the other hand, the bactericidal effect of vitamin C can also be exerted to supplement the VC required by the human body; seasoning by auxiliary materials such as sweeteners and flavors, the sensory quality is good, the spray is convenient to use, suitable for various groups of people, avoids the waste of kelp resources, and realizes the high-value utilization of kelp.

[0034] The kelp spray (which can be used as a nasal spray or an oral spray) of the present invention has good functional activity, can effectively inhibit Staphylococcus aureus, assist in inhibiting Escherichia coli and Candida albicans, and has a broad-spectrum inhibitory effect on influenza A virus. The present invention is of great significance for the development and utilization of kelp-related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the determination of the basic components of kelp.

[0036] Figure 2 This is a schematic diagram of the effect of KPS obtained by different extraction and drying methods on MDCK cell viability.

[0037] Figure 3 Schematic diagram of the effect of KPS prepared from air-dried and fresh kelp on MDCK cell viability.

[0038] Figure 4 This is a schematic diagram of the antiviral effect of KPS obtained by different extraction and drying methods.

[0039] Figure 5 Schematic diagram of the antiviral effect of KPS obtained by hydrolysis with different hydrolases.

[0040] Figure 6 It is a schematic diagram of the antiviral effect of KPS prepared under the final process conditions observed under a microscope.

[0041] Figure 7 It is the inhibitory effect of KPS prepared under the final process conditions on influenza A virus.

[0042] Figure 8 This is a schematic diagram of the effects of additives in different proportions on sensory quality.

[0043] Fig. 9 This is a schematic diagram of the weight changes of PR8-infected mice from day 0 to day 14.

[0044] Fig.10 This is a schematic diagram of the survival rate of PR8-infected mice from day 0 to day 14.

[0045] Fig.11 It is a schematic diagram of the lung index of mice in each experimental group on the 4th day.

[0046] Fig.12 It is a schematic diagram of spleen index of mice in each experimental group on the 4th day.

[0047] Fig.13 This is a schematic diagram of HE staining of lung tissue sections of mice in each experimental group on the 4th day.

[0048] Fig.14 This is a schematic diagram of the viral NA titer in the lung tissue homogenate of mice in each experimental group on the 4th day.

[0049] Fig.15 It is a schematic diagram of the levels of inflammatory factors in the lung homogenate of mice in each experimental group on the 4th day.

[0050] Fig.16 This is a schematic diagram for evaluating the antibacterial effect of kelp polysaccharide nasal administration.

[0051] Fig.17 This is a schematic diagram of the antibacterial activity of kelp polysaccharide nasal spray against three types of bacteria.

[0052] Fig.18 This is a schematic diagram of the UV spectrum scan of KPS.

[0053] Fig.19 It is a schematic diagram of the components of KPS.

[0054] Fig. 20 This is a schematic diagram comparing the molecular weights of kelp polysaccharides prepared by water extraction and enzymatic hydrolysis.

[0055] Fig.21 It is a schematic diagram of the HPLC chromatogram of the monosaccharide composition in KPS.

[0056] Fig. 22 This is a schematic diagram of the molar percentage of each monosaccharide in KPS. DETAILED DESCRIPTION

[0057] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0058] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0059] The materials used in the present invention are not limited to those described in the following embodiments. Any technician in the field can make appropriate changes to the brand or source of the materials required for the present invention without departing from the spirit and scope of the present invention. The kelp can also be obtained from the local market.

[0060] The main reagents and material sources of the embodiments of the present invention are as follows:

[0061]

[0062]

[0063] Example 1

[0064] 1. Determination of the basic components of kelp

[0065] Experimental method: After washing the surface impurities and salt of the kelp with clean water, remove the surface moisture with absorbent paper and set aside. The moisture content in the sample was determined by the method in "GB5009.3-2016 Determination of Water in Food"; the ash content in the sample was determined by the method in "GB5009.4-2016 Determination of Ash in Food"; the protein content in the sample was determined by the method in "GB5009.5-2016 Determination of Protein in Food"; the fat content in the sample was determined by the method in "GB5009.6-2016 Determination of Fat in Food"; the total sugar content in the sample was determined by the method in "GB / T9695.31-2008 Determination of Total Sugar Content in Meat Products".

[0066] The results of the determination of the basic components of kelp are as follows Figure 1 As shown in the figure, it can be seen that the water content in kelp is extremely high, accounting for the majority, the sugar content in kelp dry matter is relatively high, and the protein and fat content is extremely low, which is consistent with previous research results, and also shows that kelp is a high-quality raw material for extracting polysaccharides.

[0067] 2. Preparation of Laminaria japonica polysaccharide by different preparation methods

[0068] Fresh kelp was purchased from the market and pre-processed for subsequent experimental steps:

[0069] (1) Cleaning: Soak the fresh kelp in clean water to wash away surface impurities and any small amount of salt that may be present;

[0070] (2) Pretreatment: After the cleaned kelp is washed with absorbent paper to remove the surface moisture, it is cut with a knife to facilitate subsequent processing;

[0071] (3) Drying: The pre-treated kelp is air-dried in a drying oven at 45 to 60° C. for 24 h or dried until its moisture content is less than 10%;

[0072] (4) Crushing: The air-dried kelp is crushed with a high-speed crusher for 1 to 2 minutes until it is in a clear powdery state. In the examples of the present invention, kelp polysaccharides are prepared by using different extraction methods (hydrolase enzymatic hydrolysis, water extraction) and drying methods (freeze drying, spray drying), and each process is as follows:

[0073] 1. Enzymolysis: Take the prepared kelp powder, add 1.5% enzyme (cellulase, pectinase, papain and composite enzyme are used respectively in the embodiment of the present invention) according to the weight of the raw material, mix the kelp and pure water at a solid-liquid ratio of 1g:60mL and homogenize for 2min, perform water bath enzymolysis at 50-55°C for 3.0-3.5h, and after the enzymolysis time is over, place the kelp enzymolysis solution in a 100°C water bath to inactivate the enzyme for 10-15min; centrifuge the inactivated kelp enzymolysis solution at 8000r / min for 15-20min, and take the supernatant after centrifugation; filter the supernatant to remove the remaining kelp residue, take the supernatant and dry it, and the powder obtained after drying is kelp polysaccharide.

[0074] 2. Water extraction method: Mix the prepared kelp powder with pure water in a solid-liquid ratio of 1g:60mL and homogenize for 2 minutes. Stir the homogenized kelp slurry in a 70°C water bath for 3.5 hours, then collect the supernatant by centrifugation (8000r / min, 15min), filter the supernatant to remove the remaining kelp residue, take the final water extraction supernatant and dry it. The powder obtained after drying is kelp polysaccharide.

[0075] 3. Freeze drying: The supernatant prepared by enzymatic hydrolysis or water extraction is pre-frozen at -50°C in a vacuum freeze dryer for 8 hours, and then maintained in a vacuum state before freeze drying is completed. Under the vacuum state, the temperature is first increased to -10°C at a gradient of 10°C / h, and then maintained at -10°C for 4.5 hours before being increased to -5°C, and then maintained for 5.5 hours before the temperature is increased to 0°C, and then maintained at 0°C and 5°C for 6 hours respectively, and then maintained at 10°C and 15°C for 4.5 hours respectively, and further maintained at 20°C for 6 hours before reaching 25°C, dried at 25°C for 12 hours, then heated to 35°C, and finally dried at 35°C for 1 hour to complete freeze drying, and finally obtain kelp polysaccharide powder.

[0076] 4. Spray drying: Use a peristaltic pump to suck 500 mL of the supernatant prepared by enzymatic hydrolysis or water extraction into the spray dryer, and then the supernatant is atomized by an atomizer. The sprayed water mist combines with the 170°C high-temperature airflow during the descending process to dry and dehydrate, and finally collect the kelp polysaccharide powder.

[0077] Example 2 Effects of laminarin polysaccharides prepared by different preparation methods and different materials on MDCK cell viability 1 Effects of laminarin polysaccharides prepared by different preparation methods on MDCK cell viability

[0078] 1.1 Experimental methods

[0079] 1.1.1 Preparation of KPS by different extraction and drying methods

[0080] Cellulase was used to hydrolyze kelp at its optimum temperature (55°C) to obtain kelp polysaccharide. Water extraction was used to extract kelp polysaccharide. Dry kelp polysaccharide (KPS) was obtained by freeze drying and spray drying.

[0081] 1.1.2 Evaluate the effects of KPS obtained by different extraction and drying methods on MDCK cell viability:

[0082] The MTT method was used to determine the effect of KPS on MDCK cell viability. MDCK cell suspension was added to a 96-well plate at a concentration of 5×10 3 / mL, add 0.1mL to each well, and culture for 24h at 37℃ and 5% CO2. Prepare KPS into a series of dilutions, replace the culture medium of the monolayer cells with the dilutions, set up normal cell controls, and then culture in a 37℃, 5% CO2 incubator for 48h; add 20μL, 5mg / mL tetramethylthiazolium (MTT) solution to each well, continue to culture for 4h, then discard the culture medium, add 150μL of dimethyl sulfoxide (DMSO) to each well, shake for 10min, and after the crystals are completely dissolved, use an enzyme marker to measure the absorbance value (OD value) at a wavelength of 490nm. Set up 3 parallels for each concentration, and use the average value to evaluate the cytotoxicity of kelp polysaccharides obtained by different extraction and drying methods to MDCK. Cell viability is calculated according to the following formula:

[0083]

[0084] Where: OD0——absorbance of each well of DMSO;

[0085] OD1——absorbance value of each well in the drug treatment group;

[0086] OD2——absorbance of each well in the negative control group.

[0087] 1.2 Experimental Results

[0088] The results of the effects of KPS on MDCK cell viability are as follows Figure 2As shown in the results, the KPS obtained by freeze drying and spray drying had no obvious cytotoxicity to MDCK in the concentration range of 0-5 mg / mL, but the cytotoxicity increased with the increase of concentration. When the concentration was 10 mg / mL, the activity of MDCK cells after the KPS obtained by freeze drying was 43.96±6.51%, while the activity of MDCK cells after the KPS obtained by spray drying was 24.98±2.15%. Therefore, the KPS obtained by freeze drying had little effect on the cell activity of MDCK. This may be because the high temperature environment of the spray drying method destroyed the structure of the polysaccharide, causing the properties of the polysaccharide to change and produce substances that are not conducive to cell survival. In addition, the cell activity of MDCK cells after the KPS prepared by enzymatic hydrolysis was 43.65±5.35%, so the effects of KPS prepared by water extraction and enzymatic hydrolysis on the activity of MDCK cells were not much different.

[0089] 2 Effects of KPS prepared from air-dried kelp and fresh wet kelp on MDCK cell viability

[0090] 2.1 Experimental methods

[0091] Clean fresh wet kelp was air-dried in a drying oven (45-60°C) for 24 hours, crushed by a grinder, and then hydrolyzed by cellulase at its optimal temperature (55°C) to obtain laminarin. At the same time, fresh wet kelp was crushed by a grinder and subjected to the same enzymatic treatment. Finally, dry laminarin (KPS) was obtained by freeze-drying. The MTT method was used to evaluate the effects of KPS prepared from air-dried kelp and fresh wet kelp on the cell viability of MDCK cells.

[0092] 2.2 Experimental Results

[0093] The results are as follows Figure 3 As shown, the two kelp polysaccharides had low cytotoxicity to MDCK cells at 0-5 mg / mL, but showed obvious cytotoxicity at 10 mg / mL. At 5 mg / mL, the cell viability of MDCK cells after KPS from fresh wet kelp was 78.89±6.43%, showing weak cytotoxicity, while the cell viability of MDCK cells after KPS from air-dried kelp was 91.39±12.09%. In summary, the kelp polysaccharide prepared after air-drying has less cytotoxicity.

[0094] Example 3 Antiviral activity of kelp polysaccharides prepared by different extraction / drying methods and hydrolysis with different hydrolases 1 Antiviral activity of kelp polysaccharides prepared by different extraction / drying methods

[0095] 1.1 Experimental methods

[0096] Cellulase was used to hydrolyze dry kelp powder at its optimum temperature (55°C) to obtain kelp polysaccharide. Water extraction was used to extract kelp polysaccharide. Dry kelp polysaccharide (KPS) was obtained by freeze drying and spray drying.

[0097] 1.2 Antiviral activity detection

[0098] The antiviral activity of KPS obtained by different extraction and drying methods was evaluated, and the inhibition rate of KPS against influenza A virus was determined by cytopathic effect (CPE) inhibition experiment. After the canine kidney cells (MDCK) in the 96-well plate grew into a monolayer, they were inoculated with PR8 influenza A virus solution with a virus infection multiplicity (MOI) of 0.1, then incubated at 37°C for 1h, the supernatant was discarded, 200μL of KPS solution with a concentration of 1mg / mL was added, and then incubated at 37°C and 5% CO2 for 48h. The drug Ribavirin (C8H 12 N4O5) was used as the positive control group (50 μg / mL), and a normal cell group and a virus control group were set up at the same time. Finally, 4% paraformaldehyde was used for fixation and crystal violet staining was used for 30 minutes. The absorbance value of each well was measured at a wavelength of 570 nm by an ELISA reader. The virus inhibition rate was calculated according to the following formula:

[0099]

[0100] Among them: OD a ——Absorbance value of drug-treated group;

[0101] OD b ——Absorbance value of virus control group;

[0102] OD c ——Absorbance value of normal cell control group.

[0103] The antiviral effects of KPS (1 mg / mL) obtained by different methods are as follows Figure 4 As shown, the antiviral effect of KPS obtained by spray drying is poor. Among the KPS prepared by enzymatic hydrolysis, the inhibition rate of KPS (1 mg / mL) prepared by freeze drying on PR8 virus is 61.62±2.13%, which significantly exceeds the effect of spray drying (13.67±4.16%, p<0.05); similarly, the inhibition rate of KPS (1 mg / mL) prepared by enzymatic hydrolysis-freeze drying on PR8 virus is significantly higher than that of the water extraction-freeze drying experimental group (p<0.05).

[0104] 2 Antiviral activity of KPS hydrolyzed by different hydrolases

[0105] 2.1 Experimental methods

[0106] Cellulase (55℃, pH 5.5, 3.5h), pectinase (55℃, pH 3.5, 3.5h), papain (50℃, pH 6.5, 3.5h) and compound enzyme (50℃, pH 6.0, 3.5h) were used to hydrolyze kelp at the optimal temperature and pH of the hydrolase, and finally dried kelp polysaccharide (KPS) was obtained by freeze drying. The inhibition rate of KPS against influenza A virus PR8 was determined by cytopathic effect (CPE) inhibition experiment.

[0107] 2.2 Experimental Results

[0108] The results are as follows Figure 5 As shown, the inhibitory effects of KPS obtained by hydrolysis with different hydrolases on PR8 virus were significantly different. The inhibition rate of KPS (500 μg / mL) obtained by hydrolysis with papain on PR8 virus was 90.21±3.12%, which was comparable to the effect of ribavirin (50 μg / mL) and significantly exceeded the antiviral effect of KPS obtained by hydrolysis with other hydrolases (p < 0.05).

[0109] Example 4 Verification of the in vitro anti-influenza effect of KPS

[0110] From the results of the above examples, it can be seen that the best preparation process of laminarin is: hydrolyze with papain to obtain enzymatic solution, and finally obtain laminarin (KPS) capable of inhibiting influenza A virus by freeze drying. The final verification was carried out by cytopathic inhibition experiment (CPE). After the canine kidney cells (MDCK) in the 96-well plate grew into a monolayer, they were inoculated with PR8 influenza A virus solution with a virus infection multiplicity (MOI) of 0.1, then incubated at 37°C for 1h, the supernatant was discarded, 200μL of laminarin solution with a concentration of 1mg / mL was added, and then incubated at 37°C and 5% CO2 for 48h, the cytopathic condition of MDCK was observed under a microscope (60×), and the final inhibition rate of KPS on PR8 virus was calculated.

[0111] The results are as follows Figure 6 As shown, under the microscope, normal MDCK cells showed complete, regular and plump cell structures, while MDCK cells infected with PR8 virus showed serious pathological phenomena such as rounding, shedding and aggregation. After KPS treatment, the aggregation of MDCK cells was reduced, some independent cells appeared, and the cell pathological condition improved, turning to a regular and plump direction.

[0112] Figure 7The results showed that the inhibition rates of KPS (1 mg / mL) against influenza A viruses H1N1, PR8, NWS and H3N2 were 83.25±3.74%, 69.33±9.12%, 89.14±4.45% and 73.54±2.33%, respectively, indicating that KPS has a broad-spectrum anti-influenza activity in vitro.

[0113] According to relevant literature reports, papain, as a biocatalyst, breaks peptide bonds and opens cell walls, allowing more active polysaccharides to be dissolved. Although spray drying is a low-cost drying method, the excessively high temperature used in this drying method may change the structure of polysaccharides, resulting in changes or loss of activity. Therefore, the kelp polysaccharide (KPS) obtained by hydrolyzing air-dried kelp with papain and freeze-drying the hydrolyzate can significantly inhibit the activity of influenza A virus.

[0114] Example 5 Effects of different additive ratios on the sensory quality of kelp polysaccharide nasal spray - single factor experiment

[0115] The addition ratio of each component in this embodiment is the mass percentage of each component to the total mass of the spray.

[0116] 1. Preparation of semi-finished products of kelp polysaccharide spray with different formulations

[0117] Referring to the previous experimental method, since kelp polysaccharide has basically no cytotoxicity to MDCK cells in the concentration range of 0-5 mg / mL, its addition ratio is set to 0.5% unchanged, and the addition ratios of mint flavor, xylitol, and strawberry flavor are 0.3%, 8%, and 0.5%, respectively, and 0.25%, 0.5%, 0.75%, 1.0%, and 1.25% vitamin C are added, respectively, and finally supplemented with pure water, ultrasonically dissolved, mixed and filtered with a sieve, aseptically canned into a vial, and then sterilized by ultra-high temperature instantaneous sterilization to obtain the semi-finished kelp polysaccharide spray. Explore the effects of different addition ratios of vitamin C on the sensory quality of the spray and determine the optimal amount of vitamin C.

[0118] Furthermore, the addition ratios of vitamin C, xylitol and strawberry flavor were kept at 0.75%, 8% and 0.5% respectively, and mint flavor was added at 0.1%, 0.2%, 0.3%, 0.4% and 0.5% respectively. After supplementing with water, the semi-finished product of kelp polysaccharide spray was prepared, and the effects of mint flavor in different addition ratios on the sensory quality of the spray were explored to determine the optimal addition amount of mint flavor.

[0119] Further, the addition ratios of vitamin C, mint flavor and strawberry flavor were kept at 0.75%, 0.3% and 0.5% respectively, 4%, 6%, 8%, 10% and 12% of xylitol were added respectively, and the semi-finished product of kelp polysaccharide spray was prepared after supplementing with water, and the effects of different addition ratios of xylitol on the sensory quality of the spray were explored, and the optimal addition amount of xylitol was determined. Further, the addition ratios of vitamin C, mint flavor and xylitol were kept at 0.75%, 0.3%, and 8% respectively, 0.3%, 0.4%, 0.5%, 0.6% and 0.7% of strawberry flavor were added respectively, and the semi-finished product of kelp polysaccharide spray was prepared after supplementing with water, and the effects of different addition ratios of strawberry flavor on the sensory quality of the spray were explored, and the optimal addition amount of strawberry flavor was determined.

[0120] 2. Effects of different additive ratios on the sensory quality of sprays

[0121] 1. Sensory quality evaluation

[0122] Sensory evaluation: 9 sensory evaluators who had received sensory training were invited to conduct sensory evaluation on the kelp polysaccharide nasal spray in the food sensory evaluation room and record the sensory scores. The team members conducted sensory evaluation on the spray from four aspects: color, texture, smell and taste according to the sensory evaluation rules (see Table 1). The sensory evaluation was conducted on the semi-finished oral spray, and the average value was taken as the sensory score result.

[0123] Table 1 Sensory evaluation details

[0124]

[0125] 2. Evaluation results

[0126] Effects of different additive ratios on the sensory quality of sprays Figure 8 As shown in the figure, when the sensory score of the spray reaches the highest, the addition ratios of vitamin C, mint flavor, xylitol and strawberry flavor are 0.75%, 0.3%, 8% and 0.5% respectively. The pH of vitamin C in aqueous solution is acidic, and a higher addition ratio of vitamin C will give people a strong sense of sourness, resulting in poor sensory quality. When the mint flavor is excessive, the taste will become bitter and the smell is extremely pungent, and as the amount of xylitol added continues to increase, the sensory quality of color decline and bitter taste will be produced. In addition, strawberry flavor has little effect on the sensory quality of the spray, but excessive strawberry flavor will cause the smell to be too strong and the taste to be too sweet. Therefore, the addition ratio of additives is an important determinant of the sensory quality of the product. Combined with the results of the single factor experiment, the addition amounts of vitamin C, mint flavor, xylitol and strawberry flavor were kept in the range of 0.5-1.0%, 0.2-0.4%, 6-10% and 0.4-0.6%, respectively, and subsequent orthogonal tests were carried out to determine the optimal addition ratio.

[0127] The preparation method of kelp polysaccharide nasal spray (Antiflukelp) is as follows:

[0128] (1) Mixing and dissolving: Weigh the required weights of kelp polysaccharide, vitamin C, and sweetener in the order of proportion. Mint essence and strawberry essence can be added in the required amount by a micropipette. Finally, add purified water and dissolve evenly by ultrasonication. The ultrasonic frequency is 45 khz and the ultrasonic time is 20 to 30 min.

[0129] (2) Packaging: The mixed spray concentrate is filtered through a 200-mesh sieve to remove insoluble impurities, and then packaged in a spray bottle. Finally, ultra-high temperature instantaneous sterilization is performed at 136-150°C for 5-10 seconds to obtain the kelp polysaccharide nasal spray.

[0130] Example 6 Effect of different additive ratios on product sensory quality - orthogonal test

[0131] Based on sensory scores, L9(3 4 ) Orthogonal experiment was used to further determine the addition ratio of vitamin C, xylitol, mint flavor and strawberry flavor (based on the single factor experimental results). The orthogonal experimental factor design is shown in Table 2.

[0132] Table 2 Orthogonal experimental factor design

[0133]

[0134] The results are shown in Table 3. It can be seen that the effect of vitamin C on the sensory results of the spray is very obvious. Among the four factors, the order of the strength of the factors affecting the sensory organs of the spray is vitamin C>xylitol>mint flavor>strawberry flavor. The results of K value analysis show that the best addition ratio combination of vitamin C, xylitol, mint flavor and strawberry flavor is A1B1C3D2. The addition ratios of kelp polysaccharide, vitamin C, xylitol, mint flavor, strawberry flavor and purified water are 0.5%, 0.5%, 10.0%, 0.2%, 0.5% and 88.3% respectively. After mixing, ultra-high temperature instantaneous sterilization is performed and the mixture is filled into a spray bottle under a sterile environment to obtain kelp polysaccharide nasal spray (Antiflukelp). At this time, the sensory quality of the spray is the best.

[0135] Table 3 Effects of different additive ratios on product sensory quality

[0136] Test No. A Vitamin C B Mint flavor C Xylitol DStrawberry flavor Sensory Rating 1 1 1 1 1 73.89 2 1 2 2 2 71.39 3 1 3 3 3 74.33 4 2 1 2 3 69.83 5 2 2 3 1 70.06 6 2 3 1 2 71.89 7 3 1 3 2 69.67 8 3 2 1 3 67.56 9 3 3 2 1 64.11 K1 219.61 213.39 213.34 208.06 / K2 211.78 209.01 205.33 212.95 / K3 201.34 210.33 214.06 211.72 / k1 73.20 71.13 71.11 69.35 / k2 70.59 69.67 68.44 70.98 / k3 67.11 70.11 71.35 70.57 / R 6.09 1.46 2.91 1.22 /

[0137] Example 7 In vivo experiment of Laminaria polysaccharide nasal spray (Antiflukelp) against influenza virus

[0138] 1. Experimental plan

[0139] 96 SPF female mice (14-16 g) were selected, weighed one by one, and randomly divided into 8 groups according to their body weight (the specific experimental grouping is shown in Table 4), with 12 mice in each group. The experiment began after 1 day of adaptation.

[0140] In the survival experiment, mice were anesthetized with 30% ulose, and then they were breathing rapidly. Each mouse was quickly infected with PR8 virus (2LD 50 / mice); the normal control group was given the same volume of saline as a control, and the anti-influenza drug Baloxavir (Baloxavir, C 24 H 19 F2N3O4S) and the commercially available anti-influenza product red algae polysaccharide (RAP) nasal spray were used as positive controls. The day of virus inoculation was counted as day 0. Baloxavir (10 mg / kg / day), RAP (25 μg / day), KPS (2.5 or 10 mg / kg / day) and Antiflukelp (2.5 or 10 mg / kg / day) were intranasally administered 2 hours after virus infection, once a day for a total of 5 days. Body weight and mortality were recorded for 14 consecutive days.

[0141] In the pathological experiment, mice in each group were infected with PR8 virus (2LD 50 / mouse), and then 2h after virus infection, Baloxavir (10mg / kg / day), RAP (25μg / day), KPS (2.5 or 10mg / kg / day), and Antiflukelp (2.5 or 10mg / kg / day) were administered intranasally for 3 consecutive days. The mice were weighed and killed on the 4th day, and the lung and spleen indexes were calculated according to the weight of lung and spleen tissues. After formaldehyde fixation and paraffin embedding of lung tissues (3 samples / group), hematoxylin-eosin (H&E) staining was used for pathological analysis of lung tissues. Lung tissues were homogenized with PBS (1×), and the virus titer in lung homogenates was determined by NA titer method, and ELISA detection kits were used to detect changes in CXC chemokine ligand 1 (CXCL1), chemokine ligand 5 (RANTES), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) in lung homogenates.

[0142] The study was conducted in strict accordance with the protocol approved by the Animal Care and Use Committee of Ocean University of China (approval number: OUC-SMP-2023-07-03). Mice were given adequate feed and drinking water during their upbringing. At the end of the experiment, mice were humanely euthanized or killed at 14 dpi.

[0143] Table 4 Animal experiment groups

[0144]

[0145] 2. Index determination

[0146] Mouse survival rate and weight change: After inoculation with the virus, the death and weight of mice were recorded every day for 14 consecutive days, and the weight recovery after administration was compared (when the weight of the mouse dropped by more than 25% relative to the weight on the day of inoculation, it was considered dead), the survival rate of the mice was calculated, and the weight change and survival rate curves were drawn. The weight change and survival rate were calculated according to the following formulas,

[0147]

[0148]

[0149] The results are as follows Fig. 9 , Fig.10 As shown, the proportion of the initial body weight of mice in the placebo control group was significantly reduced (55.56%), and the proportion of the initial body weight of mice in the Baloxavir (10mg / kg / day) and RAP (25μg / day) treatment groups was 102.32±5.71% and 98.95±3.36%, respectively. It can be seen that the commercially available antiviral products have a significant effect on inhibiting the weight loss of mice infected with viruses. The body weight proportions of the low-dose group (2.5mg / kg / day) and high-dose group (10mg / kg / day) of KPS were 94.28±6.38% and 98.41±2.61%, respectively, which is consistent with the effect of antiviral products. The proportion of the initial body weight of mice in the low-dose group (2.5mg / kg / day) and high-dose group (10mg / kg / day) of Antiflukelp were 101.47±4.75% and 101.24±3.29%, respectively, which even exceeded the effect of the commercial product red algae polysaccharide nasal spray (25μg / day). In the survival rate results, the final survival rate of mice in the placebo group was only 14.29%, which was extremely low. It can be seen that the virus had a very high degree of damage to mice. The survival rates of the Baloxavir (10mg / kg / day) and RAP (25μg / day) treatment groups were 100% and 57.14%, respectively. The effect of antiviral drugs was still very significant. The current antiviral products have a certain effect on improving the survival rate of mice infected with the virus. The high-dose treatment group of KPS (10mg / kg / day) and Antiflukelp (10mg / kg / day) can increase the survival rate of mice to 71.43%. It can be seen that the administration of kelp polysaccharides and kelp polysaccharide nasal spray can improve the survival of mice infected with the virus.

[0150] Mouse lung and spleen index and lung pathology analysis: On the 4th day after infection, 3 mice were taken from each group and killed by cervical dislocation. The complete lung and spleen tissues were taken out and weighed. The lung index and spleen index of each group of mice were calculated based on the weight of the mice. A small part of the lung tissue (3 parts / group) was taken for formaldehyde fixation and paraffin embedding, and then H&E staining analysis was performed to observe the lung lesions. The lung / spleen index was calculated according to the following formula:

[0151]

[0152] According to relevant research reports, the lungs become edematous due to viral infection, and the weight of lung tissue increases, which leads to an increase in lung index, such as Fig.11 As shown in the results, the lung index of the placebo control group was 1.19±0.15%, significantly higher than that of the normal control group (0.63±0.02%, p<0.05), while the lung index of the Baloxavir (10 mg / kg / day) and RAP (25 μg / day) treatment groups was 0.67±0.13% and 0.83±0.10%, significantly lower than that of the placebo control group (p<0.05). KPS and Antiflukelp had a significant inhibitory effect on the increase of the lung index of virus-infected mice, and it was dose-dependent. The low-dose group of KPS (2.5 mg / kg / day) The lung indexes of the high-dose group of KPS (10 mg / kg / day) and Antiflukelp (10 mg / kg / day) were 0.79±0.11% and 0.77±0.10%, respectively, which reached the effect of the Baloxavir (10 mg / kg / day) treatment group. Therefore, Laminaria polysaccharide nasal spray (Antiflukelp) has a good protective effect on the lung tissue of mice infected with influenza virus. Fig.12 It can be seen that the spleen indexes of the Baloxavir (10mg / kg / day) and RAP (25μg / day) treatment groups were 0.28±0.01% and 0.34±0.16%, respectively. The spleen indexes of KPS and Antiflukelp in the high-dose treatment group (10mg / kg / day) were 0.30±0.08% and 0.30±0.11%, respectively. This is slightly different from the spleen index of the normal control group (0.29±0.02%) that was not infected with the virus, but it is still lower than the spleen index after virus infection (0.46±0.05%). Previous research data showed that influenza viruses mainly enter the lungs through the respiratory tract, infect the lung tissue and cause lung lesions, and the damage to the spleen tissue is relatively small. Fig.13As shown, compared with the uninfected normal control group, mice infected with PR8 virus had extremely severe lung lesions, inflammatory cell infiltration, lung enlargement, severe deformation and depression of alveoli, and thickening of alveolar walls. After treatment with KPS and Antiflukelp in the low-dose group (2.5 mg / kg / day), lung tissue lesions were reduced, alveoli began to recover, alveolar walls became thinner, and inflammatory cells in the lungs were reduced. The therapeutic effects of KPS (10 mg / kg / day) and Antiflukelp (10 mg / kg / day) in the high-dose group were equivalent to the therapeutic level of Baloxavir (10 mg / kg / day).

[0153] Determination of virus titer in mouse lungs: Mice were killed on the 4th day after virus infection, and lung tissues were removed for preparation of lung tissue homogenate. The virus titer in the lung homogenate was determined using the NA titer method (the NA titer in the lung tissue homogenate in the placebo control group was taken as 100%), thereby evaluating the inhibitory ability of kelp polysaccharide nasal spray on viral proliferation.

[0154] Depend on Fig.14 It can be seen that the viral NA titers of the Baloxavir (10 mg / kg / day) and RAP (25 μg / day) treatment groups were 29.49±7.83% and 43.25±6.60%, respectively. It can be seen that both Baloxavir and Rhodopsin nasal spray can effectively reduce the viral NA titer in the lungs (p < 0.05); KPS and Antiflukelp also significantly inhibited the proliferation of lung viruses (p < 0.05), and a high dose of Antiflukelp had a significant effect on reducing the viral titer in the lungs (p < 0.05). The final viral NA titer of the Antiflukelp (10 mg / kg / day) group was 36.61±1.42%, which is comparable to the effect of the commercially available antiviral product Rhodopsin nasal spray (25 μg / day) experimental group.

[0155] Cytokine determination: After homogenizing the mouse lung tissue, the changes in chemokines (CXCL1, RANTES) and inflammatory factors (IL-6, TNF-α) in the mouse lung tissue homogenate were determined according to the method specified in the ELISA kit instructions. The results are as follows: Fig.15 shown.

[0156] Previous research results show that after influenza virus infects the lungs, it will induce a sharp increase in the levels of chemokines and inflammatory factors in the lungs. Therefore, the ultimate anti-influenza effect of kelp polysaccharide nasal spray is verified by measuring the level of cytokines induced by the lung virus. Fig.15As shown in the figure, the concentrations of CXCL1, RANTES, IL-6 and TNF-α in the normal control group were 105.50±15.74pg / mL, 1043.08±24.01pg / mL, 122.02±17.16pg / mL and 134.34±1.59pg / mL, respectively. Consistent with the research report, the cytokine levels in the lungs of mice increased significantly after influenza virus inoculation (p<0.05). The high-dose group (10mg / kg / day) of KPS and Antiflukelp had the most significant inhibitory effect on the increase of virus-induced cytokines. Compared with the placebo control group, the CXCL1 level in the Antiflukelp (10mg / kg / day) treatment group was significantly reduced from 327.82±24.31pg / mL to 71.53±40.02pg / mL (p<0.05), and the RANTES level was significantly reduced from 15 The level of IL-6 decreased significantly from 178.06±3.43pg / mL to 141.73.05±5.77pg / mL (p<0.05), and the level of TNF-α decreased significantly from 211.12±20.18pg / mL to 147.14±6.84pg / mL (p<0.05). Although the low-dose group (2.5 mg / kg / day) reduced the level of virus-induced cytokines to a certain extent, the effect was not significant enough. The effect of the high-dose group (10 mg / kg / day) of kelp polysaccharide nasal spray was not even lower than that of the antiviral product red algae polysaccharide nasal spray (25 μg / day). The above results fully verified that kelp polysaccharide nasal spray (Antiflukelp) can have a good anti-influenza effect on virus-infected mice, and also explained that downregulating the level of virus-induced cytokines is an important way to inhibit influenza virus infection of the lungs of mice.

[0157] Example 8 Antibacterial activity of kelp polysaccharide nasal spray

[0158] 1. Observation of the antibacterial effect of kelp polysaccharide nasal spray by plate coating method

[0159] Experimental method: Gram-positive bacteria Escherichia coli (E.coli), Gram-negative bacteria Staphylococcus aureus (S.aureus) and fungi Candida albicans (Candida albicans) were selected as experimental strains. After the strains were cultured to the logarithmic growth phase, the bacterial solution was diluted 1000 times with liquid culture medium in a sterile 5mL test tube, and then mixed with kelp polysaccharide nasal spray 1:1 (v / v). After shaking for 2 hours in an incubator at the optimal culture temperature for the strains, 20μL of the mixed solution was fully applied on the solid culture medium, and the culture medium was inverted and cultured for 24 hours. After the culture was completed, the growth of the colonies was observed, and sterile water was used as the negative control of the antibacterial experiment.

[0160] The results are as follows Fig.16 As shown, the total number of colonies in the plates of the three bacteria after coating with the kelp polysaccharide nasal spray (Antiflukelp) tended to decrease compared with the negative control group. This also preliminarily indicated that the spray had a certain antibacterial effect, but it was not very obvious, and the specific inhibition rate of the kelp polysaccharide nasal spray on the three bacteria could not be obtained through the total number of colonies.

[0161] 2. Determination of antibacterial activity of kelp polysaccharide nasal spray by microdilution method

[0162] Experimental method: To further determine the antibacterial rate of kelp polysaccharide nasal spray, its antibacterial activity was determined by microdilution method. After the strain was cultured in liquid culture medium to the logarithmic growth phase, its concentration was adjusted to about 2×10 5 CFU / mL and set aside. Take 50μL of bacterial solution in a 96-well plate and add an equal volume of Antiflukelp sample. In the experiment, sterile ultrapure water was used instead of the sample as a negative control, and a blank experiment was performed. Each group performed 6 parallel experiments. After all groups have completed the addition of drugs, the 96-well plate (with a cover) was placed in a shaker at the optimal culture temperature of the strain (37 or 28°C, 180 or 150rpm) and incubated for 16 to 18 hours. Then, the absorbance was measured at a wavelength of 600nm using an enzyme reader. The antibacterial rate of the sample was calculated according to the following formula: The antibacterial rate was calculated according to the following formula,

[0163]

[0164] Where: OD0——the absorbance value of each well in the blank group (without inoculation of bacterial solution),

[0165] OD1——Absorbance value of each well in the negative control group (sterile water),

[0166] OD2——Absorbance value of each well in the experimental group (Antiflukelp).

[0167] Depend on Fig.17It can be seen that the kelp polysaccharide nasal spray has a very high antibacterial effect on Staphylococcus aureus, with an inhibition rate of 97.82±0.51%. It also has significant inhibitory activity against Escherichia coli and Candida albicans, with inhibition rates of 61.02±5.96% and 73.46±8.00%, respectively, and has a certain inhibitory activity against Candida albicans, with an inhibition rate of 53.36±4.38%. The above results show that the kelp polysaccharide nasal spray has a broad-spectrum antibacterial activity.

[0168] Influenza viruses enter the lungs from the respiratory tract, spread in the alveoli, and infect lung cells, causing serious lung lesions. Neuraminidase (NA), an important glycoprotein of influenza viruses, can cut the glycosidic bonds between sialic acids, separate the virus from the cell membrane, promote the release of the virus, spread the virus between cells, and continuously produce new sub-viruses to infect other host cells. Influenza viruses use neuraminidase (NA) to spread in the lungs, induce an increase in the level of lung inflammatory factors, and ultimately lead to lung inflammation. Therefore, preventing influenza virus infection of the lungs is a reliable way to reduce related diseases caused by severe influenza.

[0169] In this experiment, kelp polysaccharide nasal spray can significantly reduce the NA titer of IAV-infected lungs, effectively prevent the virus from spreading further in the body, and significantly reduce the level of lung inflammatory factors induced by the virus, preventing lung lesions caused by increased inflammatory factors, and then inhibiting various flu symptoms caused by lung lesions. At the same time, the experiment also confirmed that kelp polysaccharide nasal spray has a very significant inhibitory effect on Staphylococcus aureus. According to research reports, after IAV infects the lungs, most of them will induce pneumonia complications mainly caused by Staphylococcus aureus. This also shows that kelp polysaccharide nasal spray can reduce the damage of the virus to the human body by inhibiting the occurrence of pneumonia induced by influenza A virus infection. In addition, kelp polysaccharide nasal spray also has a certain inhibitory activity against Escherichia coli and Candida albicans, and can be used as a backup product to inhibit various diseases caused by bacterial and fungal infections.

[0170] The kelp polysaccharide nasal spray of the present invention belongs to kelp products, fully utilizes kelp, solves the problem of low utilization rate of kelp processing waste materials, and improves the high-value utilization of kelp. At the same time, the polysaccharide is extracted by traditional enzymatic hydrolysis, and its anti-influenza activity is verified, providing a theoretical basis and technical support for the research of anti-influenza virus drugs. The present invention utilizes the advantages of additives such as xylitol and vitamin C to develop a nasal spray that can comprehensively prevent influenza virus and pathogenic bacteria infection, is easy to use, has good sensory quality, and is suitable for various groups of people.

[0171] Example 9 Analysis of Laminaria polysaccharides

[0172] 1 Preparation and UV spectral scanning of pure kelp polysaccharide

[0173] 1.1 Experimental methods

[0174] The enzymatic hydrolysis-alcohol precipitation method was used to extract relatively purified kelp polysaccharides. Take the prepared kelp powder, add 1.5% papain according to the weight of the raw material, mix the kelp and pure water at a ratio of 1g:60mL and homogenize for 2 minutes, enzymolyze in a water bath at 50℃ for 3.5 hours, and after the enzymatic hydrolysis time, inactivate the enzyme in a water bath at 100℃ for 10 minutes; centrifuge the inactivated kelp hydrolysate at 8000r / min for 15 minutes, take the supernatant and filter to remove the remaining kelp residue, concentrate the supernatant to one-third of the original volume by rotary evaporator, and then mix the concentrated kelp hydrolysate with three times the volume of 95% ethanol and precipitate at 4℃ for 24 hours. Then centrifuge the mixed solution at 10000r / min for 15 minutes, discard the supernatant, take the precipitate and dissolve it in ultrapure water to obtain a crude polysaccharide solution. As the protein is removed, chloroform and n-butanol are mixed evenly at a ratio of 4:1 (v / v) to obtain a Sevage solution, and then the Sevage solution is added to the crude polysaccharide solution until the proportion of the Sevage solution in the system reaches 80%, and then the solution is transferred to a separatory funnel and shaken vigorously until stratification occurs, the lower organic solvent and denatured protein are removed, and the upper aqueous phase is retained. The protein removal operation of the aqueous phase is repeated 3 to 5 times until no protein layer appears, and then the organic solvent in the aqueous phase is removed by a rotary evaporator, and then the solution is transferred to a 3500Da dialysis bag for dialysis for 72 hours (water is replaced every 12 hours), and finally the dialysate is freeze-dried to obtain purified kelp polysaccharide (KPS).

[0175] The freeze-dried powder of kelp polysaccharide KPS was prepared into a solution with a concentration of 1 mg / mL with ultrapure water, and then the solution was scanned by ultraviolet spectrophotometer in the wavelength range of 190-800 nm. Laminarin standard was used as the control of the experiment.

[0176] 1.2 Experimental Results

[0177] The results are as follows Fig.18 As shown in the results, kelp polysaccharide has a maximum ultraviolet absorption wavelength in the range of 190-202nm, and has an absorption peak at 195nm with kelp polysaccharide, which is similar to the results reported in previous studies. At the same time, the ultraviolet spectrum scanning results show that KPS has no obvious absorption peaks at 260 and 280nm, indicating that there are basically no protein and nucleic acid impurities in KPS, which indicates that the extracted kelp polysaccharide has a high purity.

[0178] 2 Determination of the composition of kelp polysaccharides

[0179] 2.1 Determination indicators and experimental methods

[0180] 2.1.1 Total sugar content determination method

[0181] Accurately weigh 0.05g polysaccharide sample and mix it with 1mL 5% trichloroacetic acid solution, vortex oscillate for 2min, centrifuge at 8000r / min for 15min, collect the supernatant, draw 0.5mL sample supernatant into a test tube, add 0.25mL 6% phenol solution and 1.25mL concentrated sulfuric acid in sequence, shake well and react in a 100℃ water bath for 5min, cool to room temperature, and measure the absorbance at 490nm. Weigh dry glucose and dissolve it in ultrapure water to prepare standard solutions of various concentrations. Referring to the determination method of polysaccharide samples, measure the absorbance of the standard solution synchronously, and draw a standard curve with polysaccharide concentration (mg / mL) and corresponding absorbance as horizontal and vertical coordinates respectively. According to the absorbance value and conversion factor of the sample, the total sugar content in KPS is obtained. Ultrapure water is used instead of the sample as a blank control, and the average value of three parallel tests is used as the result of each group.

[0182] 2.1.2 Determination of uronic acid content

[0183] Weigh 0.477g of sodium tetraborate decahydrate reagent and dissolve it in concentrated sulfuric acid and dilute to 50mL to obtain a borax-concentrated sulfuric acid solution; weigh 25mg of carbazole reagent and dissolve it in 20mL of anhydrous ethanol to obtain a carbazole solution. Accurately pipette 0.5mL of borax-concentrated sulfuric acid solution and 0.1mL of polysaccharide solution into a glass tube, vortex and oscillate evenly, react in a 100℃ water bath for 10min, cool to room temperature, add 0.02mL of carbazole solution, shake well, react in a 100℃ water bath for 15min, cool to room temperature again, and measure the absorbance at 530nm. At the same time, the absorbance of the D-glucuronic acid standard is measured simultaneously according to the same determination method as the sample, and the uronic acid standard curve is drawn, and the uronic acid content of KPS is determined by substituting it into the standard curve.

[0184] 2.1.3 Determination of sulfate content

[0185] Weigh 108.91 mg of potassium sulfate and dissolve it in 1M hydrochloric acid solution and make up to 100 mL. Shake well to obtain a sulfate standard stock solution with a concentration of 0.6 mg / mL. Accurately pipette 0, 0.02, 0.06, 0.1, 0.12, 0.14, 0.18 and 0.2 mL of the stock solution into test tubes, add hydrochloric acid to make up to 0.2 mL respectively, add 3.8 mL of trichloroacetic acid and 1 mL of 1% barium chloride solution, mix well, let stand for 25 min, and measure the absorbance A1 at a wavelength of 360 nm. Repeat the same operation using 1 mL of 5% gelatin solution instead of barium chloride solution to measure the absorbance A2. Draw a standard curve with sulfate content (mg) as the horizontal axis and A1-A2 as the vertical axis. At the same time, weigh 20 mg of KPS sample into a stoppered test tube, add 10 mL of 1 M hydrochloric acid solution and hydrolyze at 110°C for 8 h. After cooling, centrifuge at 8000 r / min for 15 min to obtain the supernatant as the sample solution to be tested. Pipette 0.2 mL of the sample solution and measure the absorbance (A1-A2) according to the method for preparing the standard curve. Calculate the sulfate content in KPS according to the standard curve. Repeat each group three times and take the average value to obtain the final result.

[0186] 2.1.4 Protein content determination method

[0187] Prepare Folin-phenol reagent A solution according to the instructions of the Folin-phenol kit, and prepare a KPS solution with a concentration of 2.5 mg / mL, then mix 2 mL of polysaccharide solution with 10 mL of reagent A, shake evenly and react at 20-25°C for 10 minutes, and finally add 1 mL of Folin-phenol reagent B solution (Folin-phenol), shake well and react at 20-25°C for 30 minutes, and then measure the absorbance (OD value) at a wavelength of 500 nm. At the same time, perform synchronous tests with standard protein solutions of different concentrations and draw a standard curve to obtain the protein content in KPS. Three parallel groups were set in the experiment, and ultrapure water was used instead of the sample as a blank control.

[0188] 2.2 Experimental Results

[0189] The composition analysis of KPS is as follows Fig.19 As shown, it can be seen that the total sugar content of kelp polysaccharide obtained by papain enzymatic hydrolysis and freeze drying is 83.69%, protein impurities are less, and the purity of KPS is high. In addition, the glucuronic acid and sulfate contents in KPS are 20.25±0.33% and 23.49±0.45%, respectively, which confirms that KPS is a typical sulfated acidic polysaccharide.

[0190] 3 Structural analysis of kelp polysaccharides

[0191] 3.1 Molecular weight

[0192] 3.1.1 Experimental methods

[0193] The molecular weight (MW) of KPS was determined by high performance liquid chromatography equipped with a multi-angle laser scattering instrument and a refractive index detector. With Na2SO4 solution as the mobile phase, the polysaccharide (10 mg) was dissolved in 0.1M Na2SO4 solution to prepare a 1 mg / mL solution. After passing through a 0.22 μm filter membrane, 100 μL of the sample solution was injected into a TSK gel column (7.8×300 mm) at an eluent flow rate of 0.45 mL / min, a refractive index increment dn / dc of 0.135 mL / g, and a column temperature of 30°C. Laminarin standard was used as a control for the experiment.

[0194] 3.1.2 Experimental Results

[0195] The results are as follows Fig. 20 As shown in Figure 2, the molecular weight high performance liquid chromatography (HPLC) graphs of laminarin and KPS are very similar, and the molecular weight characterization results are also relatively close. The molecular weight of the relatively purified laminarin (KPS) obtained by enzymatic hydrolysis-alcohol precipitation is 7.642×10 5 Da, while the molecular weight of kelp polysaccharide obtained by water extraction and alcohol precipitation is 1.128×10 6 Da, which indicates that among the large molecular weight polysaccharides, combined with the previous results analysis, kelp polysaccharides with a molecular weight of several hundred kDa have more significant anti-influenza activity than kelp polysaccharides with a molecular weight of several thousand kDa.

[0196] 3.2 Monosaccharide composition

[0197] 3.2.1 Experimental methods

[0198] KPS was dissolved in water to prepare a polysaccharide solution with a concentration of 10 mg / mL, and 200 μL of the solution was added to an ampoule, and 1 mL of 2M trifluoroacetic acid was added. After digestion for 8 hours, it was blown dry with nitrogen. The dried sample was added to an EC C18 reverse phase column (4.6 mm × 150 mm) to analyze the sugar content. The HPLC chromatogram of the monosaccharide components was recorded using an Agilent 1260 HPLC system. Mobile phase A was 18% acetonitrile and 82%, 0.05 M phosphate buffer, and mobile phase B was 35% acetonitrile and 65%, 0.05 M phosphate buffer. The column temperature was 30°C, and the elution column flow rate was 0.04 mL / min. The monosaccharide composition was determined using lactose as an internal standard.

[0199] 3.2.2 Experimental Results

[0200] The results of monosaccharide composition analysis of KPS are as follows: Fig.21 and Fig. 22 As shown, from Fig.21From the monosaccharide composition chromatogram, we can see that KPS is mainly composed of 6 monosaccharides, namely mannose (Man), glucosamine (GlcN), glucose (Glc), galactose (Gal), xylose (Xyl) and fucose (Fuc). Quantitative analysis shows that the molar percentages of these 6 monosaccharides are 8.29%, 7.67%, 8.81%, 7.37%, 3.09% and 64.20%, respectively, which indicates that the most important monosaccharide in KPS is fucose.

Claims

1. Use of laminarin or an antiviral sterilizing liquid containing laminarin in the preparation of an antiviral product, characterized in that: The virus is an influenza virus; The antiviral and bactericidal liquid contains an effective amount of laminarin and one or more pharmaceutically acceptable excipients; The preparation method of kelp polysaccharide comprises the following steps: taking kelp, crushing it, adding it into water, adding papain to fully hydrolyze it, inactivating the enzyme after the hydrolysis, centrifuging, taking the supernatant and freeze-drying it to obtain kelp polysaccharide.

2. The use according to claim 1, characterized in that: The enzymatic hydrolysis method comprises: adding papain in an amount of 1-2% of the mass of kelp powder, adding water in a solid-liquid ratio of 1:30-100, performing water bath enzymatic hydrolysis at 50-55° C. for 3.0-4.0 h, and inactivating the enzyme in the kelp enzymatic hydrolyzate after the enzymatic hydrolysis time is over.

3. The use according to claim 1, characterized in that: The auxiliary materials include aromatics, sweeteners, and antioxidants; the aromatics include essences and spices; and the antioxidants include vitamin C.

4. The use according to claim 3, characterized in that: The antiviral and bactericidal liquid comprises the following components in parts by weight: 0.4-0.6 parts of kelp polysaccharide, 0-1.25 parts of vitamin C, 0-1.2 parts of aromatics, 0-10 parts of sweeteners, and 87-99.6 parts of purified water.

5. The use according to claim 4, characterized in that: The antiviral sterilization liquid comprises the following components in parts by weight: 0.4-0.6 parts of kelp polysaccharide, 0.25-1.25 parts of vitamin C, 0.1-1.0 parts of aromatics, 6-10 parts of sweeteners, and 87-99.6 parts of purified water; The aromatic agent is selected from mint essence, strawberry essence, passion fruit essence, and lavender essence; The sweetener is selected from xylitol, glucose, erythritol and steviol glycoside.

6. The use according to claim 5, characterized in that: The antiviral and bactericidal liquid comprises the following components in parts by weight: 0.4-0.6 parts of kelp polysaccharide, 0.5-1.0 parts of vitamin C, 0.2-0.4 parts of mint essence, 6-10 parts of sweetener, 0.4-0.6 parts of strawberry essence, and 87.5-92.5 parts of purified water.

7. The use according to claim 1, characterized in that: The virus is influenza A virus, including H1N1, PR8, NWS and H3N2; The products include medicines, disinfectants or health products; the dosage form is spray or solution.

8. The use according to claim 7, characterized in that: The dosage form is a nasal spray or an oral spray.

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