Preparation method of Sargassum fusiformis segmented polysaccharide and its application in preparation of anti-RSV drugs

Abstract

The present invention relates to the field of medical technology, and provides a method for preparing a segmented polysaccharide of Sargassum fusiformis and an application thereof in preparing an anti-RSV drug. The segmented polysaccharide of Sargassum fusiformis is prepared by the following steps: (1) preparing crude Sargassum fusiformis polysaccharide; (2) preparing an intermediate product; (3) preparing Sargassum fusiformis polysaccharide; (4) dissolving the Sargassum fusiformis polysaccharide in a solvent, passing the supernatant through different molecular retention plates, and then freeze-drying to obtain the segmented polysaccharide of Sargassum fusiformis with a corresponding molecular weight. The segmented polysaccharide of Sargassum fusiformis prepared by the present invention has a significant inhibitory effect on RSV, and its drug inhibition index (TI) can reach more than 4000.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a method for preparing a segmented Sargassum fusiformis polysaccharide and an application thereof in preparing an anti-RSV drug. Background Art

[0002] Sargassum fusiformis, a plant of the genus Sargassum in the family Sargassum of the phylum Phaeophyceae, is also known as hexagonal vegetable, sea barley, jade seaweed, etc. Its algae is yellow-brown in color and is one of the main plant sources of the Chinese medicine "seaweed". my country is rich in seaweed resources, which have high nutritional and medicinal value and large morphological differences. The height of mature Sargassum fusiformis is 30 to 50 cm. The origin of Sargassum fusiformis is mainly distributed in warm temperate waters such as the Bohai Sea (such as Liaoning), the southeast coast (such as Zhejiang and Fujian), and the Yellow Sea (such as Shandong) in China. It is also distributed in Japan, South Korea, North Korea and other countries. Sargassum fusiformis tastes bitter, salty and cold. It has the effects of softening and dispersing lumps, promoting diuresis and reducing swelling, purging heat and reducing phlegm. It is often used in clinical Chinese medicine to treat scrofula, testicular swelling and pain, phlegm and edema and other diseases.

[0003] The main chemical component of Sargassum fusiformis is Sargassum polysaccharide, which accounts for more than 50% of the dry algae. Modern pharmacological experiments have shown that Sargassum polysaccharide has biological activities such as anti-oxidation, anti-aging, anti-fatigue, anti-tumor, anti-virus, antibacterial, hypoglycemic and growth promotion. Sargassum polysaccharide is a mixture of acidic polysaccharides with similar composition structures, and the molecular weight is mostly below 3500kDa. Under current technical conditions, the Sargassum polysaccharides that can be separated mainly include fucoidan sulfate, alginate, brown algae polysaccharide sulfate and brown algae starch.

[0004] Respiratory syncytial virus (RSV) is an RNA virus of the family Paramyxovirus, which was isolated from chimpanzees with rhinitis. It is called RSV because it can cause diseased cells to fuse into multinucleated giant cells. RSV is common in the spring and winter seasons in temperate regions and in the rainy season in tropical regions. Susceptible populations include infants, young children, and the elderly, who have poor resistance. RSV has the characteristics of long outbreak time and wide epidemic area. The main transmission route of RSV is droplet transmission, followed by RSV inoculation into the nasal mucosa, eye mucosa and other parts of the body by contaminated fingers, thereby inducing infection.

[0005] Due to the lack of research on infant immune function, maternal genetic differences in infant immunity, and the existence of uncontrollable factors such as infant and elderly susceptible populations' immune response to RSV, there is currently no vaccine or specific medicine for the treatment of RSV. Western medicine treatments such as bronchodilator salbutamol and anti-inflammatory drugs such as glucocorticoids can be used as auxiliary medications for RSV infection, but in clinical practice, there are adverse reactions such as affecting growth and development and bone metabolism. Traditional Chinese medicine and compound preparations have unique advantages in antiviral treatment due to their low toxicity and side effects. Many experiments have shown that traditional Chinese medicine can downregulate gene expression, reduce the expression of inflammatory factors, and can also exert its anti-RSV effect by regulating the body's immunity.

[0006] The applicant first discovered that Sargassum polysaccharide has a significant inhibitory effect on RSV and EV71 viruses, and applied for patent CN201610763357.4. The Sargassum powder was mixed with distilled water, boiled, and extracted by changing the liquid. After the filtrate after extraction was concentrated, 5% trichloroacetic acid was added, and the protein was removed by centrifugation. The obtained solution was then dialyzed and frozen, and 95% ethanol was added to precipitate the polysaccharide. The precipitate was centrifuged and vacuum dried to obtain Sargassum polysaccharide powder. The Sargassum polysaccharide obtained above was subjected to an inhibition test, and the results showed that its drug inhibition index TI reached 955.43, which had a significant inhibitory effect on the virus. However, in the process of in-depth research on the antiviral mechanism of Sargassum, the applicant found that the mixture Sargassum polysaccharide prepared by the above method had a large molecular weight span, with a minimum molecular weight of less than 1kDa and a maximum molecular weight of several thousand kDa, which directly affected the activity of Sargassum polysaccharide and weakened its inhibitory effect on RSV, which was manifested as insufficient drug inhibition index. Summary of the invention

[0007] Therefore, in response to the above-mentioned defects and shortcomings, the present invention provides a method for preparing segmented polysaccharides of Sargassum fusiformis and its application in the preparation of anti-RSV drugs, which significantly improves the inhibitory effect on RSV, and the TI can reach more than 4000. It has both theoretical and practical significance for promoting the practical application of anti-RSV drugs, and at the same time lays the foundation for the antiviral action mechanism of Sargassum fusiformis.

[0008] To achieve the above object, the present invention is achieved through the following technical solutions:

[0009] The preparation method of Sargassum fusiformis segmented polysaccharide comprises the following steps:

[0010] (1) Mixing Sargassum fusiformis powder and distilled water, stirring and heating, heating to boiling and continuing stirring for 2 to 4 hours, taking the filtrate, concentrating, and freeze-drying to obtain Sargassum fusiformis crude polysaccharide;

[0011] (2) Mixing the crude polysaccharide of Sargassum fusiformis with distilled water, heating to 50-70° C., stirring for 20-60 min and then standing, slowly adding anhydrous ethanol after the solution becomes clear, stirring evenly for 1-3 h, standing overnight, filtering and drying the next day to obtain an intermediate product;

[0012] (3) adding water to the intermediate product and stirring to dissolve it, then adding Sevage reagent, stirring and mixing thoroughly and centrifuging, then filtering to remove the protein at the junction of the water layer and the organic solvent layer, repeating the above operation five times, collecting the organic layer, concentrating, and freeze-drying to obtain Sargassum fusiformis polysaccharide;

[0013] (4) The Sargassum fusiformis polysaccharide is dissolved in a solvent, and the supernatant is passed through different molecular retention plates and then freeze-dried to obtain Sargassum fusiformis segmented polysaccharides of corresponding molecular weights.

[0014] A further improvement is that the material-liquid ratio in step (1) is 1:100-150.

[0015] A further improvement is that the material-liquid ratio in step (2) is 1:15-30.

[0016] A further improvement is that the amount of anhydrous ethanol added in step (2) is 30 to 40 times the mass of the crude polysaccharide of Sargassum fusiformis.

[0017] A further improvement is that the unit size of the molecular retention plate in step (4) is selected as: 1 kDa, 3 kDa, 5 kDa, 10 kDa, 100 kDa.

[0018] The present invention also relates to the use of the Sargassum fusiformis segmented polysaccharide prepared by the above method in the preparation of anti-RSV drugs.

[0019] By adopting the above technical solution, the beneficial effects of the present invention are:

[0020] 1. The present invention prepares segmented polysaccharides of different molecular weights from Sargassum fusiformis through an extraction process, and conducts an anti-toxic test to show that they all have a significant inhibitory effect on RSV, especially the TI of the segmented polysaccharides of Sargassum fusiformis with a molecular weight of more than 100 kDa exceeds 4000, proving the hypothesis of the applicant that the activity of Sargassum fusiformis polysaccharides with a large molecular weight span is affected, weakening its inhibitory effect on RSV. The test results of the present invention provide an experimental basis for treating RSV infectious diseases.

[0021] 2. The extraction method of the present invention is simple, efficient, and practical, and is easy to promote. In particular, the molecular interception technology is used to separate the Sargassum polysaccharide into Sargassum segmented polysaccharides of different molecular weights, and the separation is more efficient and convenient. DETAILED DESCRIPTION

[0022] The following will describe the implementation methods of the present invention in detail in conjunction with specific embodiments, so that the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0023] If not otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents or instruments used without indicating the manufacturer are all conventional reagent products available on the market.

[0024] Example

[0025] The preparation method of Sargassum fusiformis segmented polysaccharide comprises the following steps:

[0026] (1) Mixing Sargassum fusiformis powder and distilled water at a solid-liquid ratio of 1:150, stirring and heating, heating to boiling and continuing stirring for 3 hours, taking the filtrate, concentrating, and freeze-drying to obtain Sargassum fusiformis crude polysaccharide;

[0027] (2) 100 g of Sargassum fusiformis crude polysaccharide was mixed with distilled water at a solid-liquid ratio of 1:20, heated to 60°C, stirred for 30 min and then allowed to stand. After the solution was clarified, 4 L of anhydrous ethanol was slowly added dropwise, stirred evenly for 2 h, allowed to stand overnight, and filtered and dried the next day to obtain an intermediate product.

[0028] (3) Take 10 g of the intermediate product, add water and stir to dissolve, then add Sevage reagent (10 ml of chloroform, 2 ml of n-butanol), stir thoroughly and mix well, then centrifuge, then filter to remove the protein at the junction of the water layer and the organic solvent layer, repeat the above operation five times, collect the organic layer, concentrate, and freeze-dry to obtain Sargassum fusiformis polysaccharide;

[0029] (4) The Sargassum polysaccharide was dissolved in a solvent, and the supernatant was passed through 1 kDa, 3 kDa, 5 kDa, 10 kDa, and 100 kDa molecular retention plates in sequence, and then freeze-dried to obtain Sargassum segmented polysaccharides of corresponding molecular weights, which were named CSF-R1, CSF-R2, CSF-R3, CSF-R4, CSF-R5, and CSF-R6 in sequence. The mass of each Sargassum segmented polysaccharide based on 100 g of Sargassum polysaccharide is shown in Table 1.

[0030] Table 1 Quality of polysaccharides from various Sargassum fusiformis segments

[0031] Molecular weight Below 1 kDa 1-3 kDa 3-5 kDa 5-10 kDa 10-100 kDa 100kDa or more name CSF-R1 CSF-R2 CSF-R3 CSF-R4 CSF-R5 CSF-R6 Mass / g 68.55 1.20 0.15 2.21 0.57 4.16

[0032] Anti-RSV Activity Experiment of Sargassum fusiformis Polysaccharide

[0033] Cell lines and virus strains:

[0034] All were provided by the Microbiology Laboratory of the Institute of Basic Medicine, Shandong Academy of Medical Sciences. Hep-2 (human laryngeal carcinoma epithelial cells) were obtained from the Cell Bank of the Chinese Academy of Sciences; RSV (respiratory syncytial virus, long strain, introduced from the Virus Laboratory of the Institute of Virology, Chinese Academy of Preventive Medicine in October 2000).

[0035] Cytotoxicity assay:

[0036] According to the method in the literature (Wang Xiaoyan, Zhang Meiying, Wang Yafeng, etc. Study on the in vitro anti-coxsackievirus B3, herpes simplex virus type 1 and hepatitis B virus of betel nut extract, Shizhen Chinese Medicine, 2008; 19 (12): 2954-2955), the cells cultured on the 96-well plate grew to a monolayer, and the positive drug and the fragmented polysaccharides of Sargassum fusiformis of various molecular weights, CSF-R1, CSF-R2, CSF-R3, CSF-R4, CSF-R5, CSF-R6, were added, and 12 concentrations were diluted 2-fold from the first well with cell maintenance medium, and inoculated on the 96-well plate, and 3 replicates were set for each dilution. The plates were cultured at 37°C and 5% CO2, and the CPE was observed under a microscope, and the drug toxicity was continuously observed. After MTT staining, the TC values ​​of the positive drug and each fragmented polysaccharide of Sargassum fusiformis were calculated. 50 (50% toxic concentration). TC 50 =[Antilog (log value of drug dilution higher than 50% lesion rate - pd)] × C 首孔药物浓度 Shuanghuanglian and ribavirin were used as positive control drugs (10 mg / mL).

[0037] RSV inhibition experiment

[0038] According to the method in the literature (Wang Xiaoyan, Zhang Meiying, Wang Yafeng, et al. Study on the in vitro anti-Coxsackievirus B3, herpes simplex virus type 1 and hepatitis B virus of betel nut extract, Shizhen Chinese Medicine, 2008; 19 (12): 2954-2955), a certain concentration of Hep-2 cell suspension was spotted on a 96-well plate, and when the cells grew into a monolayer, a 2-fold concentration gradient dilution of positive drugs and Sargassum fusiformis polysaccharides CSF-R1, CSF-R2, CSF-R3, CSF-R4, CSF-R5, CSF-R6 were added with cell maintenance medium starting from the first well, 2-fold dilutions were made into 12 concentrations, and the plates were inoculated on a 96-well plate (50 μL / well), and each dilution was repeated for 3 wells. The plates were cultured at 37°C and 5% CO2, and the cell pathological changes were observed under a microscope until the virus control CPE reached 90% and then the culture was terminated. Cell control and virus control were set, Shuanghuanglian and ribavirin were used as positive control drugs (10 mg / mL). The dilution of CPE50% was regarded as the half effective concentration (EC 50 ).EC 50 =[Antilog(log value of drug dilution higher than 50% survival rate - pd)]×C首孔药物浓度 According to the half-toxic concentration (TC 50 ) and the drug's median effective concentration (EC 50 ), the two were compared to obtain the inhibition index (TI), and those with TI greater than 4 were judged to be effective. The results are shown in Table 2.

[0039] Test results

[0040] 1.TC 50 Determination: Observe the cells of the control group under a microscope to see that they have good morphology and grow densely attached to the wall. The cells of the drug group are broken and show a poisoned morphology. The OD value is measured and the TC of the drug is calculated according to the formula 50 .

[0041] 2. Drug antiviral test: Microscopic observation showed that the cells in the control group had good morphology and were densely attached to the wall, while the RSV virus control group showed more than 90% CPE within 24 hours. The main characteristics of RSV CPE in Hep-2 were wall thickness, rounding, and adhesion fragmentation.

[0042] Table 2 Results of the toxicity inhibition test of Sargassum fusiformis segmented polysaccharides

[0043]

[0044]

[0045] As can be seen from the above table, Shuanghuanglian and ribavirin are significantly weaker than the segmented polysaccharide of Sargassum fusiformis in inhibiting RSV, and the activities of segmented polysaccharides of Sargassum fusiformis with different molecular weights are different, and the difference in the antitoxicity index is very obvious, with the minimum value being only 183.33 and the maximum value being 4364.86. The present invention provides an experimental basis for the clinical use of Sargassum fusiformis polysaccharide in the treatment of viral infectious diseases, provides certain guiding significance for the development of drugs against RSV virus, and has important reference value.

[0046] In the present invention, the material-liquid ratio in step (1) is within the range of 1:100 to 1:150, and the same technical effect can be achieved, and is not limited to the ratio given in the embodiment. Similarly, the material-liquid ratio in step (2) is within the range of 1:15 to 1:30, and the addition amount of anhydrous ethanol is 30 to 40 times the mass of the crude polysaccharide of Sargassum fusiformis.

[0047] The above records are only embodiments of the present invention. Any modification or change made by a person familiar with the present technology using the present invention shall fall within the patent scope claimed by the present invention and shall not be limited to those disclosed in the embodiments.

Claims

1. An application of a segmented polysaccharide of Sargassum fusiformis in the preparation of an anti-RSV drug, characterized in that: The method for preparing the segmented polysaccharide of Sargassum fusiformis comprises the following steps: (1) mixing Sargassum fusiformis powder with distilled water, stirring and heating, heating to boiling and continuing stirring for 2 to 4 hours, taking the filtrate, concentrating, and freeze-drying to obtain Sargassum fusiformis crude polysaccharide; (2) Mixing the crude polysaccharide of Sargassum fusiformis with distilled water, heating to 50-70° C., stirring for 20-60 min and then standing, slowly adding anhydrous ethanol after the solution becomes clear, stirring evenly for 1-3 h, standing overnight, filtering and drying the next day to obtain an intermediate product; (3) Add water to the intermediate product and stir to dissolve it, then add Sevage reagent, stir thoroughly and mix well, then centrifuge. Then, the protein at the interface between the water layer and the organic solvent layer is removed by filtration, and the above operation is repeated five times, and the organic layer is collected, concentrated, and freeze-dried to obtain Sargassum fusiformis polysaccharide; (4) Dissolving the Sargassum fusiformis polysaccharide in a solvent, and passing the supernatant through different molecular retention plates in sequence, wherein the unit size of the molecular retention plates is selected as: 1 kDa, 3 kDa, 5 kDa, 10 kDa, and 100 kDa, and then freeze-drying is performed to collect the Sargassum fusiformis segmented polysaccharides with a molecular weight of more than 100 kDa.

2. The use of a segmented polysaccharide of Sargassum fusiformis according to claim 1 in the preparation of an anti-RSV drug, characterized in that: In step (1), the material-liquid ratio is 1:100-150.

3. The use of a segmented polysaccharide of Sargassum fusiformis according to claim 1 in the preparation of an anti-RSV drug, characterized in that: In step (2), the material-liquid ratio is 1:15-30.

4. The use of a segmented polysaccharide of Sargassum fusiformis in the preparation of an anti-RSV drug according to claim 1, characterized in that: The amount of anhydrous ethanol added in step (2) is 30 to 40 times the mass of the crude polysaccharide of Sargassum fusiformis.

Images