CN114316081B

CN114316081B - A sulfated polysaccharide of Botrytis longipedicularis with SARS-CoV-2 inhibiting activity, and its preparation method and application

Info

Publication number: CN114316081B
Application number: CN202111481802.5A
Authority: CN
Inventors: 宋爽; 游颖; 温成荣; 艾春青; 董秀萍; 付颖寰; 王立龙; 祁立波
Original assignee: Dalian Polytechnic University
Current assignee: Dalian Polytechnic University
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2023-03-28
Anticipated expiration: 2041-12-07
Also published as: CN114316081A

Abstract

The invention discloses a vitis vinifera sulfated polysaccharide with SARS-CoV-2 inhibition activity and a preparation method and application thereof, belonging to the technical field of active polysaccharide. The backbone of the polysaccharide is composed of → 6) -beta-D-Manp (1 → where there is a side chain at C2 of Man, the side chain includes → 3) -beta-Galp (1 → and Xyl, the sulfate group is at C4 of Gal or at C2 and C4, the molecular weight is in the range of 2000-4000kDa, wherein galactose, mannose and xylose account for 45-55%, 30-45% and 10-15% of the monosaccharide, respectively. The invention takes the caulerpa lentillifera as a raw material and is prepared by the purification steps of hot water extraction, enzymolysis, alcohol precipitation, protein removal, chromatography, ultrafiltration and the like. The polysaccharide obtained by the invention can effectively inhibit the infection of SARS-CoV-2, and has no influence on the cell activity; can be used as inhibitor for inhibiting new coronavirus.

Description

A sulfated polysaccharide of Botrytis longipedicularis with SARS-CoV-2 inhibiting activity, and its preparation method and application

Technical Field

The invention relates to a vitis vinifera sulfated polysaccharide with SARS-CoV-2 inhibition activity and a preparation method and application thereof, belonging to the technical field of active polysaccharide, in particular to a polysaccharide with SARS-CoV-2 inhibition activity, an extraction method and application thereof in functional foods, medicines and daily necessities for SARS-CoV-2 inhibition.

Background

Caulerpa lentillifera (Caulerpa lentillifera) belongs to Chlorophyta, lupeophyceae, alternaria, and is mainly distributed on the tropical Pacific coast of Okinawa, malaysia, philippines, vietnam, etc., and in recent years, the introduction of culture has also begun in the areas of Fujian, hainan, taiwan, etc. of China. The grape is named as a sea grape because the appearance is glittering, translucent and plump green globule; the fish roe paste has a taste similar to salmon roe without fishy smell, is called as 'green caviar', contains various amino acids and vitamins required by human body, has high dietary fiber content, low total fat content and zero cholesterol, and is an ideal health food. As a novel edible green alga, development and utilization of caulerpa lentillifera resources are receiving attention.

Since the outbreak of the novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 virus infection, the novel coronavirus pneumonia has been declared as an emergent public health incident of international concern and a global pandemic by WHO, so far, more than 2 hundred million people are infected, and the physical and psychological health of human is seriously damaged. Accordingly, there is a need to develop antiviral substances that have the ability to mitigate and/or prevent viral transmission. Studies have shown that sulfated polysaccharides have inhibitory effects on many viruses and have been shown to be effective in preventing SARS-CoV-2 infection, such as fucoidan, carrageenan, heparin, and the like. At present, the COVID-19 epidemic is still continuous and more effective SARS-CoV-2 inhibitor is needed to be found for the further variation of the strains with stronger infectivity and more harm, so as to reduce or prevent the spread of the epidemic.

Disclosure of Invention

[ problem ] to

The technical problem to be solved by the present invention is the lack of effective substances for inhibiting novel coronaviruses.

[ solution ]

In order to solve the technical problems, the invention provides a vitis vinifera dunaliella sulphated polysaccharide (CLSP-2), which is prepared from fresh vitis vinifera dunaliella as a raw material through protease enzymolysis, hot water extraction, ethanol precipitation, purification and other processes. The main chain structure is → 6) -beta-D-Man- (1 →,12.5 μ g/mL, and the SARS-CoV-2 can be effectively inhibited.

In order to achieve the above objects, the present invention provides a botryococcus longissimus sulphated polysaccharide CLSP-2 with inhibitory activity against the novel coronavirus SARS-CoV-2, the polysaccharide having a main chain consisting of → 6) - β -D-Manp (1 → where the C2 position of Man is present as a side chain comprising → 3) - β -Galp (1 → and Xyl, the sulfate group being at the C4 position or at the C2 and C4 positions of Gal, and having a molecular weight in the range of 2000-4000kDa, wherein the galactose content is 45-55% of the CLSP-2 monosaccharide composition, the mannose content is 30-45% of the CLSP-2 monosaccharide composition, and the xylose content is 10-15% of the CLSP-2 monosaccharide composition.

In one embodiment of the present invention, the Man is mannose, the Gal is galactose, and the Xyl is xylose.

The second object of the present invention is to provide a method for preparing the sulfated polysaccharide having SARS-CoV-2 inhibitory activity, comprising the steps of:

s1, raw material pretreatment: taking caulerpa lentillifera, removing impurities, and pulping to obtain homogenate;

s2, enzymolysis: adding neutral protease into the homogenate obtained in the step S1, and carrying out enzymolysis to obtain an enzymolysis liquid;

s3, hot water leaching: heating the enzymolysis liquid in the step S2 to 80-95 ℃, leaching for 1-4h, cooling, and centrifuging to obtain supernatant;

s4, adding a certain proportion of water into the precipitate obtained in the step S3; repeating the steps of S2 and S3, and combining the supernatants;

s5, alcohol precipitation: taking the supernatant obtained in the step S4, adding an ethanol solution with the volume fraction of 80-100% after rotary evaporation and concentration, standing, and centrifuging to obtain a precipitate;

s6, impurity removal: washing the precipitate obtained in the step S5 with ethanol, adding water for redissolving, removing protein by using a Sevag method, and then dialyzing to remove salt to obtain a crude sugar solution;

s7, freeze-drying: freeze-drying the crude sugar solution obtained in the step S6 to obtain crude polysaccharide of the botrytis macrorrhiza;

s8, purification: and (2) dissolving the crude polysaccharide obtained in the step (S7) in water, purifying by using a DEAE-52 cellulose anion exchange column, eluting by 2-5 column volumes of deionized water and 0.1-2.0M NaCl solution respectively in sequence, collecting the eluted part of the 0.8M NaCl solution, dialyzing, ultrafiltering by using an ultrafiltration tube with the interception amount of 3k-100kDa, collecting the intercepted solution, and freeze-drying to obtain CLSP-2.

In a preferable mode, in the step S2, the enzymolysis temperature is 30-60 ℃, and the enzymolysis lasts for 1-4 h.

Preferably, in step S2, the neutral protease is added in an amount of 500-5000U per 100mL of homogenate.

Preferably, in step S5, the rotary evaporation is performed to concentrate the solution to 20% to 60% of the original volume.

Preferably, in step S5, 300-400mL of ethanol solution with volume fraction of 80% -100% (v/v) is added to each 100mL of the concentrated solution.

In a preferred embodiment, the method specifically comprises:

s1, raw material pretreatment: washing fresh caulerpa lentillifera with flowing water to remove silt impurities, and pulping by a refiner to obtain homogenate;

s2, enzymolysis: adding 500-5000U of neutral protease into each 100mL of homogenate obtained in the step S1, and carrying out magnetic stirring enzymolysis for 1-4h at the temperature of 30-60 ℃ to obtain an enzymolysis liquid;

wherein the addition amount of the neutral protease is 500-5000U of neutral protease added into each 100mL of homogenate;

s3, hot water leaching: heating the enzymolysis liquid obtained in the step S2 to 80-95 ℃, leaching with hot water for 1-4h while inactivating the enzyme, cooling to room temperature, and centrifuging to obtain supernatant;

s4, adding deionized water in a certain proportion into the precipitate obtained in the step S3; repeating the steps S2 and S3 once, and combining the supernatants;

s5, alcohol precipitation: taking the supernatant obtained in the step S4, concentrating the supernatant to a certain volume by rotary evaporation, adding 300-400mL of ethanol solution with the volume fraction of 80-100% (v/v) into each 100mL of the supernatant, standing the mixture for more than 2 hours at the temperature of 2-8 ℃, and centrifuging the mixture to obtain a precipitate;

wherein, the rotary evaporation is concentrated to 20 to 60 percent of the original volume;

s6, impurity removal: washing the precipitate obtained in the step S5 with ethanol, redissolving the precipitate with deionized water, removing protein by a Sevag method, and then desalting by dialysis to obtain a crude sugar solution;

s7, freeze-drying: freeze-drying the crude sugar solution obtained in the step S6 to obtain crude polysaccharide of the caulerpa lentillifera;

s8, purification: and (2) dissolving the crude polysaccharide obtained in the step (S7) in deionized water, purifying by using a DEAE cellulose anion exchange column, eluting 2-5 column volumes by using the deionized water and 0.1-2.0M NaCl solution respectively in sequence, collecting the eluted part of the 0.8M NaCl solution, performing dialysis, performing ultrafiltration by using an ultrafiltration tube with the interception amount of 3k-100kDa, collecting the intercepted solution, and performing freeze-drying to obtain CLSP-2.

In a preferable mode, the stirring speed in the step S2 is 100-500 rpm/min, and the stirring is carried out for 1-4h;

preferably, the centrifugation in step S3 is specifically: centrifuging at 4-30 deg.c and 3000-10000 r/min for 5-60 min.

Preferably, the centrifugation in step S5 is: centrifuging at 3000-15000 r/min for 5-60 min.

In a preferred embodiment, the impurity removal in step S6 is specifically: washing the precipitate with ethanol, adding 1-20 mL of ethanol with the concentration of more than 50% (v/v) into each gram of precipitate for washing, pouring out the ethanol solution, repeating the washing operation for 1-5 times, dissolving the obtained precipitate with 10-20 mL of water per gram, and using a solvent with the volume ratio of 4-6:1 chloroform: mixing n-butanol solution, centrifuging to obtain water layer solution, and repeating the step for 3-5 times to remove protein; then dialyzing with flowing tap water and deionized water (each 1ml sample in the dialysis bag is dialyzed with 20ml deionized water) respectively for more than 24 hours by using a 500-10000 Da dialysis bag, and the obtained solution is a crude sugar solution.

Preferably, the lyophilization conditions in step S7 are: the temperature of a cold trap of the freeze dryer is-60 ℃, the vacuum degree is 1-10 Pa, and the freeze-drying time of the sample is more than or equal to 4h.

Preferably, the post-dialysis lyophilization in step S8 specifically includes: ultrafiltering the eluted part of the 0.8M NaCl solution by using an ultrafiltration tube with 3k-100kDa, dialyzing the trapped solution by using a dialysis bag with 500-10000 Da for more than 24 hours by using running water and deionized water respectively to obtain a purified polysaccharide solution; freeze-drying the polysaccharide solution to obtain CLSP-2; wherein, each milliliter of dialysis internal solution is dialyzed by using 20ml of deionized water correspondingly; the parameters of the lyophilization are as follows: the temperature of a cold trap of the freeze dryer is minus 60 ℃, the vacuum degree is 1 to 10Pa, and the freeze-drying time of the polysaccharide solution is more than or equal to 4 hours.

Unless otherwise specified, the room temperature in the invention refers to 25 ℃.

The invention also provides the application of the sulfated polysaccharide for inhibiting the novel coronavirus SARS-CoV-2 in preparing anti-coronavirus medicaments, wherein the coronavirus comprises the novel coronavirus SARS-CoV-2.

Preferably, the anti-coronavirus is used for preventing or treating pneumonia caused by coronavirus, and comprises the S protein combined with the surface of SARS-CoV-2 virus and preventing SARS-CoV-2 virus from invading cells of the organism.

Preferably, the sulfated polysaccharide capable of inhibiting the novel coronavirus SARS-CoV-2 can be used for preparing medicaments, foods and daily chemical products for preventing or treating coronavirus infection.

The invention also provides a medicament for preventing or treating coronavirus infection, which takes the sulfated polysaccharide capable of inhibiting the novel coronavirus SARS-CoV-2 as an active ingredient for inhibiting the coronavirus from entering into cells of an organism.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials, including: the composition comprises one or more of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrating agent, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a flow aid, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic, an anti-adhesive, an integration agent, a permeation accelerator, a pH value regulator, a buffering agent, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent, a deflocculating agent, a filter aid, a release retardant and the like.

In a preferred mode, the dosage form of the medicament can be: spray, aerosol, powder spray, lotion, ointment, liniment, nasal spray, effervescent tablet, gargle, powder, emulsion, suspension, solution.

The invention provides an application of sulfated polysaccharide for inhibiting novel coronavirus SARS-CoV-2 in preparing protective articles for preventing coronavirus infection, wherein the protective articles comprise: hand lotion, hand sanitizer and mouthwash.

The beneficial effects of the invention are:

the novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 virus infection seriously jeopardizes the public health and social stability of society. Therefore, it is important to develop an antiviral substance that can reduce and/or prevent viral transmission and effectively inhibit viral invasion. The invention uses in vitro virus neutralization test to detect the activity of SARS-CoV-2 in vitro by using the Caulerpa lentillifera polysaccharide with different concentrations through an immunofluorescence method. The result shows that the Caulerpa lentillifera polysaccharide prepared by the invention has remarkable activity of inhibiting SARS-CoV-2 virus from invading host cells, the IC50 value is 48.48 mu g/mL, and the Caulerpa lentillifera polysaccharide still has remarkable anti-SARS-CoV-2 activity when the concentration is as low as 12.5 mu g/mL.

Drawings

FIG. 1 is a plot of the relative molecular mass distribution of the Botrytis longipedicularis polysaccharide of example 1;

FIG. 2 contains the standards of example 1: rha (rhamnose), fuc (fucose), xyl (xylose), man (mannose), glc (glucose), gal (galactose), galA (galacturonic acid), glcA (glucuronic acid) and botryococcus longissimus polysaccharides;

FIG. 3 is the monosaccharide composition of the supernatant and precipitate of the Botrytis longipedicularis polysaccharide of example 1 after weak acid hydrolysis;

FIG. 4 is the HPLC-MS of oligosaccharide fragment produced by acid hydrolysis in example 1 ⁿ Analyzing the result; after PMP derivatization, wherein A represents a hexasaccharide fragment obtained after acid hydrolysis, B represents a pentasaccharide fragment obtained after acid hydrolysis, C represents a tetrasaccharide fragment obtained after acid hydrolysis, and D represents a trisaccharide fragment obtained after acid hydrolysis;

FIG. 5 is an embodimentExample 1 HPLC-MS of oligosaccharide fragments after photocatalytic degradation ⁿ The analysis result is that after PMP derivatization, wherein A represents galactose containing two sulfuric acid groups, B represents xylose containing two sulfuric acid groups, C represents xylose containing a single sulfuric acid group, and D represents galactose containing a single sulfuric acid group;

FIG. 6 is the Caulerpa lentillifera polysaccharide of example 1 ¹ H NMR spectrum;

FIG. 7 is a spectrum of the Botrytis longipedicularis polysaccharide DEPT 135 from example 1;

FIG. 8 is a HSQC spectrum of Caulerpa lentillifera polysaccharide of example 1;

FIG. 9 is the Caulerpa lentillifera polysaccharide of example 1 ¹ H- ¹ H COSY spectrogram;

FIG. 10 is a TOCSY spectrum of the Caulerpa lentillifera polysaccharide of example 1;

FIG. 11 is a chart of HSQC spectra of Staphylophyta nodosa polysaccharide of example 1 after desulfurization;

FIG. 12 is a possible structural diagram of the polysaccharide of Caulerpa lentillifera in example 1;

FIG. 13 is the effect of Caulerpa lentinan on HELA cell viability in example 2;

FIG. 14 is the effect of Caulerpa lentillifera polysaccharide on the amount of SARS-CoV-2 virus in example 3.

Detailed Description

The following detailed description of the present invention, taken in conjunction with the accompanying drawings and examples, is provided to enable the invention and its various aspects and advantages to be better understood. However, the specific embodiments and examples described below are for illustrative purposes only and are not limiting of the invention.

The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The invention provides a botryococcus longipediculus sulphated polysaccharide, a botryococcus longipediculus sulphated polysaccharide CLSP-2 capable of inhibiting a novel coronavirus SARS-CoV-2, wherein the main chain of the polysaccharide is formed by → 6) -beta-D-Manp (1 → the C2 position of Man is provided with a side chain, the side chain comprises → 3) -beta-Galp (1 → and Xyl, the sulfate group is positioned at the C4 position or the C2 and C4 positions of Gal, the molecular weight range is 2000-4000kDa, wherein the galactose content accounts for 45-55% of the CLSP-2 monosaccharide composition, the mannose content accounts for 30-45% of the CLSP-2 monosaccharide composition, and the xylose content accounts for 10-15% of the CLSP-2 monosaccharide composition.

The invention also provides an extraction method of the polysaccharide, wherein the extraction method comprises the following steps:

s1, raw material pretreatment: washing fresh caulerpa lentillifera with flowing water, removing silt impurities, and pulping by a refiner to obtain homogenate;

s2, enzymolysis: adding neutral protease into the homogenate obtained in the step S1 per 100mL of the homogenate, and carrying out magnetic stirring enzymolysis for 1-4h at the temperature of 30-60 ℃ to obtain an enzymolysis liquid;

wherein the addition amount of the neutral protease is that 500-5000U of neutral protease is added into each 100mL of homogenate;

s3, hot water leaching: heating the enzymolysis liquid in the step S2 to 80-95 ℃, leaching with hot water for 1-4h while inactivating the enzyme, cooling to room temperature, and centrifuging to obtain supernatant;

s4, adding deionized water in a certain proportion into the precipitate obtained in the step S3; repeating the steps of S2 and S3, and combining the supernate;

s8, purification: dissolving the crude polysaccharide obtained in the step S7 in deionized water, using and purifying, sequentially eluting 2-5 column volumes by using deionized water, 0.1M NaCl, 0.2M NaCl, 0.3M NaCl, 0.4M NaCl, 0.5M NaCl, 0.6M NaCl, 0.7M NaCl, 0.8M NaCl, 0.9M NaCl, 1.0M NaCl and 1.2M NaCl solutions respectively, collecting the eluted part of the 0.8M NaCl solution, dialyzing, selecting an ultrafiltration tube with the interception amount of 3k-100kDa for ultrafiltration, collecting the interception liquid, and freeze-drying to obtain CLSP-2.

The invention also provides the application of the polysaccharide in the preparation of functional food, daily necessities or medicines for inhibiting SARS-CoV-2 virus activity.

Replication of viruses requires the host cell to supply materials, energy and sites of replication, and must therefore be performed in living cells. After entering the body, the virus is adsorbed on the surface of sensitive cells, and then genetic material is injected into host cells through penetration and shelling, so that the virus is replicated and assembled to cause infection of the body.

In the invention, SARS-CoV-2 virus is separated from the body of COVID-19 patient, CLSP-2 with different concentration is added in the coculture with HELA cell in vitro, the DPAI staining method is adopted to carry out quantitative analysis to the living cell, the immunofluorescence test is utilized to analyze the virus number, and the activity of CLSP-2 for inhibiting SARS-CoV-2 virus is determined.

Phenol-sulfuric acid method:

1. drawing of standard music

Accurately weighing 20mg of glucose standard substance dried to constant weight at 105 ℃ into a 500mL volumetric flask, adding water to constant volume to scale, shaking up, respectively sucking 0.00, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20, 1.40, 1.60 and 1.80mL of the solution into 9 test tubes, and supplementing the solution to 2.00mL with deionized water. Then, 1.0mL of 6.0% phenol solution and 5.0mL of concentrated sulfuric acid were added, mixed, left to stand for 20min, and then the absorbance was measured at 490nm using a spectrophotometer. A standard curve was drawn using 2.0mL of deionized water as a blank, the sugar content as the abscissa, and the absorbance at 520nm as the ordinate.

2. Sample sulfate group content determination

And (3) determination of sample content: preparing 2mg/mL of the polysaccharide solution by using deionized water, diluting in a gradient manner, sucking 2.0mL of the dilution of the sample to be detected, determining the absorbance value at 490nm according to the operation steps, and calculating the polysaccharide content of the sample through a standard curve.

Gelatin turbidimetry:

1. drawing of standard music

Will K ₂ SO ₄ Drying in a 105 deg.C oven to constant weight, accurately weighing 108.75mg, adding 1.0M HCl to constant volume in 100ml volumetric flask, shaking to obtain K ₂ SO ₄ The concentration of the standard solution, i.e., sulfate group, was 0.6mg/mL. 0.00, 0.04, 0.08, 0.12, 0.16 and 0.20mL of standard solution were pipetted into a 10mL glass tube, each tube was made up to 0.2mL with 1M HCl, and 3.8mL of trichloroacetic acid was added to each tube. 0.5g of gelatin and 1.0g of BaCl were taken ₂ Dissolving in 100mL deionized water, heating for dissolving, centrifuging, and collecting supernatant to obtain BaCl ₂ -gelatin turbidimetric solution. 1.0mLBaCl was added to each tube ₂ -gelatin turbidimetric solution, mixed well, left to stand for 20min, absorbance at 360nm was measured and recorded as A1. The turbid solution was then replaced by 1.0mL of gelatin solution and the absorbance measured was designated A2. The results obtained were repeated three times, and a standard curve was prepared using the results obtained, with the abscissa being milligrams of sulfate groups and the ordinate being a = A1-A2, to obtain a linear regression equation.

2. Determination of sulfate radical content of sample

Weighing 4.0mg of above polysaccharide sample, placing into a test tube with a plug containing 2.0mL of 1.0mol/L HCl, sealing, hydrolyzing at 110 deg.C for 8h, cooling to room temperature, centrifuging for 10min (8,000r/min), sucking 0.2mL (three parallel parts) of each sample solution, and determining its absorbance value according to standard curve operation method. And calculating the sulfate group content in the sample according to the standard curve.

TSK gel chromatography:

the relative molecular mass was determined by high performance gel permeation chromatography. The gel chromatographic column is TSK-gel G4000PWxl (7.5 mm multiplied by 30.0 cm), and is matched with a differential refractometer, and the temperature of the detector and the temperature of a column box are both 30 ℃. The mobile phase was ammonium acetate buffer (0.1 mol/L, pH 6.0) at a flow rate of 0.4mL/min. Dextran (relative molecular mass of 5, 12, 25, 50, 150, 410 and 670 kDa) is used as standard substance, dissolved in mobile phase, 10 μ L of sample is injected, retention time is determined, retention time of chromatographic peak is used as abscissa, lg Mw is used as ordinate, and linear regression equation is obtained.

Determination of the relative molecular mass of the samples: the polysaccharide was mixed with a mobile phase to give a 2mg/mL solution, which was sampled at 10. Mu.L, and the retention time of the chromatographic peak was measured. The relative molecular mass of each sample was calculated from the standard curve.

Analysis of monosaccharide composition by liquid chromatography:

1. preparation of standards

Accurately weighing 5mg of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and fucose standard substances into a hydrolysis tube, respectively adding 1mL of ammonia water for dissolving, respectively placing 100 mu L of the standard substances into a new hydrolysis tube, adding 800 mu L of 0.3M PMP methanol solution, and carrying out PMP derivatization analysis, wherein the product is used for liquid chromatography analysis.

2. Preparation of samples

Accurately weighing 5mg of the polysaccharide sample, adding 1mL of 2M trifluoroacetic acid, hydrolyzing at 120 ℃ for 3h, blowing nitrogen or centrifugally concentrating to remove hydrolysate after the hydrolysis is finished, and performing PMP derivatization, wherein the derivatized product is used for liquid chromatography analysis.

3. Liquid chromatography conditions:

chromatographic conditions are as follows: silgreen ODS C18 column (250 mm. Times.4.6 mm,5 μm); the column temperature is 30 ℃; mobile phase 20mmol ammonium acetate-acetonitrile (78, V/V; the flow rate was 1mL/min.

Example 1

A method for preparing Caulerpa lentillifera polysaccharide with SARS-CoV-2 virus inhibiting activity comprises the steps of:

s1, raw material pretreatment: washing fresh caulerpa lentillifera (1000 g) with running water to remove impurities such as silt, and pulping with a refiner to obtain homogenate (1000 mL);

s2, enzymolysis: adding 0.1g of 5 ten thousand U/g neutral protease into the homogenate obtained in the step S1, and magnetically stirring at 50 ℃ and 100rpm for enzymolysis for 2 hours to obtain an enzymolysis solution (1000 mL);

s3, hot water leaching: heating the enzymolysis solution (1000 mL) obtained in the step S2 to 90 ℃, leaching with hot water for 2h while inactivating the enzyme, cooling to room temperature, centrifuging to obtain a supernatant, adding 1000mL of deionized water into the precipitate, repeating the steps S2 and S3, and combining the supernatants (2000 mL);

s4, alcohol precipitation: taking the supernatant fluid obtained in the step S3, carrying out rotary evaporation concentration to 500mL, adding 2000mL of ethanol solution with volume fraction of 95% (v/v), standing for 12h at 4 ℃, and centrifuging to obtain a precipitate;

s5, impurity removal: washing the precipitate obtained in the step S5 with ethanol, redissolving with 200mL of deionized water, removing protein by a Sevag method (in a Sevag reagent, chloroform: n-butyl alcohol =4: 1), and then putting the precipitate into a 3kDa dialysis bag for dialysis and desalination to obtain a crude sugar solution;

s6, freeze-drying: and (5) freeze-drying the crude sugar solution obtained in the step (S5), wherein the freeze-drying conditions are as follows: the temperature of a cold trap of a freeze dryer is-60 ℃, the vacuum degree is 1-10 Pa, and the sample freeze-drying time is 24h to obtain the crude polysaccharide of the caulerpa lentillifera;

s7, purification: dissolving the crude polysaccharide (1 g) obtained in the step S6 in 20mL of deionized water, loading a sugar solution onto a DEAE-cellulose column balanced by 1000mL of deionized water, wherein the column volume is 500mL, eluting 3 column volumes respectively by using deionized water, 0.2M NaCl, 0.5M NaCl, 0.8M NaCl and 1.2M NaCl solution in sequence, collecting the eluted part of the 0.8M NaCl solution, putting the eluted part into a 3500Da dialysis bag, dialyzing for 48 hours by using running tap water, and dialyzing for 24 hours by using the deionized water; and finally, selecting an ultrafiltration tube with the interception amount of 100kDa for ultrafiltration, collecting the intercepted liquid, putting the intercepted liquid into a cold trap at the temperature of minus 60 ℃ and the vacuum degree of 1 to 10Pa, and freeze-drying for 48 hours to obtain CLSP-2.

The CLSP-2 prepared in the example has a total sugar content of 70.8% measured by a phenol-sulfuric acid method; the sulfuric acid group content of the CLSP-2 prepared by the embodiment is 22.7 percent by adopting a gelatin turbidimetry method; the molecular weight of CLSP-2 obtained by this example was 3985kDa as determined by TSK gel chromatography, as shown in FIG. 1.

The monosaccharide composition of the CLSP-2 obtained in the example was analyzed by liquid chromatography, and the results are shown in FIG. 2, in which CLSP-2 consists of galactose, mannose and xylose, wherein galactose content is 45-55%, mannose content is 30-45%, and xylose content is 10-15%.

Polysaccharide is degraded in a certain mode, and the analysis of the degraded oligosaccharide fragment is helpful for the analysis of polysaccharide structure. In this example, CLSP-2 was hydrolyzed with weak acid (0.2M TFA) and centrifuged to obtain a supernatant andprecipitating, collecting a part of supernatant and precipitate, performing complete acid hydrolysis, performing PMP derivatization, and analyzing monosaccharide composition in supernatant and precipitate by liquid chromatography (FIG. 3); the other part of the supernatant is subjected to HPLC-MS after PMP derivatization ⁿ Oligosaccharide fragment analysis (figure 4). As known in the literature, the side chain is easily broken by weak acid hydrolysis, i.e., the supernatant is in a side chain structure and precipitates in a main chain structure. FIG. 3 shows that the CLSP-2 side chain consists mainly of galactose, mannose and a small amount of xylose; the backbone consists of mannose. As can be seen in fig. 4, after weak acid hydrolysis, four hexose fragments appeared, indicating that they may be present in the side chain with CLSP-2.

It is known in the literature that the high temperature conditions of acid hydrolysis tend to cause the sulfate groups to be detached from the sugar chains, and information on the positions of the sulfate groups in the sugar chains cannot be obtained. In the embodiment, CLSP-2 is degraded by photocatalytic oxidation, and the specific degradation and analysis steps are as follows:

1. photocatalytic degradation of vitis vinifera sulfated polysaccharide

1) Dissolving the sulfated polysaccharide of Caulerpa lentillifera in water to obtain polysaccharide solution with concentration of 5mg/ml. Adding 0.01g of photocatalytic reaction catalyst TiO with particle size of 25nm into 20ml of Botrytis longipedicularis sulfated polysaccharide solution ₂ . While adding TiO2, the polysaccharide solution was stirred to ensure uniform distribution of the catalyst in the solution, while 0.64mL of H was added ₂ O ₂ So as to improve the efficiency of the photocatalytic reaction;

2) And (3) carrying out photocatalytic degradation on the solution to be degraded under simulated sunlight (500W xenon lamp). In the degradation process, the solution to be degraded is stirred while illumination is carried out so as to ensure that the catalyst is uniformly distributed in the solution, the photocatalytic degradation is carried out for 0.5 hour, and the solution after degradation is obtained after the reaction is finished;

3) Centrifuging the degraded solution at 10000g for 15min, and taking supernatant;

4) The supernatant is derivatized by PMP to obtain a derivatized product for HPLC-MS ⁿ And (6) analyzing.

2. Derivatization product HPLC-MS ⁿ Analysis of

Analysis was performed on an LXQ linear ion trap mass spectrometer equipped with an electrospray ion source (ESI) and a photodiode array detector PAD, XCalibur software operating system.

(1) Chromatographic conditions

Using a TSKgel-Amide-80 (

20X

150,3 μm) chromatography column; the flow rate is 0.2mL/min; mobile phase a was 20mM ammonium acetate in water pH =6.0 and mobile phase B was acetonitrile in a ratio of 78.

(2) Conditions of Mass Spectrometry

An ion source ESI source; the spraying voltage is 4.5kV; the capillary temperature was 275 ℃; the capillary voltage is 37V; sheath gas: 40AU; auxiliary gas: 10AU; detecting in a negative ion mode; the scanning mode is Full scanning (Full Scan); the scan range is 100-2000 (m/z).

Derivatization by PMP and HPLC-MS ⁿ The analysis yielded a sulfate-containing oligosaccharide fragment, as shown in FIG. 5. From FIGS. 5A, D, it can be seen that 1 and or 2 sulfate groups are present in one hexose residue, and similarly from FIGS. B, C, 1 and or 2 sulfate groups are also present on one xylose residue, indicating more than one site of sulfation on the same sugar residue in CLSP-2.

FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11 are for CLSP-2 ¹ H NMR, DEPT 135, HSQC (Heteronuclear Single Qauntum Coherence), ¹ H- ¹ H COSY (chemical shift correlation Spectrum) ¹ H- ¹ HSQC spectra of H Chemical Shift correction Spectroscopy), TOCSY (Total Correlation Spectroscopy), and CLSP-2 desulfurization product dS-CLSP-2. The HSQC spectrum (FIG. 8) can reflect the coupling relationship between directly connected H and C nuclei, wherein about 103ppm of 2 carbon signals are signals of terminal carbon atoms, the configuration is beta-type, and 60ppm of C-6 signals. Bonding with ¹ H- ¹ H-COSY (FIG. 9) and TOCSY spectra (FIG. 10) assign chemical shifts of sugar ring protons, and then HSQC is performed to further assign chemical shifts of carbon. Finally, accurate attribution of all hydrocarbon signals is achieved (table 1). By comparing the chemical shifts of CLSP-2 before and after desulfurization, it can be seen that mannose is mainly linked by → 6) - β -D-Man- (1 →, galactose → 3) - β -D-Gal- (1 → linkage and a sulfate group is present at the C4 position.

In this example, the results of methylation analysis of CLSP-2 and dS-CLSP-2 are shown in Table 2. As can be seen from the table, CLSP-2 is mainly linked by → 6) -Manp- (1 → and → 3, 4) - β -D-Gal- (1 →, the result of which is consistent with nuclear magnetism, and contains a portion → 2, 6) -Manp- (1 → and → 2,3, 4) -Galp- (1 →, in combination with the results of weak acid hydrolysis and photocatalytic degradation, it is known that the CLSP-2 main chain consists of → 6) -Manp- (1 → a side chain exists at the C2 position of Man, the side chain consists of → 3) - β -D-Gal- (1 → a sulfate group exists mainly at the C4 position of galactose or at the C2 and C4 positions and a small amount of Xyl exists in the side chain of CLSP-2, and the structure of CLSP is presumed as shown in fig. 12.

TABLE 1CLSP-2 and dS-CLSP-2 ¹ H and ¹³ chemical shift of C NMR

TABLE 2CLSP-2 and dS-CLSP-2 methylation analysis results

Example 2

This example was used to evaluate the toxicity assay of the vitis vinifera dunaliella polysaccharide CLSP-2 prepared in example 1 on host cells. The specific experimental method is as follows:

taking 200 μ L of density at logarithmic growth phase of 1 × 10 ⁴ HELA cells were seeded in 96-well culture plates per mL and cultured for 24 hours in a complete medium (DMEM medium containing 1% (v/v) of a mixed solution of penicillin streptomycin (Corning, MT30002 CI) and 10% (v/v) of high-grade fetal bovine serum). Using 10% DMEM to prepare different concentrations of CLSP-2 solution: 12.5 25, 50 and 100 mu g/mL, sucking out complete culture medium after 24 hours of culture, adding 150 mu L of culture medium containing or not containing CLSP-2 and 50 mu L of SARS-CoV-2 virus solution into each well, and setting 6 multiple wells for each group; at 37 ℃ C, 5% CO ₂ After incubation for 16h in an incubator, immunofluorescence was performedLight assay, DAPI staining for cytotoxicity analysis. As shown in fig. 13, the above four concentrations had no effect on cell viability.

Example 3

Inoculating HELA cells inoculated with SARS-CoV-2 virus and 12.5, 25, 50, 100 μ g/mL CLSP-2 at 37 deg.C, 5% ₂ After incubation for 16h in an incubator, immunofluorescence analysis was performed, the specific procedure was as follows:

sucking out culture solution, adding 100 μ L of formaldehyde solution into each well, treating at-20 deg.C for more than 20min, and fixing cells; after fixation, 200 μ L of 3% BSA (PBS configuration) was added per well, blocking was performed, 80 μ L of 3% BSA diluted neocoronal recovery patient serum was added per well (1. The intensity of green fluorescence represents the number of viruses, and the intensity of blue fluorescence represents the number of cells.

As shown in FIG. 14, four different concentrations of CLSP-2 were effective in inhibiting the activity of SARS-CoV-2 virus with an IC50 value of 48.48. Mu.g/mL

Example 4 preparation of Caulerpa lentillifera polysaccharide nasal spray

In a sterile environment, 40 g of sodium chloride, 100 g of citric acid, 100 g of sodium citrate and 1g of benzalkonium chloride are respectively dissolved by 10 times of purified water with stirring. 10 g of Botrytis longipedicularis polysaccharide was dissolved with 500mL of purified water with stirring. Mixing the above solutions, diluting with purified water to 10L, filtering with 0.5 μm filter membrane, and packaging.

Example 5 preparation of Ampelopsis longipedicularis polysaccharide hand lotion

The formula of the hand lotion is shown in the following table:

TABLE 1 hand lotion ingredient Table

Heating oil phase (stearic acid, monoglyceride stearate, isopropyl palmitate, vaseline, white mineral oil, cetyl alcohol, methyl hydroxybenzoate, and propyl hydroxybenzoate) under stirring for dissolving. Dissolving Caulerpa lentillifera polysaccharide in water, adding glycerol and triethanolamine, heating, stirring, and dissolving. The aqueous phase was then slowly poured into the oil phase with vigorous stirring. After the treatment of the homogenizer, heating and stirring are started, allantoin, dimethyl-p-chlorophenol and essence are added when the temperature is cooled to 50 ℃, and the materials are discharged when the temperature is continuously stirred and cooled to 30 ℃. And (5) ageing for 2-3 days without change, and packaging after the inspection is qualified.

Example 6 preparation of Caulerpa lentillifera polysaccharide oral liquid

Decocting 1.6 kg of fructus Lycii and 1.6 kg of arillus longan in 16L of water, maintaining with slow fire for 1 hr, filtering to obtain filtrate, concentrating to 12L, adding 60 g of Botrytis longipedicularis polysaccharide, 12 g of pectin, and 840 g of Mel, stirring, naturally cooling, standing for 3 hr to precipitate completely. Taking a filtrate by adopting filter pressing, adding 910 g of white sugar, 52 g of citric acid, 6.5 g of salt and 26 g of vitamin C, uniformly mixing, performing pressure filtration by using diatomite, performing filtration by using two stages of microporous filter membranes with the pore diameter of 5 micrometers and 0.5 micrometer, performing pasteurization (80 ℃ for 30 minutes) on the filtrate, and canning.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A botryococcus longissima sulphated polysaccharide CLSP-2 with inhibitory effect on the novel coronavirus SARS-CoV-2, wherein the backbone of said polysaccharide is constituted by → 6) -beta-D-Manp (1 → Man, there are side chains at position C2, said side chains including → 3) -beta-Galp (1 → and Xyl, the sulphate group is at position C4 or at positions C2 and C4 of Gal, the molecular weight is in the range of 2000-4000kDa,

wherein, the galactose content accounts for 45-55% of the CLSP-2 monosaccharide composition, the mannose content accounts for 30-45% of the CLSP-2 monosaccharide composition, and the xylose content accounts for 10-15% of the CLSP-2 monosaccharide composition.

2. The method for preparing the botryococcus longissimus sulfated polysaccharide CLSP-2 of claim 1, comprising the steps of:

s6, impurity removal: washing the precipitate obtained in the step S5 with ethanol, adding water for redissolving, removing protein by a Sevag method, and then dialyzing to remove salt to obtain a crude sugar solution;

3. The preparation method according to claim 2, wherein in the step S2, the enzymolysis temperature is 30-60 ℃, and the enzymolysis time is 1-4h; the addition amount of the neutral protease is 500-5000U of neutral protease added into each 100mL of homogenate.

4. The method according to claim 2, wherein in step S5, the rotary evaporation is performed to a concentration of 20% to 60% of the original volume.

5. The method according to any one of claims 2 to 4, wherein in step S5, from 300 to 400mL of an 80% to 100% (v/v) volume fraction ethanol solution is added per 100mL of the concentrate.

6. The method according to any one of claims 2 to 4, wherein the method specifically comprises:

s2, enzymolysis: adding 500-5000U of neutral protease into each 100mL of homogenate obtained in the step S1, and carrying out magnetic stirring enzymolysis for 1-4h at the temperature of 30-60 ℃ to obtain an enzymolysis liquid; wherein the addition amount of the neutral protease is that 500-5000U of neutral protease is added into each 100mL of homogenate;

s4, adding deionized water in a certain proportion into the precipitate obtained in the step S3; repeating the steps of S2 and S3 once, and combining the supernatant;

s5, alcohol precipitation: taking the supernatant obtained in the step S4, concentrating to a certain volume by rotary evaporation, adding 300-400mL of ethanol solution with volume fraction of 80% -100% (v/v) into each 100mL of the supernatant, standing for more than 2h at 2-8 ℃, and centrifuging to obtain a precipitate; wherein, the rotary evaporation is concentrated to 20 to 60 percent of the original volume;

s8, purification: and (4) dissolving the crude polysaccharide obtained in the step (S7) in deionized water, using and purifying, sequentially eluting 2-5 column volumes by using deionized water and 0.1-2.0M NaCl solution respectively, collecting the eluted part of the NaCl solution with the concentration of 0.8M, performing ultrafiltration by using an ultrafiltration tube with the interception amount of 3k-100kDa after dialysis, collecting the intercepted liquid, and performing freeze-drying to obtain CLSP-2.

7. Use of the botryococcus longissimus sulphated polysaccharide CLSP-2 of claim 1 or the method of manufacture of any one of claims 2 to 6 in the manufacture of a medicament against coronavirus, wherein the coronavirus comprises the novel coronavirus SARS-CoV-2.

8. A pharmaceutical or protective product for the prevention or treatment of coronavirus infection, comprising vitis vinifera sulfated polysaccharide CLSP-2 according to claim 1.

9. The pharmaceutical or protective product according to claim 8, wherein said pharmaceutical further comprises pharmaceutically acceptable excipients comprising: solvent, propellant, emulsifier, colorant, adhesive, disintegrating agent, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, coating material, aromatic, anti-adhesive, integrating agent, penetration enhancer, pH value regulator, plasticizer, surfactant, defoaming agent, thickening agent, coating agent, humectant, absorbent, diluent, flocculating agent and deflocculating agent, filter aid and release retardant.

10. Use of the botryococcus longissima sulfated polysaccharide CLSP-2 of claim 1 in the manufacture of a protective product, food product, daily chemical product for the protection against coronavirus infection.