Selenized low-molecular-weight sargassum fusiforme polysaccharide as well as preparation method and application thereof in virus resistance

Abstract

The invention discloses selenized low-molecular-weight sargassum fusiforme polysaccharide with an efficient antiviral effect and a preparation method and application thereof. The preparation method comprises the following steps: uniformly mixing low-molecular-weight sargassum fusiforme polysaccharide, selenium inorganic salt and a reducing agent, oscillating, keeping out of light, fully reacting, carrying out selenylation modification on the sargassum fusiforme polysaccharide, and dialyzing after the reaction is completed, so as to obtain the selenylation low-molecular-weight sargassum fusiforme polysaccharide. The prepared selenized low-molecular-weight sargassum fusiforme polysaccharide has a remarkable antiviral effect on various viruses, no organic solvent is involved in the preparation process, the process is safe and environmentally friendly, the cost is low, operation is easy, and the selenized low-molecular-weight sargassum fusiforme polysaccharide can be widely applied to antiviral preparations.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a seleniumized low-molecular-weight Sargassum fusiforme polysaccharide, a preparation method thereof, and an application thereof in antiviral treatment. Background Art

[0002] Sargassum fusiforme (scientific name: Hizikia fusiforme) is a species of Sargassum with both edible and medicinal values, widely distributed in the Bohai Sea, the Yellow Sea and the southeastern coastal areas of China, as well as the coastal areas of Korea, South Korea and Japan. With the progress of the artificial cultivation technology of Sargassum fusiforme in areas such as Wenzhou, Zhejiang in China, Sargassum fusiforme has currently become one of the main large-scale economic seaweeds in China. Polysaccharide is one of the important active ingredients in Sargassum fusiforme, with a variety of biological activities, mainly including antioxidant, immunomodulatory, anti-tumor, hypoglycemic, anticoagulant, anti-inflammatory and antiviral activities. At present, the hot water extraction method is the most widely used method for preparing Sargassum fusiforme polysaccharide. It has less damage to the structure of Sargassum fusiforme polysaccharide and is easy to operate and produce on a large scale. However, the extraction rate of this method is low, and the obtained polysaccharide has a large molecular weight, poor water solubility and a darker color. It has been reported that the average molecular weight of Sargassum fusiforme polysaccharide prepared by this method is 224 kDa, and its appearance is usually in the form of dark brown flakes, showing a dark amber color after dissolving in water. Degradation can usually improve the activity of polysaccharide while reducing the molecular weight.

[0003] Viruses are small and relatively simple non-cellular organisms that seriously threaten human health. They are closely related to the occurrence of infectious diseases, immune disorders, tumors, etc. The research on the application of polysaccharides in antiviral treatment has unique advantages such as being less likely to produce drug resistance. At present, the antiviral activity mechanism of polysaccharides mainly includes the following four aspects: (1) directly killing viruses; (2) inhibiting the virus replication process (including invasion, adsorption, etc.); (3) activating the immune regulation system; (4) reducing immune stress. In the application of Sargassum fusiforme polysaccharide in antiviral treatment, it has been found that Sargassum fusiforme polysaccharide has an inhibitory effect on subgroup J of avian leukosis virus (ALV-J), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infectious bursal disease virus (IBDV), enterovirus 71 (EV71) and respiratory syncytial virus (RSV). In the application of low-molecular-weight seaweed polysaccharides in antiviral treatment, only Li Pengcheng et al. (patent application number: CN201611187764.1) reported the anti-avian leukosis virus (ALV) activity of low-molecular-weight seaweed polysaccharides.

[0004] In summary, there are few reports on the antiviral application of low-molecular-weight Sargassum fusiforme polysaccharides at present, and there is no report on the antiviral application of seleniumized low-molecular-weight Sargassum fusiforme polysaccharides. Summary of the Invention

[0005] The object of the present invention is to provide a selenium - modified low - molecular - weight Sargassum fusiforme polysaccharide, its preparation method and its application in anti - virus in view of the above problems.

[0006] In order to achieve its object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a preparation method of a selenium - modified low - molecular - weight Sargassum fusiforme polysaccharide, which includes the following steps: taking low - molecular - weight Sargassum fusiforme polysaccharide, selenium inorganic salt and a reducing agent, mixing them evenly, oscillating and reacting in the dark sufficiently, performing selenium modification on the Sargassum fusiforme polysaccharide, and performing dialysis after the reaction is completed to obtain the selenium - modified low - molecular - weight Sargassum fusiforme polysaccharide;

[0008] The selenium inorganic salt is selected from sodium selenite and sodium thiosulfate;

[0009] The reducing agent is selected from ascorbic acid and glutathione.

[0010] The average molecular weight of the low - molecular - weight Sargassum fusiforme polysaccharide is 10 - 50 kDa;

[0011] The low - molecular - weight Sargassum fusiforme polysaccharide is prepared by combining ultraviolet light and hydrogen peroxide.

[0012] In the described preparation method, the concentrations of each substance in the mixed solution of low - molecular - weight Sargassum fusiforme polysaccharide, selenium inorganic salt and reducing agent are: low - molecular - weight Sargassum fusiforme polysaccharide 0.5 - 2.5 mg / mL, selenium inorganic salt 10 - 50 mmol / L, reducing agent 40 - 80 mmol / L; the reaction time in the dark is 1 - 6 h.

[0013] Preferably, the concentrations of each substance in the mixed solution of low - molecular - weight Sargassum fusiforme polysaccharide, selenium inorganic salt and reducing agent are: low - molecular - weight Sargassum fusiforme polysaccharide 1.0 - 2.0 mg / mL or 1.0 - 1.5 mg / mL, selenium inorganic salt 10 - 30 mmol / L or 15 - 25 mmol / L, reducing agent 60 - 80 mmol / L, and react in the dark for 3 - 5 h.

[0014] Preferably, after dialysis, freeze - drying is performed to obtain a freeze - dried powder of the selenium - modified low - molecular - weight Sargassum fusiforme polysaccharide.

[0015] The second aspect of the present invention provides a preparation method of a selenium - modified low - molecular - weight Sargassum fusiforme polysaccharide, which is prepared by using the preparation method described in any one of the above.

[0016] The second aspect of the present invention provides the application of the above - mentioned selenium - modified low - molecular - weight Sargassum fusiforme polysaccharide in the preparation of anti - virus preparations.

[0017] The application described above, wherein the viruses include RNA viruses and DNA viruses. The RNA viruses include vesicular stomatitis virus (VSV) and influenza virus, and the DNA viruses include herpes simplex virus and pseudorabies virus (PRV). The influenza virus includes influenza A virus WSN, and the herpes simplex virus includes herpes simplex virus type 1 (HSV-1).

[0018] In the application described above, the working concentration of the selenium-enriched low molecular weight Sargassum fusiforme polysaccharide is 10 - 50 μg / ml, preferably 10 - 20 μg / ml.

[0019] In the application described above, preferably, the virus is HSV-1 or VSV.

[0020] The beneficial effects of the present invention are as follows:

[0021] Fully exploring the nutritional and functional properties of seaweed products and enhancing the market competitiveness and added value of products are crucial for the development of the seaweed industry. In the present invention, taking low molecular weight Sargassum fusiforme polysaccharide as the object, a selenium-enriched low molecular weight seaweed polysaccharide is prepared by the method of combining ascorbic acid with sodium selenite. It is found that its antiviral effect is significantly better than that of the non-selenium-enriched low molecular weight seaweed polysaccharide, and it has broad application prospects. The preparation process of the selenium-enriched low molecular weight Sargassum fusiforme polysaccharide in the present invention does not involve organic solvents, and the process is safe, low-cost and easy to operate, which will contribute to the development of seaweed-derived broad-spectrum antiviral preparations. Description of the Drawings

[0022] Figure 1 shows the influence of polysaccharide concentration on the selenium-enriched low molecular weight Sargassum fusiforme polysaccharide: (A) apparent characteristics of the reaction solution, (B) particle size distribution and selenium loading.

[0023] Figure 2 is the electron microscope image and energy spectrum scanning analysis chart of the selenium-enriched low molecular weight Sargassum fusiforme polysaccharide.

[0024] Figure 3 shows the influence of selenium-enriched and non-selenium-enriched low molecular weight Sargassum fusiforme polysaccharide on the gene replication of different viruses: (A) RNA viruses (VSV-G virus and WSN virus), (B) DNA viruses (HSV-1 virus and PRV virus).

[0025] Figure 4 shows the influence of selenium-enriched and non-selenium-enriched low molecular weight Sargassum fusiforme polysaccharide on the protein replication of different viruses: (A) RNA viruses (VSV-G virus and WSN virus), (B) DNA viruses (HSV-1 virus and PRV virus). Detailed Embodiments

[0026] The technical solution of the present invention will be described in detail below through embodiments, but the technical solution of the present invention is not limited to the following embodiments. The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0027] The low-molecular-weight Sargassum fusiforme polysaccharide in the embodiment of the present invention is prepared by referring to the preparation of Sargassum fusiforme polysaccharide (PSF) in "Study on the anti-inflammatory effect and mechanism of Sargassum fusiforme polysaccharide degraded by UV / H2O2" (Doctoral dissertation of South China University of Technology, Chen Xiaoyong, 2021), Chapter 2, "2.3.1 Preparation of Sargassum fusiforme polysaccharide", and then PSF is prepared into PSF-T2 (i.e., low-molecular-weight Sargassum fusiforme polysaccharide) according to "3.3.1 Preparation of Sargassum fusiforme polysaccharides with different degradation degrees" in Chapter 3.

[0028] Example 1. Preparation of selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide

[0029] The selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide was prepared by the method of combining ascorbic acid with sodium selenite. The specific method is as follows:

[0030] Take an appropriate amount of low-molecular-weight Sargassum fusiforme polysaccharide prepared by ultraviolet light combined with hydrogen peroxide (average molecular weight ~17.31 kDa) and make polysaccharide aqueous solutions with concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL respectively. Then add a certain amount of sodium selenite and ascorbic acid respectively, so that their final concentrations in the polysaccharide solution are 20 mmol / L and 80 mmol / L respectively. After mixing, place it on a shaker and react in the dark for 4 h. After the reaction is completed, take a photo for record. Finally, dialyze the solution with a dialysis bag to remove small molecules (such as unreacted sodium selenite, ascorbic acid) and unbound elemental selenium generated. After dialysis is completed, freeze-dry to obtain the freeze-dried powder of selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide.

[0031] Use a nano-particle size and Zeta potential analyzer and a flame graphite furnace integrated atomic absorption spectrophotometer to measure the particle size and selenium content of the selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide, and perform scanning electron microscopy and energy spectrum analysis on the selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide under specific conditions.

[0032] As Figure 1 shown in A, after the reaction is completed, when there is no low-molecular-weight Sargassum fusiforme polysaccharide, the solution is red; after adding low-molecular-weight Sargassum fusiforme polysaccharide, the color of the solution becomes lighter, and when the polysaccharide concentration reaches 2.5 mg / mL, it is orange. As Figure 1As can be seen from B, after adding low-molecular-weight Sargassum fusiforme polysaccharide, the average particle size of the particles is less than 100 nm, and the particle size is lower than that without addition. When the polysaccharide concentration increases to 2.5 mg / mL, the particle size becomes larger. In addition, within the range of 0-2.5 mg / mL polysaccharide concentration, with the increase of the concentration, the selenium content of selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide first increases and then decreases. The selenium contents of the low-molecular-weight Sargassum fusiforme polysaccharide samples at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL are 13.79, 24.86, 42.21, 38.71, 22.45, and 10.57 mg / g, respectively.

[0033] It can be seen from Figure 2 that the results of scanning electron microscope observation show that selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide is flaky and its surface is not smooth. The distribution map of energy spectrum surface scanning for selenium confirms the existence of selenium in selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide.

[0034] Example 2: Effects of Selenium-Enriched and Unselenium-Enriched Low-Molecular-Weight Sargassum fusiforme Polysaccharides against RNA Viruses

[0035] After Vero cells (African green monkey kidney cells) were inoculated into a 12-well cell culture plate at 1.5×10 5 cells / well, they were placed in a constant temperature incubator at 37°C containing 5% CO2 and cultured with complete medium containing 10% fetal bovine serum for 12 h. The experimental group cells were pretreated with different concentrations of selenium-enriched or unselenium-enriched low-molecular-weight Sargassum fusiforme polysaccharides for 6 h, and the control group (i.e., WT) was added with an equal volume of ultrapure water. Then, the viruses VSV-G and WSN (MOI (multiplicity of infection) = 1) were respectively inoculated into the cells and incubated for 24 h. The supernatant was discarded, the cells were washed with PBS, and the cell RNA samples were collected. The effects of pretreatment with selenium-enriched and unselenium-enriched low-molecular-weight Sargassum fusiforme polysaccharides on the gene replication of different viruses (VSV-G and WSN) were detected by real-time fluorescence quantitative PCR technology. In addition, the cell protein samples were collected, and the effects of pretreatment with selenium-enriched and unselenium-enriched low-molecular-weight Sargassum fusiforme polysaccharides on the protein replication of different viruses (VSV-G and WSN) were detected by immunoblotting technology.

[0036] It can be seen from Figure 3 (A) that the results of real-time fluorescence quantitative PCR analysis show that selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide has a significant inhibitory effect on the expression of both the VSV-G replication-related gene (G gene) and the WSN replication-related gene (M1 gene). It is worth noting that there are differences in the inhibitory effects of its expression on the replication-related genes of the two. Compared with WSN, its inhibitory effect on the expression of the VSV-G replication-related gene is better. Although unselenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide has an inhibitory effect on the expression of both the VSV-G replication-related gene (G gene) and the WSN replication-related gene (M1 gene), the inhibitory effect is worse than that of selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide.

[0037] As can be seen from Figure 4 (A), the inhibitory effect of selenium - low molecular weight Sargassum fusiforme polysaccharide on the expression of RNA virus replication - related proteins is similar to that of real - time fluorescence quantitative PCR analysis. It has an obvious inhibitory effect on the expression of VSV - G replication - related proteins and WSN replication - related proteins, and there are differences in the inhibitory effects on the two. Compared with WSN, its inhibitory effect on the expression of VSV - G replication - related proteins is better. Although the non - selenium - low molecular weight Sargassum fusiforme polysaccharide has an inhibitory effect on the expression of both VSV - G replication - related proteins and WSN replication - related proteins, its inhibitory effect is worse than that of the selenium - low molecular weight Sargassum fusiforme polysaccharide. The selenium - low molecular weight Sargassum fusiforme polysaccharide at a concentration of 10 - 20 μg / mL can achieve the virus inhibition effect of 50 μg / mL non - selenium - low molecular weight Sargassum fusiforme polysaccharide. For VSV - G, the effect of 20 μg / mL selenium - low molecular weight Sargassum fusiforme polysaccharide is equivalent to or even better than that of 50 μg / mL non - selenium - low molecular weight Sargassum fusiforme polysaccharide, while 20 μg / mL non - selenium - low molecular weight Sargassum fusiforme polysaccharide does not show a significant inhibitory effect on VSV - G; the antiviral activity of Sargassum fusiforme polysaccharide after seleniumization is greatly improved.

[0038] Example 3: Effects of Selenium - and Non - Selenium - Low Molecular Weight Sargassum fusiforme Polysaccharides against DNA Viruses

[0039] Vero cells (African green monkey kidney cells) were seeded at 1.5×10 5 cells per well in a 12 - well cell culture plate, then placed in an incubator at 37°C with 5% CO2 and cultured with complete medium containing 10% fetal bovine serum for 12 h. The experimental group cells were pretreated with different concentrations of selenium - or non - selenium - low molecular weight Sargassum fusiforme polysaccharides for 6 h, and the control group (i.e., WT) was added with an equal volume of ultrapure water. Then, the viruses HSV - 1 and PRV (MOI (multiplicity of infection) = 1) were respectively inoculated into the cells and incubated for 24 h. The supernatant was discarded, the cells were washed with PBS, and cell RNA samples were collected. The effects of pretreatment with selenium - and non - selenium - low molecular weight Sargassum fusiforme polysaccharides on the gene replication of different viruses (HSV - 1 and PRV) were detected by real - time fluorescence quantitative PCR technology. In addition, cell protein samples were collected, and the effects of pretreatment with selenium - and non - selenium - low molecular weight Sargassum fusiforme polysaccharides on the protein replication of different viruses (HSV - 1 and PRV) were detected by immunoblotting technology.

[0040] As can be seen from Figure 3As can be seen from (B), the results of real-time fluorescence quantitative PCR analysis showed that selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide had a significant inhibitory effect on the expression of genes related to HSV-1 replication (gB gene) and genes related to PRV replication (gD gene). It is worth noting that there were also differences in its inhibitory effect on the expression of replication-related genes of the two. Compared with HSV-1, its inhibitory effect on the expression of PRV replication-related genes was better. Although non-selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide had an inhibitory effect on the expression of genes related to HSV-1 replication (gB gene) and genes related to PRV replication (gD gene), its inhibitory effect was worse than that of selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide.

[0041] As can be seen from Figure 4 (B), the inhibitory effect of selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide on the expression of proteins related to HSV-1 and PRV replication was similar to that of real-time fluorescence quantitative PCR analysis. It had an obvious inhibitory effect on the expression of proteins related to HSV-1 replication and proteins related to PRV replication, and there were differences in its inhibitory effect on the two. Compared with PRV, its inhibitory effect on the expression of proteins related to HSV-1 replication was better. Although non-selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide had an inhibitory effect on the expression of proteins related to HSV-1 replication and proteins related to PRV replication, its inhibitory effect was worse than that of selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide. Selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide at a concentration of 10-20 μg / ml could achieve the virus inhibitory effect of 50 μg / ml non-selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide, or even better; for HSV-1, the effect of 10 μg / ml selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide was equivalent to or even better than that of 50 μg / ml non-selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide, while 20 μg / ml non-selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide did not show a significant HSV-1 inhibitory effect; for PRV, 20 μg / ml selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide showed a significant inhibitory effect, while 50 μg / ml non-selenium-enriched low-molecular-weight Sargassum fusiforme polysaccharide did not show a significant PRV inhibitory effect; the antiviral activity of Sargassum fusiforme polysaccharide was greatly improved after selenium enrichment.

Claims

1. A method for preparing selenized low molecular weight Sargassum fusiformis polysaccharide, characterized in that: The steps include: taking low molecular weight Sargassum fusiformis polysaccharide, selenium inorganic salt and reducing agent, mixing them evenly, shaking and protecting from light for full reaction, performing selenization modification on the Sargassum fusiformis polysaccharide, and performing dialysis after the reaction is completed to obtain selenized low molecular weight Sargassum fusiformis polysaccharide; The selenium inorganic salt is selected from sodium selenite and sodium thiosulfate; The reducing agent is selected from ascorbic acid and glutathione.

2. The preparation method according to claim 1, characterized in that: The average molecular weight of the low molecular weight Sargassum fusiformis polysaccharide is 10 to 50 kDa; The low molecular weight Sargassum fusiformis polysaccharide is prepared by combining ultraviolet light with hydrogen peroxide.

3. The preparation method according to claim 1, characterized in that: The concentrations of various substances in the mixed solution of low molecular weight Sargassum fusiformis polysaccharide, inorganic selenium salt and reducing agent are: low molecular weight Sargassum fusiformis polysaccharide 0.5-2.5 mg / mL, inorganic selenium salt 10-50 mmol / L, reducing agent 40-80 mmol / L; the light-proof reaction time is 1-6 hours.

4. The preparation method according to claim 3, characterized in that: The concentrations of the various substances in the mixed solution of low molecular weight Sargassum fusiformis polysaccharide, inorganic selenium salt and reducing agent are: low molecular weight Sargassum fusiformis polysaccharide 1.0-2.0 mg / mL or 1.0-1.5 mg / mL, inorganic selenium salt 10-30 mmol / L or 15-25 mmol / L, reducing agent 60-80 mmol / L, and the reaction is carried out in the dark for 3-5 hours.

5. The preparation method according to claim 1, characterized in that: The product was lyophilized after dialysis to obtain selenized low molecular weight Sargassum fusiformis polysaccharide lyophilized powder.

6. A method for preparing selenized low molecular weight Sargassum fusiformis polysaccharide, characterized in that: The preparation method is described in any one of claims 1 to 5.

7. Use of the selenized low molecular weight Sargassum fusiformis polysaccharide according to claim 6 in the preparation of antiviral preparations.

8. The use according to claim 7, characterized in that: The viruses include RNA viruses and DNA viruses. The RNA viruses include vesicular stomatitis virus VSV and influenza virus. The DNA viruses include herpes simplex virus and pseudorabies virus PRV. The influenza virus includes influenza A virus WSN; The herpes simplex virus includes herpes simplex virus type 1 HSV-1.

9. The use according to claim 7, characterized in that: The working concentration of the selenized low molecular weight Sargassum fusiformis polysaccharide is 10-50 μg / ml, preferably 10-20 μg / ml.

10. The use according to claim 9, characterized in that: The virus is HSV-1 or VSV.

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