An antiviral SVA virus Sargassum fusiforme polysaccharide and its preparation method and application

Abstract

The present invention discloses a Sargassum fusiforme polysaccharide against SVA virus, its preparation method and application. The purified Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa has the best anti-SVA virus effect, and is characterized by high anti-SVA virus activity and low cytotoxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extracts and their applications, and particularly relates to a Sargassum fusiforme polysaccharide against Senecavirus A (SVA), a preparation method thereof, and an application thereof. Background Art

[0002] The statements herein only provide background art related to the present invention, and do not necessarily constitute prior art.

[0003] Senecavirus A (SVA), also known as Seneca valley virus (SVV), can cause swine vesicular diseases in pigs, mainly affecting neonatal piglets, resulting in symptoms such as reduced appetite, lethargy, ulcers in the mouth, nose, and coronary band of the hoof in neonatal piglets, and even death of piglets in severe cases, which has a great impact on the pig farming industry. In addition, mice and flies can also carry the virus, and SVA neutralizing antibodies have been detected in humans, cattle, sheep and other animals. SVA was discovered by the American company Neotropix in 2002 when culturing adenovirus 5 vector in cells and was named SVV-001. This virus belongs to the family Picornaviridae, and other viruses in the same family include foot-and-mouth disease virus, etc. In 2015, swine vesicular disease caused by SVA was first discovered in Guangdong Province in China. Subsequently, swine vesicular disease cases and SVA were isolated in Hubei Province, Shandong Province, Heilongjiang Province and other places.

[0004] As a new virus, the transmission route, host range, etc. of SVA have not been fully understood yet, and there are no commercial vaccines and specific drugs. Once an outbreak occurs, it will cause great harm. Currently, the research on SVA mainly focuses on vaccine development, and there is less research on drugs. Developing active ingredients of natural products is an important way for antiviral drug research and development. Polysaccharides are widely present in animals, plants, bacteria and fungi, and have multiple biological activities. Some of these polysaccharides exhibit certain antiviral activities, can antagonize multiple links in the virus life cycle, and can also play an antiviral role by regulating immune responses, inflammatory responses, etc. They are currently a research hotspot for antiviral drugs.

[0005] Sargassum fusiforme belongs to the Phaeophyta in seaweeds, the order Fucales, the family Sargassaceae, and has aliases such as antler tip and seaweed bud. It is mainly distributed in the southeastern coastal areas of China and the edges of the Yellow Sea and the Bohai Sea, with a large yield and a wide source. "Shennong Ben Cao Jing" records the dietary therapy properties and usage methods of Sargassum fusiforme. Modern research shows that Sargassum fusiforme polysaccharide has antiviral effects against avian leukosis virus subgroup J, respiratory syncytial virus, herpes simplex virus, enterovirus 71, coxsackievirus, etc. There is currently no patent or literature reporting the application of Sargassum fusiforme polysaccharide in anti-SVA virus. Summary of the Invention

[0006] This invention explores the extraction, isolation, anti-SVA virus activity and mechanism of Sargassum fusiforme polysaccharide. The results show that it has a significant inhibitory effect on the virus. However, during the in-depth study of the anti-SVA virus mechanism of Sargassum fusiforme by the inventor, it is found that the molecular weight of the Sargassum fusiforme polysaccharide in the mixture prepared by the traditional method has a large span, with the lowest molecular weight below 1 kDa and the highest molecular weight reaching several thousand kDa, which not only affects the anti-SVA virus activity but also enhances the cytotoxicity.

[0007] The purpose of the embodiment of this invention is to provide a Sargassum fusiforme polysaccharide with anti-SVA virus activity, its preparation method and application.

[0008] To achieve the above purpose, the embodiment of this invention provides the following technical solutions:

[0009] A preparation method of a Sargassum fusiforme polysaccharide with anti-SVA virus activity includes:

[0010] Step 1: Wash and dry Sargassum fusiforme, mix it with distilled water, heat and keep boiling for 2 - 4 h, and collect the clear liquid; concentrate the clear liquid and freeze-dry it to obtain the crude Sargassum fusiforme polysaccharide.

[0011] Step 2: Take the crude Sargassum fusiforme polysaccharide, dissolve it with distilled water, take the supernatant after centrifugation, and pass the supernatant through a 100 kDa molecular cut-off membrane; rinse the molecular cut-off membrane with distilled water, collect the Sargassum fusiforme polysaccharide solution with a molecular weight above 100 kDa, concentrate the solution and freeze-dry it to obtain the crude Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa.

[0012] Purify the crude Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa obtained in Step 2 to obtain the purified Sargassum fusiforme polysaccharide.

[0013] In some embodiments, the purification uses the water extraction and alcohol precipitation method or the Sevag method to remove proteins to obtain the purified Sargassum fusiforme polysaccharide.

[0014] Sargassum fusiforme is mainly distributed in the southeastern coastal areas of China and the edges of the Yellow Sea and the Bohai Sea, with a large output and wide sources. The cost of extracting Sargassum fusiforme polysaccharide from Sargassum fusiforme is lower. In this application, the crude Sargassum fusiforme polysaccharide is extracted and prepared from Sargassum fusiforme by the distillation method. The antiviral effect of Sargassum fusiforme polysaccharide gradually enhances with the increase of the molecular weight. During the purification process of the crude polysaccharide, the fat-soluble components decrease, resulting in a reduction in cytotoxicity. After purification, the cytotoxicity to cells decreases. Through experiments, the inventor finds that the purified Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa prepared in this application has the best antiviral effect and has the characteristics of high anti-SVA virus activity and low cytotoxicity.

[0015] The water extraction and alcohol precipitation method is as follows: Add 100 g of crude polysaccharide to a 5-L three-necked flask, add 2 L of water, heat to 60 °C, stir until the polysaccharide is completely dissolved, slowly add 4 L of absolute ethanol dropwise at 60 °C, stir for 2 h, and then settle overnight. Filter the settled polysaccharide, dry it at 70 °C, and repeat 4 times.

[0016] The Sevag method for protein removal: Add 10 g of dried crude polysaccharide to a 500-mL three-necked flask, add 250 mL of water, stir to dissolve, add 10 mL of chloroform and 2 mL of n-butanol, stir vigorously for 2 h, centrifuge, and separate the denatured protein at the junction of the aqueous layer and the solvent layer. Repeat the operation five times, collect the organic layer, concentrate, and freeze-dry.

[0017] In some embodiments, the material-liquid ratio of dried Sargassum fusiforme to distilled water in step 1 is 0.5 - 5∶150, preferably 1∶150;

[0018] Or, after mixing Sargassum fusiforme and distilled water in step 1, heat to boiling for 3 h. Optimize the extraction method of Sargassum fusiforme polysaccharide to increase the yield of Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa.

[0019] In some embodiments, the molecular cut-off plate in step 2 is an ultrafiltration membrane. Preferably, the pore size of the ultrafiltration membrane is 50 nm. Filter the dissolved Sargassum fusiforme polysaccharide solution with an ultrafiltration membrane with a pore size of 50 nm to obtain Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa, which has high anti-SVA virus activity and low cytotoxicity.

[0020] In some embodiments, the material-liquid ratio of crude Sargassum fusiforme polysaccharide to distilled water in step 2 is 1:10 - 30, preferably 1:22.

[0021] In some embodiments, the crude Sargassum fusiforme polysaccharide in step 2 is dissolved with distilled water at 30 - 70 °C. This speeds up the dissolution rate and also prevents the destruction of the activity of Sargassum fusiforme polysaccharide due to too high a temperature of the distilled water.

[0022] In some embodiments, the centrifugation speed in step 2 is 5000 r / min, and centrifuge for 10 min.

[0023] On the other hand, the present invention also provides the Sargassum fusiforme polysaccharide prepared by the above preparation method.

[0024] The present invention explores the extraction, separation, anti-SVA virus activity and mechanism of Sargassum fusiforme polysaccharide, and the results show that it has a significant inhibitory effect on the virus. However, during the in-depth study of the anti-SVA virus mechanism of Sargassum fusiforme by the applicant, it is found that the molecular weight of the mixed Sargassum fusiforme polysaccharide prepared by the above method has a large span, the lowest molecular weight is below 1 kDa, and the highest molecular weight can reach several thousand kDa, which not only affects the anti-SVA virus activity but also enhances the cytotoxicity.

[0025] The inventors first discovered through research that the anti-SVA virus effect of Sargassum fusiforme polysaccharide gradually increases with the increase in molecular weight, and the toxicity to cells decreases after purification. The Sargassum fusiforme polysaccharide with a molecular weight of more than 100 kDa prepared in this application has the characteristics of high anti-SVA virus activity and low cytotoxicity.

[0026] On the other hand, the present invention also provides an application of Sargassum fusiforme polysaccharide in the preparation of anti-SVA virus products.

[0027] On the other hand, the present invention also provides an application of Sargassum fusiforme polysaccharide in the preparation of drugs for swine vesicular disease.

[0028] The larger the molecular weight of the polysaccharide, the stronger the anti-SVA activity. The polysaccharide concentration and anti-SVA activity show a dose-effect relationship. The therapeutic index of the purified polysaccharide against SVA (MOI = 0.1) - infected PK-15 cells is 412. The inhibition rate of the virus load of 1 mg / mL polysaccharide against SVA (MOI = 10 - 1 - 104) - infected PK-15 cells is between 99.999% and 90%. The Sargassum fusiforme polysaccharide prepared in this application has reduced cytotoxicity to cells after purification. Sargassum fusiforme polysaccharide shows activity in inhibiting SVA adsorption or penetration into cells and inhibiting virus replication. The activity of inhibiting virus adsorption or penetration is stronger than that of inhibiting virus replication. In addition, the polysaccharide also has a certain preventive effect on the virus. The antiviral effect of the polysaccharide is strongly dependent on the concentration. The high-concentration drug shows inhibitory activity against different concentrations of SVA, and after incubation with low-concentration virus (MOI = 0.1) for 24 h, it loses its activity to infect cells.

[0029] One or more technical solutions provided in the embodiments of the present invention have at least the following technical effects or advantages:

[0030] 1. Sargassum fusiforme polysaccharide shows activity in inhibiting SVA adsorption or penetration into cells and inhibiting virus replication. The activity of inhibiting virus adsorption or penetration is stronger than that of inhibiting virus replication. In addition, the polysaccharide also has a certain preventive effect on the virus, and has a good effect on preventing and treating SVA virus infection and swine vesicular disease caused by SVA. The therapeutic index of the purified polysaccharide against SVA (MOI = 0.1) - infected PK-15 cells is 412. The inhibition rate of the virus load of 1 mg / mL polysaccharide against SVA (MOI = 10 - 1 - 104) - infected PK-15 cells is between 90% and 99.999%.

[0031] 2. Both the polysaccharide concentration and the SVA concentration show a dose-effect relationship with the anti-SVA activity. The anti-virus effect of Sargassum fusiforme polysaccharide gradually increases with the increase in molecular weight, and the toxicity to cells decreases after purification. The Sargassum fusiforme polysaccharide with a molecular weight of more than 100 kDa obtained in this application has the characteristics of high anti-SVA virus activity and low cytotoxicity.

[0032] 3. Sargassum fusiforme polysaccharide has the characteristics of wide source, and has the advantages of high activity, low toxicity, low side effects and low tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0034] Figure 1 It is a schematic diagram of different administration methods when detecting the action mode of Sargassum fusiforme polysaccharide on viruses;

[0035] Figure 2 It is a comparison chart of the cytotoxicity and antiviral activity of Sargassum fusiforme crude polysaccharides with different molecular weights;

[0036] Among them,

[0037] (a) and (b) are respectively the cytotoxicity and antiviral activity diagrams of Sargassum fusiforme crude polysaccharides with a molecular weight below 1 kDa,

[0038] (c) and (d) are respectively the cytotoxicity and antiviral activity diagrams of Sargassum fusiforme crude polysaccharides with a molecular weight of 1 kDa - 3 kDa,

[0039] (e) and (f) are respectively the cytotoxicity and antiviral activity diagrams of Sargassum fusiforme crude polysaccharides with a molecular weight of 3 kDa - 10 kDa,

[0040] (c) and (d) are respectively the cytotoxicity and antiviral activity diagrams of Sargassum fusiforme crude polysaccharides with a molecular weight of 10 kDa - 100 kDa,

[0041] (c) and (d) are respectively the cytotoxicity and antiviral activity diagrams of Sargassum fusiforme crude polysaccharides with a molecular weight above 100 kDa;

[0042] Figure 3 It is a comparison chart of the cytotoxicity of Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa after purification;

[0043] Figure 4 It is a comparison chart of the antiviral activity of Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa after purification;

[0044] Among them,

[0045] (a) is a comparison chart of the virus titer of Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa after purification at different concentrations and ribavirin at 0.5 mg / mol,

[0046] (b) is a comparison chart of the antiviral activity of Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa after purification at different concentrations and ribavirin at 0.5 mg / mol,

[0047] (c) Comparison chart of the antiviral activity of purified Sargassum fusiforme polysaccharide above 100 kDa at 1 mg / mol against different virus inoculation amounts;

[0048] Figure 5 Effect diagram of the inhibition of virus expression by different administration methods. Detailed implementation manners

[0049] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0050] Aiming at the problem that there is little research on drugs for SVA in the prior art and there is no effective drug, the purpose of the embodiments of the present invention is to provide an application of Sargassum fusiforme polysaccharide in anti-SVA virus for the prevention and treatment of SVA virus infection.

[0051] In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:

[0052] A preparation method of Sargassum fusiforme polysaccharide, comprising,

[0053] Step 1: Wash and dry Sargassum fusiforme, mix it with distilled water, heat and keep boiling for 2 - 4 h, and collect the clear liquid; concentrate the clear liquid and freeze-dry it to obtain crude Sargassum fusiforme polysaccharide;

[0054] Step 2: Take the crude Sargassum fusiforme polysaccharide, dissolve it with distilled water, take the supernatant after centrifugation, and pass the supernatant through a 100KDa molecular retention plate; rinse the molecular retention plate with distilled water, collect the crude Sargassum fusiforme polysaccharide solution with a molecular weight above 100 kDa, concentrate the solution, and freeze-dry it to obtain crude Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa.

[0055] Example 1

[0056] A preparation method of crude Sargassum fusiforme polysaccharide, comprising:

[0057] Step 1: Wash and dry Sargassum fusiforme, extract it with hot water, the material-liquid ratio is 1∶150, heat and boil for 3 h, concentrate the extract and freeze-dry it to obtain crude Sargassum fusiforme polysaccharide.

[0058] Step 2: Weigh 100 g of crude Sargassum fusiforme polysaccharide, add it to 2200 mL of distilled water at 50 °C and dissolve it. After centrifugation (5000 r / min, 10 min), take the supernatant. Use a peristaltic pump to pass the supernatant through molecular retention membranes with cut-off molecular weights of 1 kDa, 3 kDa, 10 kDa, and 100 kDa respectively. The supernatant is divided into fractions below 1 kDa, 1 kDa - 3 kDa, 3 kDa - 10 kDa, 10 kDa - 100 kDa, and above 100 kDa. Collect the solutions of each fraction, concentrate them, and store them after freeze-drying. Obtain crude Sargassum fusiforme polysaccharides with molecular weights below 1 kDa, 1 kDa - 3 kDa, 3 kDa - 10 kDa, 10 kDa - 100 kDa, and above 100 kDa.

[0059] Purify the crude Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa by the water extraction and alcohol precipitation method: Add 100 g of crude polysaccharide to a 5 L three-necked flask, add 2 L of water, heat to 60 °C, stir until the polysaccharide is completely dissolved, slowly add 4 L of absolute ethanol dropwise at 60 °C, stir for 2 h, and then let it settle overnight. Filter the settled polysaccharide, dry it at 70 °C, and repeat 4 times to obtain the purified Sargassum fusiforme polysaccharide of each fraction.

[0060] Example 2

[0061] A preparation method of crude Sargassum fusiforme polysaccharide includes:

[0062] Step 1: Wash and dry Sargassum fusiforme, extract it with hot water, with a material-liquid ratio of 0.5∶150, heat to boiling for 2 h, concentrate the extract and then freeze-dry it to obtain crude Sargassum fusiforme polysaccharide.

[0063] Step 2: Weigh 100 g of crude Sargassum fusiforme polysaccharide, add it to 3000 mL of hot water at 70 °C and dissolve it. After centrifugation (5000 r / min, 10 min), take the supernatant. Use a peristaltic pump to pass the supernatant through an ultrafiltration membrane with a pore size of 50 nm. Rinse the ultrafiltration membrane with distilled water, collect the crude Sargassum fusiforme polysaccharide solution with a molecular weight above 100 kDa, concentrate the solution, and freeze-dry it to obtain the crude Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa.

[0064] Purify the crude Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa by the water extraction and alcohol precipitation method: Add 100 g of crude polysaccharide to a 5 L three-necked flask, add 2 L of water, heat to 60 °C, stir until the polysaccharide is completely dissolved, slowly add 4 L of absolute ethanol dropwise at 60 °C, stir for 2 h, and then let it settle overnight. Filter the settled polysaccharide, dry it at 70 °C, and repeat 4 times to obtain the purified Sargassum fusiforme polysaccharide.

[0065] Example 3

[0066] A preparation method of crude Sargassum fusiforme polysaccharide includes:

[0067] Step 1: Wash and dry the Sargassum fusiformis, extract with hot water, with a solid-liquid ratio of 5:150, heat and boil for 4 hours, concentrate the extract and freeze-dry it to obtain Sargassum fusiformis crude polysaccharide.

[0068] Step 2: Weigh 100g of Sargassum crude polysaccharide, add 1000mL of hot water to dissolve, and the temperature of the hot water is 30°C, centrifuge (5000r / min, 10min), take the supernatant, and use a peristaltic pump to pass the supernatant through an ultrafiltration membrane with a pore size of 50nm. Rinse the ultrafiltration membrane with distilled water, collect the Sargassum crude polysaccharide solution with a molecular weight of more than 100kDa, concentrate the solution, and freeze-dry to obtain Sargassum crude polysaccharide with a molecular weight of more than 100kDa.

[0069] The crude polysaccharide of Sargassum fusiformis with a molecular weight of more than 100 kDa was purified by deproteinization using the Sevag method: 10 g of the dried crude polysaccharide was added to a 500 mL three-necked flask, 250 mL of water was added, and the mixture was stirred to dissolve. 10 mL of chloroform and 2 mL of n-butanol were added, and the mixture was stirred vigorously for 2 hours. The denatured protein at the junction of the water layer and the solvent layer was separated and centrifuged. The operation was repeated five times, and the organic layer was collected, concentrated, and freeze-dried to obtain purified Sargassum fusiformis polysaccharide.

[0070] Example 4 Test on the Anti-Seneca Virus Type A of Sargassum fusiformis Polysaccharide

[0071] 1 Materials and methods

[0072] 1.1 Materials

[0073] 1.1.1 Test materials

[0074] Senecavirus A (SVA) and porcine kidney cells (PK-15) were preserved in this experiment, and the various segments of Sargassum fusiformis polysaccharides prepared in Experimental Example 1 of this application were prepared.

[0075] 1.1.2 Main reagents and instruments

[0076] DMEM high-glucose medium and phosphate-buffered saline (PBS) were purchased from Dalian Meilun Biotechnology Co., Ltd.; 0.25% trypsin was purchased from Gbico; special fetal bovine serum (FBS) was purchased from Lonsera; viral DNA / RNA extraction kit, 2x Accurate Taq Master Mix (dye pLus), and reverse transcription reagent were purchased from Hunan Aikerui Bioengineering Co., Ltd.; CCK-8 kit and penicillin-streptomycin-amphotericin B solution (three antibodies) were purchased from Beijing Solebow Technology Co., Ltd.

[0077] The peristaltic pump (molecular retention plate) was purchased from Millipore Corporation; the N-130 rotary evaporator was purchased from Tokyo Rikakikai Co., Ltd.; the freeze dryer was purchased from Jinan Pusen Instrument Co., Ltd.; the microplate reader was purchased from Nanjing Detie Experimental Equipment Co., Ltd.; the Roche 480 was purchased from Roche; the automatic nucleic acid extractor was purchased from Nanjing Novoprotein Scientific Inc.

[0078] 1.2 Methods

[0079] 1.2.1 Cell culture

[0080] Take out the PK-15 cells cryopreserved in liquid nitrogen, place them in a 37 °C water bath to melt, transfer them to a 15 mL centrifuge tube after melting, add 10 mL of PBS, centrifuge at 1500 r / min for 5 min, discard the supernatant, resuspend the cells with DMEM high-glucose medium containing 10% serum and 1% triple antibody and add them to a cell culture flask, and place them in a 37 °C, 5% CO2 incubator for culture for later use.

[0081] 1.2.2 Virus titer detection

[0082] Dilute the SVA virus stock solution 10-fold with DMEM to obtain virus solutions from 10 -1 to 10 -11 . Add them to a 96-well plate filled with a monolayer of PK-15 cells, with 8 replicates for each concentration, place them in a 37 °C, 5% CO2 incubator for culture, observe every 24 h, wait until the cells in the wells no longer show cytopathic effects, record the results, and calculate the virus titer by the Reed-Muench method.

[0083] 1.2.3 Cytotoxicity detection of crude polysaccharides with different molecular weights

[0084] Passage the PK-15 cells into a 96-well plate. When the cells grow to a monolayer, add PBS and rinse 3 times. Prepare solutions of crude polysaccharides with different molecular weights (below 1 kDa, 1 kDa - 3 kDa, 3 kDa - 10 kDa, 10 kDa - 100 kDa, above 100 kDa) prepared in Example 1 at 10 mg / mL, 1 mg / mL, 0.1 mg / mL, and 0.01 mg / mL respectively and add them to the 96-well plate. Set up cell controls and blank controls, with 8 replicates for each concentration. After culturing in a 37 °C, 5% CO2 incubator for 44 h, add CCK-8 and incubate for 2 h, and measure the absorbance at OD450nm with a microplate reader. The formula for calculating cell survival rate is as follows:

[0085] Cell survival rate (%) = (OD450nm of drug group - OD450nm of blank control) / (OD450nm of cell control - OD450nm of blank control) × 100%.

[0086] 1.2.4 Detection of antiviral activities of crude polysaccharides with different molecular weights

[0087] Passage PK-15 cells into 24-well plates. When the cells grow to a monolayer, add PBS and rinse three times. Then, co-incubate crude Sargassum fusiforme polysaccharides with different molecular weights at a safe concentration and virus solution (MOI = 0.1) on the cells. Set up cell controls and virus controls, with 3 replicates for each treatment method. After incubating in a 37 °C, 5% CO₂ incubator for 2 h, discard the supernatant, add PBS and rinse three times. Then, add the Sargassum fusiforme polysaccharide solution at the same concentration again. After incubating in a 37 °C, 5% CO₂ incubator for 24 h, perform three cycles of freezing and thawing, collect the virus solution, filter it through a 0.22 μm filter membrane. Take 200 μL from each well and use a magnetic bead method virus DNA / RNA extraction kit to extract viral nucleic acids. After reverse transcription of the extracted nucleic acids, perform fluorescence quantitative (SYBR Green I) detection. Select β-actin as the internal reference, and the primer design for SVA is shown in Table 1. The fluorescence quantitative PCR program settings are as follows: pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 30 s, annealing / extension at 60 °C for 15 s, for 40 cycles. Read the fluorescence value cp at 60 °C, and the results are calculated according to the following method. ΔΔcp = (cp of the drug group - Δcp of the internal reference) - Δ (cp of the virus group - Δcp of the internal reference). Select the molecular weight range with the best antiviral effect for purification and use it for subsequent experiments. -ΔΔcp The method is calculated as follows. ΔΔcp = (cp of the drug group - Δcp of the internal reference) - Δ (cp of the virus group - Δcp of the internal reference). Select the molecular weight range with the best antiviral effect for purification and use it for subsequent experiments.

[0088] Table 1 Primer sequences

[0089]

[0090] 1.2.5 Detection of the safe concentration of the purified polysaccharide

[0091] Passage PK-15 cells into 96-well plates. When the cells grow to a monolayer, add PBS and rinse three times. Then, prepare solutions of the purified Sargassum fusiforme polysaccharide with a molecular weight above 100 KDa prepared in Example 1 at concentrations of 10 mg / mL, 8 mg / mL, 6 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, and 0.5 mg / mL with DMEM and add them to the 96-well plates. The subsequent method is the same as 1.2.4.

[0092] 1.2.6 Detection of the antiviral activity of the purified Sargassum fusiforme polysaccharide

[0093] Determine the effects of polysaccharide concentration and virus concentration on the antiviral activity of polysaccharides. The purified Sargassum fusiforme polysaccharide was diluted to 1 mg / mL, 0.5 mg / mL, 0.1 mg / mL, 0.01 mg / mL, and 0.001 mg / mL, and co - acted with SVA (MOI = 0.1) on PK - 15 cells. The method was the same as 1.2.5. Real - time fluorescence quantitative PCR was used to detect the virus load, and another 200 μL was taken from each well for virus titer determination. At the same time, the antiviral test was carried out on SVA (MOI = 0.1, 1, 10, 100, 1000, 10000) with 1 mg / mL of polysaccharide, and the virus load was measured. The method was the same as 1.2.5.

[0094] 1.2.7 Detection of the mode of action of polysaccharides on viruses

[0095] The PK - 15 cells were passaged into 24 - well plates. There were a total of seven groups in the experiment, and different drug - administration methods were used, such as Figure 1 shown. They were: administering the drug 2 h before virus inoculation (a), administering the drug simultaneously with virus inoculation (b), administering the drug simultaneously with virus inoculation (c), administering the drug 2 h after virus inoculation (d), administering the drug 6 h after virus inoculation (e). "-----" indicates that the drug is present in the culture medium. For the virus control and cell control, the culture medium was uniformly changed 2 h after virus inoculation. The virus inoculation amount was MOI = 10. After incubation for 22 h, it was frozen and thawed three times repeatedly, and the virus nucleic acid was extracted for fluorescence quantitative PCR determination.

[0096] 1.2.8 Data statistical analysis

[0097] The experimental results were analyzed using GraphPad Prism 9.0 software. One - Way ANOVA was used to analyze the results of the cytotoxicity test, the relative expression level of virus RNA and virus titer results in the antiviral activity and antiviral mode test of polysaccharides.

[0098] 2 Results are analyzed as follows:

[0099] 2.1 Extraction of crude Sargassum fusiforme polysaccharide and separation of polysaccharides with different molecular weights

[0100] Through the molecular retention experiment of crude Sargassum fusiforme polysaccharide, the mass ratios of polysaccharides in five segments below 1 kDa, 1 kDa - 3 kDa, 3 kDa - 10 kDa, 10 kDa - 100 kDa, and above 100 kDa were: 68.55∶1.20∶2.36∶0.57∶4.76. The mass of the segment below 1 kDa was the largest and the distribution was the most, followed by the segment above 100 kDa.

[0101] 2.2 Cytotoxicity and antiviral activity of crude Sargassum fusiforme polysaccharides with different molecular weights

[0102] Figure 2Cell relative survival rates and antiviral activities at non-toxic concentrations for different molecular weight ranges at 10 mg / mL, 1 mg / mL, 0.1 mg / mL, and 0.01 mg / mL. Compared with the blank (virus) control, * represents a significant difference (P < 0.05), ** represents a relatively significant difference (0.01 < P < 0.05), *** represents a very significant difference (0.001 < P < 0.01), and **** represents an extremely significant difference (P < 0.001). Figures 3 - 5 The same and will not be elaborated further.

[0103] Polysaccharides with a molecular weight below 1 kDa were cytotoxic at 10 mg / mL (P < 0.001), polysaccharides with a molecular weight of 1 - 3 kDa were cytotoxic at 10 mg / mL (P < 0.001), polysaccharides with a molecular weight of 3 - 10 kDa were cytotoxic at 0.1 mg / mL and above (P < 0.001), polysaccharides with a molecular weight of 10 - 100 kDa were cytotoxic at 0.1 mg / mL and above (P < 0.001), and polysaccharides with a molecular weight above 100 kDa were cytotoxic at 1 mg / mL and above.

[0104] TCID of SVA 50 = 10 -8.7 / 0.1 mL. When the virus inoculation amount was at MOI = 0.1, polysaccharides with a molecular weight below 1 kDa had antiviral activity at 1 mg / mL (P < 0.001), polysaccharides with a molecular weight of 1 - 3 kDa had antiviral activity at 1 mg / mL and 0.1 mg / mL (P < 0.001), polysaccharides with a molecular weight of 3 - 10 kDa and 10 - 100 kDa had antiviral activity at 0.01 mg / mL (P < 0.001), and polysaccharides with a molecular weight above 100 kDa had antiviral activity at 0.1 mg / mL and 0.01 mg / mL (P < 0.001).

[0105] Polysaccharides with a molecular weight above 100 kDa had the strongest antiviral effect at 0.1 mg / mL. Therefore, this part was selected for subsequent experiments.

[0106] 2.3 Cytotoxicity and antiviral activity of polysaccharides with a molecular weight above 100 kDa after purification

[0107] Figure 3 For the cytotoxicity of polysaccharides with a molecular weight above 100 kDa after purification, there was no cytotoxicity when the concentration of the purified polysaccharide was below 2 mg / mL (P > 0.5). The half-maximal inhibitory concentration IC 50 of the polysaccharide against PK - 15 cells calculated by Graphpad prism was 4.94 mg / mL.

[0108] Figure 4For the antiviral activity of polysaccharides with a molecular weight above 100 kDa after purification, the concentration of ribavirin was 0.5 mg / mL. The purified polysaccharides showed anti-SVA activity at a concentration of 0.01 mg / mL and above (P < 0.001). From the results of the virus titer test, when 1 mg / mL of polysaccharides and the virus (MOI = 0.1) acted on cells for 24 h, and the virus solution (containing polysaccharides) was reinoculated into the cells in a 96-well plate to measure the titer, no cytopathic effect was observed in the cells. The virus titer results were affected by the polysaccharides remaining in the solution, but were generally similar to the results of fluorescence quantitative PCR. The EC was calculated by fitting a curve according to the fluorescence quantitative results using Graphpad prism. 50 The EC for ribavirin at 0.5 mg / mL was also 0.012 mg / mL. Ribavirin at 0.5 mg / mL also had anti-SVA activity (P < 0.001), but its antiviral activity was lower than that of polysaccharides at the same concentration.

[0109] The purified 1 mg / mL polysaccharides showed obvious inhibitory effects (P < 0.001) against different virus inoculation amounts (MOI = 10 4 、10 3 、10 2 、10、1、0.1) compared with the virus control (MOI = 10 4 、10 3 、10 2 、10、1、0.1). As the virus concentration decreased, the antiviral effect gradually increased.

[0110] 2.4 Antiviral effects of polysaccharides with different administration methods

[0111] Figure 5 The figure shows the inhibitory effects of different administration methods on virus expression levels. Among them, a: administration 2 h before virus inoculation (administered throughout the process); b: administration at the same time as virus inoculation (administered throughout the process); c: administration at the same time as virus inoculation (the medium was changed 2 h after virus inoculation and incubation); d: administration 2 h after virus inoculation; e: administration 4 h after virus inoculation; f: virus control. Different administration methods (1 mg / mL) all had an antagonistic effect on the virus (P < 0.001). As the administration time was postponed, the antiviral effect gradually decreased. The antiviral effect of early administration was higher than that of simultaneous administration (P < 0.001). The antiviral effect of administration at the same time as virus inoculation (administered throughout the process) was higher than that of administration at the same time as virus inoculation (the medium was discarded 2 h after virus inoculation and incubation) and administration after virus inoculation (P < 0.001). The efficacy of administration at the same time as virus inoculation (the medium was discarded 2 h after virus inoculation and incubation) was higher than that of administration after virus inoculation (P < 0.001). The inhibitory effect of administration 2 h after virus inoculation on the virus was stronger than that of administration 6 h after virus inoculation (0.01 < P < 0.05).

[0112] In summary, although there are more than 30 antiviral drugs currently on the market, due to factors such as drug efficacy, toxicity, high prices, and drug tolerance, the shortage of antiviral drugs has not been effectively alleviated. Some studies have shown that the titer of neutralizing antibodies increases after pigs are infected with SVA, indicating that the virulence of the virus is gradually strengthening. The development of new antiviral drugs, especially those targeting viruses with immunosuppressive and immune escape characteristics, is particularly important. Seaweed polysaccharides have received increasing attention due to their high activity, wide sources, low toxicity, low side effects, and low tolerance. Currently, a variety of antiviral drugs based on seaweed polysaccharides have entered clinical or preclinical trials and are expected to become a new way for the research and development of antiviral drugs.

[0113] There are multiple ways for polysaccharides to exert antiviral effects. Among them, sulfated polysaccharides can physically adsorb to amino acid residues on the virus surface or cell surface, or bind to viral proteins to denature the viral protein coat, preventing the virus from binding to cells. Cynomorium polysaccharide itself does not have the activity against human immunodeficiency virus (HIV), but after sulfation, it can bind to amino acid residues on the surface of HIV and inhibit the entry of HIV into cells. Fucoidan can bind to the envelope S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and inhibit the entry of the virus into cells; Ecklonia kurome polysaccharide can bind to the protease of SARS-CoV-2 and inhibit virus replication. Sargassum fusiforme polysaccharide has shown inhibitory activity in the adsorption or penetration of SVA, virus replication and other activity cycles, which may be similar to the above mechanisms, but further experimental verification is needed.

[0114] The extraction methods of polysaccharides also include water extraction, acid extraction, enzyme extraction, ultrasonic extraction, etc.; the separation and purification methods also include chromatography, precipitation method and membrane filtration method, etc. Purification and structural identification are the basis and difficulties of polysaccharide medicinal research, and also the top priorities of future polysaccharide research. The antiviral effect of Sargassum fusiforme polysaccharide gradually increases with the increase of molecular weight, and the toxicity to cells decreases after purification. It may be that the decrease of liposoluble components in the purification process of crude polysaccharide leads to the decrease of cell toxicity. Sargassum fusiforme polysaccharide shows activity in inhibiting the adsorption or penetration of SVA into cells and inhibiting virus replication. The activity of inhibiting virus adsorption or penetration is stronger than that of inhibiting virus replication. Its adsorption mechanism may be related to its acidic polysaccharide structure. In addition, the polysaccharide also has a certain preventive effect on the virus, which may be related to the regulation of immune or inflammatory factors. The antiviral effect of polysaccharide is strongly dependent on concentration. High-concentration drugs show inhibitory activity against different concentrations of SVA. After incubation with low-concentration virus (MOI = 0.1) for 24 h, the virus loses its activity to infect cells. In addition, the laboratory has also conducted in vitro antiviral tests on porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus (the results are not shown). Sargassum fusiforme polysaccharide also shows antagonistic activity, but the activity is not as strong as that against SVA, indicating that Sargassum fusiforme polysaccharide has specificity for SVA and has the potential to develop broad-spectrum antiviral drugs.

[0115] Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, it is not a limitation to the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solutions of the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts are still within the protection scope of the present invention.

Claims

1. Application of Sargassum fusiforme polysaccharide in any one or more of the following: a. Application in the preparation of anti-SVA virus products; the molecular weight of the Sargassum fusiforme polysaccharide is above 100 kDa; b. Application in the preparation of drugs for treating swine vesicular disease; The preparation method of the Sargassum fusiforme polysaccharide includes the following steps: Step 1: Wash and dry Sargassum fusiforme, mix it with distilled water, heat and keep boiling for 2 - 4 h, and collect the clear liquid; concentrate the clear liquid and freeze-dry it to obtain crude Sargassum fusiforme polysaccharide; Step 2: Take the crude Sargassum fusiforme polysaccharide, dissolve it with distilled water, take the supernatant after centrifugation, and pass the supernatant through a 100 KDa molecular retention plate; rinse the molecular retention plate with distilled water, collect the crude Sargassum fusiforme polysaccharide solution with a molecular weight above 100 kDa, concentrate the solution and freeze-dry it to obtain crude Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa; Purify the crude Sargassum fusiforme polysaccharide with a molecular weight above 100 kDa obtained in Step 2 to obtain the purified Sargassum fusiforme polysaccharide.

2. The application according to claim 1, characterized in that The purification uses the water extraction and alcohol precipitation method or the Sevag method to remove proteins.

3. The application according to claim 1, characterized in that In Step 1, the material-liquid ratio of dried Sargassum fusiforme to distilled water is 0.5 - 5∶150; Or, in Step 1, after mixing Sargassum fusiforme with distilled water, heat and boil for 3 h.

4. The application according to claim 1, characterized in that In Step 1, the material-liquid ratio of dried Sargassum fusiforme to distilled water is 1∶150.

5. The application according to claim 1, characterized in that, In Step 2, the molecular retention plate is an ultrafiltration membrane.

6. The application according to claim 5, characterized in that, The pore size of the ultrafiltration membrane is 50 nm.

7. The application according to claim 1, wherein In Step 2, the material-liquid ratio of crude Sargassum fusiforme polysaccharide to distilled water is 1:10 - 30; Or, in Step 2, the crude Sargassum fusiforme polysaccharide is dissolved with distilled water at 30 - 70 °C.

8. The application according to claim 1, wherein In Step 2, the material-liquid ratio of crude Sargassum fusiforme polysaccharide to distilled water is 1:

22.

9. The application according to claim 1, wherein In Step 2, the centrifugation speed is 5000 r / min and the centrifugation time is 10 min.

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