CN116444689A - A kind of anti-RSV hijiki polysaccharide and its preparation method - Google Patents

Abstract

The invention relates to a preparation method of anti-RSV sargassum fusiforme polysaccharide, which comprises the following steps: (1) preparation of sargassum fusiforme crude polysaccharide; (2) water-soluble alcohol precipitation of sargassum fusiforme crude polysaccharide; (3) deproteinizing the sargassum fusiforme crude polysaccharide by a Sevage method; (4) medium pressure preparative liquid chromatography purification; (5) Sephadex G-200 column chromatography. The preparation method disclosed by the invention is favorable for obtaining high-purity polysaccharide, better plays the role of resisting RSV (respiratory syncytial virus) of the sargassum fusiforme polysaccharide, provides experimental basis for clinical application of the sargassum fusiforme polysaccharide in treating viral infectious diseases, provides a certain guiding significance for developing medicaments for resisting RSV viruses, and has important reference value.

Description

A kind of anti-RSV hijiki polysaccharide and its preparation method

technical field

The invention relates to the technical field of sugar extraction, in particular to an anti-RSV hijiki polysaccharide and a preparation method thereof.

Background technique

Hijiki, a plant of the genus Sargassum in the family Phaeaceae Sargassum, is also known as hexagonal, sea barley, jade seaweed, etc. Its algae are yellowish brown, and it is one of the main plant sources of traditional Chinese medicine "seaweed". my country is rich in seaweed resources, with high nutritional and medicinal value and large morphological differences. The plant height of mature hijiki is 30-50cm. The production areas of hijiki are mainly distributed in warm temperate waters such as the Bohai Sea (such as Liaoning), the southeast coast (such as Zhejiang, Fujian), and the Yellow Sea (such as Shandong), and there are also certain distributions in Japan, South Korea, North Korea and other countries. Hijiki tastes bitter, salty, and cold. It has the effects of softening and resolving stagnation, diuresis and swelling, purging heat and resolving phlegm. It is often used in traditional Chinese medicine to treat diseases such as scrofula, testicular swelling and pain, and phlegm retention edema.

The main chemical component of hijiki algae is hijiki polysaccharide, which accounts for more than 50% of the dry algae. Modern pharmacological experiments show that hijiki polysaccharide has anti-oxidation, anti-aging, anti-fatigue, anti-tumor, anti-virus, Antibacterial, hypoglycemic and growth-promoting biological activities. Hijiki polysaccharides are a kind of acidic polysaccharide mixture with similar composition and structure, and the molecular weight is mostly below 3500kDa. The hijiki polysaccharides that can be separated under the current technical conditions mainly include fucoidan sulfate, algin, and fucoidan sulfate and alginate etc. Respiratory syncytial virus (respiratory syncytial virus, RSV) is a paramyxoid RNA virus belonging to the pneumovirus. It is isolated from a chimpanzee with a nasal cold. It is called RSV because it can fuse diseased cells into multinucleated giant cells. . RSV is common in spring and winter in temperate regions and rainy season in tropical regions. Susceptible groups include infants and the elderly and other groups with poor resistance. RSV has the characteristics of long outbreak time and wide epidemic area.

Due to the lack of research on the immune function of infants, the differences in infant immunity caused by maternal inheritance, and the existence of uncontrollable factors such as the immune response of susceptible groups such as infants and the elderly to RSV, there is currently no vaccine or specific drug for the treatment of RSV. Western medicine, such as bronchodilator albuterol and anti-inflammatory drug glucocorticoid, can be used as adjuvant drugs for RSV infection, but clinically there are adverse reactions such as affecting growth and development and bone metabolism. Traditional Chinese medicine and compound preparations have unique advantages in anti-virus because of their less toxic and side effects. Many experiments have shown that traditional Chinese medicine can down-regulate gene expression, reduce the expression of inflammatory factors, and can also exert its anti-RSV effect by regulating the body's immunity.

The applicant discovered for the first time that hijiki polysaccharides had a significant inhibitory effect on both RSV and EV71 viruses, and applied for a patent CN201610763357.4, mixing hijiki powder with distilled water, boiling, changing the liquid, and extracting the extracted filtrate After concentrating, add 5% trichloroacetic acid, centrifuge to remove protein, then dialyze the obtained solution and freeze, add 95% ethanol to precipitate polysaccharide, centrifuge to collect precipitate, vacuum dry to obtain Hijiki polysaccharide powder.

Research on the separation and purification of hijiki polysaccharides will help to obtain high-purity polysaccharides and better exert the anti-RSV effect of hijiki polysaccharides. The effect of its separation and purification directly affects its activity and the analysis of the structure of the compound, and it also determines whether large-scale industrial production can be realized in the future. Over the years, the extraction methods of hijiki polysaccharides include ultrasonic method, microwave method, water immersion Extraction, enzymatic hydrolysis, etc., the obtained crude polysaccharide contains a large amount of impurities such as protein, which will affect the purity of the polysaccharide.

Contents of the invention

Therefore, in order to overcome the defects and deficiencies indicated above, the present invention provides an anti-RSV hijiki polysaccharide and a preparation method thereof.

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

An anti-RSV hijiki polysaccharide, including the following monosaccharide components: guluronic acid, mannuronic acid, glucosamine, mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, Fucose, the molar ratio of each monosaccharide component is 1.29:8.24:0.52:0.94:0.28:1.39:0.42:1.61:0.69:4.26.

After testing, the inhibitory index of the anti-RSV hijiki polysaccharide is 420.41.

The present invention also provides a preparation method of the anti-RSV hijiki polysaccharide, comprising the following steps:

(1) Mix the hijiki powder with water, start stirring and raise the temperature, heat to boil and continue stirring for 2-4 hours, take the filtrate, concentrate and freeze-dry to obtain the hijiki crude polysaccharide;

(2) Mix the hijiki crude polysaccharide with water, stir for 20-60 minutes and let it stand still. After the solution is clarified, slowly add absolute ethanol dropwise, stir evenly for 1-3 hours, let it stand overnight, and filter and bake the next day Dry, repeat the above operation four times to obtain the intermediate product I, and control the temperature during the stirring process at 50-70°C;

(3) Add water to the intermediate product I and stir to dissolve, then add Sevage reagent, stir well and 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, The intermediate product II was obtained after freeze-drying;

(4) Dissolve the intermediate product II, add it to the Flash column of the medium-pressure preparative liquid chromatograph, elute it with 2.5mol/L sodium chloride solution, control the flow rate to 6mL/min, and detect it with a UV spectrophotometer , collect fractions at a wavelength of 216nm, dialyze with a 1kDa regenerated cellulose dialysis bag, concentrate and freeze-dry to obtain intermediate product III;

(5) The intermediate product III obtained in step (4) was dissolved in water, separated by Sephadex G-200 chromatography, eluted with pure water, the flow rate was controlled at 0.5mL/min, and sulfuric acid-phenol method was used After tracking and detection, when the sugar-free fraction flows out, the fractions are collected, concentrated, and dried under reduced pressure to obtain the anti-RSV hijiki polysaccharide.

A further improvement is: the ratio of material to liquid in step (1) is 1:100-150.

A further improvement is: the ratio of material to liquid in step (2) is 1:15-30.

A further improvement is: the amount of absolute ethanol added in the step (2) is 30 to 40 times the mass of the hijiki crude polysaccharide.

A further improvement is: the packing of the medium-pressure preparative liquid chromatograph in step (4) is selected as: DE-52.

By adopting the aforementioned technical scheme, the beneficial effects of the present invention are:

The preparation method of the anti-RSV hijiki polysaccharide provided by the invention is simple, high in efficiency, strong in practicability, easy to popularize, and capable of extracting active polysaccharides of different molecular segments. The crude polysaccharides of hijiki were scanned in the wavelength range of 200-700nm by using ultraviolet spectroscopy. to scan. In the purification of DE-52 column chromatography, it was found that choosing 2.5mol/L sodium chloride solution as the mobile phase could provide an experimental basis for the separation and purification of crude polysaccharides from Sargassum hijiki, and to obtain high-purity polysaccharides. Finally, Sephadex G-200 column chromatography is used for separation to obtain hijiki polysaccharides.

Structural analysis of hijiki polysaccharides can lay a foundation for the study of its anti-RSV structure-activity relationship, and can also promote the development of anti-RSV drugs and polysaccharide drugs. However, the separation and purification of crude polysaccharides from Hijiki affect the analysis of the anti-RSV activity and structure of polysaccharides from Hijiki.

Description of drawings

Fig. 1 is the sulfuric acid-phenol method online detection spectrogram in the embodiment of the present invention;

The meanings of the marks in Figure 1 are as follows: a, SF-R1, b, SF-R2, c, SF-R3, d, SF-R4, e, SF-R5, f, SF-R6;

Figure 2 is a high performance liquid chromatogram of SF-R1 monosaccharide derivatization;

The meaning of each mark in Fig. 2 is as follows: 1, guluronic acid, 2, mannuronic acid, 3, glucosamine, 4, mannose, 5, rhamnose, 6, glucuronic acid, 7, glucose , 8, galactose, 9, xylose, 10, fucose;

Fig. 3 is the molecular weight distribution spectrogram of SF-R1;

Figure 4 is a field emission scanning electron microscope image of SF-R1;

Fig. 5 is an atomic force microscope image of SF-R1.

Detailed ways

The implementation of the present invention will be described in detail below in conjunction with specific examples, so as to fully understand and implement the process of how to apply technical means to solve technical problems and achieve technical effects in the present invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents and products used are also commercially available. The sources, trade names and, where necessary, listing of constituents of reagents used are indicated the first time they appear.

Example

A preparation method of anti-RSV hijiki polysaccharide, comprising the following steps:

(1) Mix the hijiki powder with distilled water, the ratio of solid to liquid is 1:150, start stirring and raise the temperature, heat to boil and continue to stir for 3 hours, take the filtrate, concentrate and freeze-dry to obtain the hijiki crude polysaccharide;

(2) Mix 100g of hijiki crude polysaccharide with distilled water, the ratio of solid to liquid is 1:20, stir for 30min and then let stand, after the solution is clarified, slowly add 4L of absolute ethanol dropwise, stir evenly for 2h, let stand overnight, Filtration and drying were carried out every day, and the above operation was repeated four times to obtain the intermediate product I, and the temperature during the stirring process was controlled at 60°C;

(3) Take 10 g of the intermediate product I, add water and stir to dissolve, then add Sevage reagent (10 ml of chloroform, 2 ml of n-butanol), stir well and then centrifuge, then filter to remove the protein at the junction of the water layer and the organic solvent layer, and repeat the above operation Five times, the organic layer was collected, concentrated and freeze-dried to obtain the intermediate product II;

(4) Dissolve the intermediate product II, add it to the Flash column of the medium-pressure preparative liquid chromatograph, elute it with 2.5mol/L sodium chloride solution, control the flow rate to 6mL/min, and detect it with a UV spectrophotometer , collect fractions at a wavelength of 216nm, dialyze with a 1kDa regenerated cellulose dialysis bag, concentrate and freeze-dry to obtain intermediate product III;

(5) The intermediate product III obtained in step (4) was dissolved in water, separated by Sephadex G-200 chromatography, eluted with pure water, the flow rate was controlled at 0.5mL/min, and sulfuric acid-phenol method was used After tracking and detection, when the sugar-free fraction flows out, the fractions are collected, concentrated, and dried under reduced pressure to obtain six segments of hijiki polysaccharides. Referring to Figure 1, they are named SF-R1, SF-R2, SF-R3, SF-R4, SF-R5 and SF-R6 in sequence.

In the present invention, the same technical effect can be achieved if the material-to-liquid ratio in step (1) is within the range of 1:100-1:150, and is not limited to the ratio given in the examples. Similarly, the solid-liquid ratio in step (2) is within the range of 1:15-1:30, and the addition of absolute ethanol is 30-40 times the mass of the hijiki crude polysaccharide.

Cell lines and virus strains:

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

Cytotoxicity Assay:

Refer to the method of the literature (Wang Xiaoyan, Zhang Meiying, Wang Yafeng, etc. Research on the in vitro anti-coxsackie virus B3, herpes simplex virus type 1 and hepatitis B virus of betel nut extract, Shi Zhen Guo Yi Guo Yao, 2008; 19 (12): 2954～ 2955), the cells to be cultured on a 96-well plate grow to a monolayer, add positive drugs and hijiki polysaccharides SF-R1, SF-R2, SF-R3, SF-R4, SF-R5, SF -R6, use the cell maintenance solution to dilute 12 times from the first well, inoculate on a 96-well plate, set up 3 duplicate wells for each dilution, culture at 37°C, 5% CO ₂ , under a microscope Observe their CPE and continue to observe drug toxicity. After MTT staining, calculate the TC ₅₀ (half toxic concentration) of the positive drug and each segmented polysaccharide of Hijiki. TC ₅₀ =[Antilog (log value of drug dilution above 50% lesion rate-pd)]×C _{first well drug concentration} . Shuanghuanglian and ribavirin were used as positive control drugs (10mg/mL).

Inhibition of RSV experiments

Spot a certain concentration of Hep-2 cell suspension on a 96-well plate, and when the cells grow into a single layer, add 2-fold concentration gradient dilution of the positive drug and hijiki polysaccharide SF-R1, and use the cell maintenance solution from the first At the beginning of the well, dilute 12 concentrations by 2 times, inoculate on a 96-well plate (50 μL/well), repeat 3 wells for each dilution, culture at 37 ° C, 5% CO ₂ conditions, and continue to observe the cytopathic changes under the microscope until The culture was terminated when the virus control CPE reached 90%. Set cell control, virus control, Shuanghuanglian, ribavirin as positive control drugs (10mg/mL). The 50% dilution of CPE was regarded as the half effective concentration (EC ₅₀ ) of the drug. EC ₅₀ =[Antilog (log value of drug dilution above 50% survival rate-pd)]×C _{first well drug concentration} . According to the measured drug half toxic concentration (TC ₅₀ ) and drug half effective concentration (EC ₅₀ ), the inhibitory index (TI) was obtained by comparing the two. Those with TI greater than 4 were considered effective, and the results are shown in the table below.

sample TC ₅₀ (g/ml) _EC50 (g/ml) T.I. Ribavirin 8.25×10 ^-3 2.00×10 ^-3 4.13 Shuanghuanglian 2.12×10 ^-2 4.50×10 ^-3 4.71 SF-R1 4.12×10 ^-2 9.80×10 ^-5 420.41 SF-R2 2.80×10 ^-2 2.82×10 ^-4 99.29 SF-R3 2.90×10 ^-2 3.82×10 ^-4 75.92 SF-R4 1.14×10 ^-2 2.41×10 ^-4 47.30 SF-R5 2.23×10 ^-2 3.60×10 ^-4 61.94 SF-R6 3.69×10 ^-2 3.32×10 ^-4 111.14

It can be seen from the above table that the effect of Shuanghuanglian and ribavirin on inhibiting RSV is significantly weaker than that of hijiki polysaccharide, and the molecular weight of hijiki polysaccharide is related to its antiviral effect. The TI value of SF-R1 is 420.41, and it has high anti-RSV activity. This application named it as anti-RSV hijiki polysaccharide, and studied its structure.

SF-R1 Monosaccharide Composition Determination

SF-R1 hydrolysis and derivatization (PMP derivatization high performance liquid chromatography)

Add 10 mg of SF-R1 to a centrifuge tube, add 1 mL of 4 mol/L trifluoroacetic acid solution, hydrolyze at 110 °C for 2 h under nitrogen protection, then cool to room temperature, add 1 mL of 4 mol/L NaOH solution for neutralization, Adjust the pH to neutral, and dilute to 2.5mL.

Add 200 μL of the above hydrolyzate to a test tube, add 200 μL of 0.3 mol/L NaOH solution and 200 μL of 0.5 mol/L 1-phenyl-3-methyl-5-pyrazolone (PMP) solution and mix well Finally, react at 70°C for 2 hours. After the reaction, add 200 μL of potassium dihydrogen phosphate buffer solution with a concentration of 0.1 mol/L (add ammonia water to adjust the pH to 6), add 2 mL of chloroform for extraction, collect the water layer, and extract three times. The water layer was passed through a 0.45 μm filter membrane and then put on the machine.

Standard solution preparation (PMP derivatized high performance liquid chromatography)

Take D-galactose, D-mannose, D-arabinose, D-glucuronic acid, L-rhamnose, D-galacturonic acid, L-fucose, D-glucose, D-glucosamine , D-xylose, D-mannuronic acid, L-guluronic acid 10mg each, add water to dissolve and dilute to 10mL. Add 200 μL to the test tube for derivatization.

HPLC conditions

Chromatographic column: ZORBAX Eclipse XDB-C18, 4.6×250mm, 5μm;

Detector: UV detector;

Injection volume: 20μL, flow rate: 1mL/min;

Mobile phase: A phase: 15% acetonitrile+85% concentration is 50mmol/L phosphate buffered saline solution (adding NaOH to adjust pH value is 6), B phase: 40% acetonitrile+60% concentration is 50mmol/L phosphate buffered saline solution ( Add NaOH to adjust the pH to 6).

Gradient elution program:

time/min Phase A/% Phase B/% 0 100 0 9 92 8 35 80 20 45 75 25 55 100 0

The high performance liquid chromatography of SF-R1 monosaccharide derivatization is shown in Figure 2. After hydrolysis and PMP derivatization, the monosaccharide species of SF-R1 can be measured as guluronic acid, mannuronic acid, glucosamine, mannose, rhamnose, glucuronic acid, glucose, galactose, xylose , Fucose, the molar ratio of its monosaccharide composition is: 1.29:8.24:0.52:0.94:0.28:1.39:0.42:1.61:0.69:4.26.

SF-R1 molecular weight distribution determination

Weigh 1 mg of each segment (Mw1000-50000) reference substance and sample, add mobile phase for ultrasonic dissolution, high-speed centrifugation and test on the machine.

The chromatographic conditions are:

Mobile phase: 0.1mol/L sodium nitrate solution;

Detector: differential refractive index detector;

Chromatographic column: TSKgel G4000PWXL, 7.8mm×30cm;

Flow rate: 0.5mL/min, column oven: 40°C;

The molecular weight distribution of SF-R1 is shown in the table below.

Field Emission Scanning Electron Microscope Observation of SF-R1

Dried the dried polysaccharide SF-R1, dispersed it on the conductive adhesive, pasted it on the sample plate, sprayed gold, and put the sample plate into the field emission scanning electron microscope. Observe under magnification to 1800 times.

It can be seen from Figure 4 that the polysaccharide SF-R1 has an obvious branched skeleton structure, and the end of the skeleton has a clear section.

Atomic force microscope observation of SF-R1

It can be seen from Figure 5 that the polysaccharides are in the form of large particles, ranging in size from tens of nanometers. After analysis, the roughness of polysaccharide SF-R1 is 0.6288nm (root mean square value), and the maximum height is 6nm.

According to the observation and analysis of field emission scanning electron microscope and atomic force microscope, the polysaccharide SF-R1 has a certain three-dimensional structure, and there is a three-dimensional space inside it, and the three-dimensional space can reach 1-2 μm. The anti-RSV activity of polysaccharide SF-R1 may be directly related to its spatial structure, and even the internal three-dimensional space formed by it can accommodate multiple RSVs, which is enough to adsorb a large number of RSVs, thereby preventing RSVs from infecting host cells.

The above records are only examples of using the technical content of this creation. Any modifications and changes made by those who are familiar with this technology using this creation belong to the scope of patents claimed by this creation, and are not limited to those disclosed in the examples.

Claims (7)

1. An anti-RSV sargassum fusiforme polysaccharide, characterized in that: comprises the following monosaccharide components: guluronic acid, mannuronic acid, glucosamine, mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, fucose, the molar ratio of the monosaccharide components being 1.29:8.24:0.52:0.94:0.28:1.39:0.42:1.61:0.69:4.26.

2. the anti-RSV sargassum fusiforme polysaccharide of claim 1, wherein: the anti-RSV sargassum fusiforme polysaccharide has a toxicity inhibition index of 420.41.

3. The method for preparing the anti-RSV sargassum fusiforme polysaccharide according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:

(1) Mixing the sargassum fusiforme powder with water, starting stirring, heating to boil, continuing stirring for 2-4 h, concentrating the filtrate, and freeze-drying to obtain sargassum fusiforme crude polysaccharide;

(2) Mixing the sargassum fusiforme crude polysaccharide with water, stirring for 20-60 min, standing, slowly dripping absolute ethyl alcohol after the solution is clarified, uniformly stirring for 1-3 h, standing overnight, filtering and drying the next day, repeating the above operation four times to obtain an intermediate product I, and controlling the temperature of the stirring process at 50-70 ℃;

(3) Adding water into the intermediate product I, stirring for dissolving, adding a Sevage reagent, stirring and mixing thoroughly, centrifuging, filtering to remove proteins at the junction of a water layer and an organic solvent layer, repeating the above operation for five times, collecting an organic layer, concentrating, and freeze-drying to obtain an intermediate product II;

(4) Dissolving the intermediate product II, adding the intermediate product II into a Flash column of a medium-pressure preparation liquid chromatograph, eluting with 2.5mol/L sodium chloride solution, controlling the flow rate to be 6mL/min, detecting by an ultraviolet spectrophotometer, collecting fractions with the wavelength of 216nm, dialyzing by using a 1kDa regenerated cellulose dialysis bag, concentrating, and freeze-drying to obtain an intermediate product III;

(5) Dissolving the intermediate product III obtained in the step (4) in water, separating by using sephadex G-200 chromatography, eluting by using pure water, controlling the flow rate to be 0.5mL/min, adopting a sulfuric acid-phenol method for tracking detection, collecting fractions when no sugar component flows out, concentrating, and drying under reduced pressure to obtain the RSV-resistant sargassum fusiforme polysaccharide.

4. The method for preparing the anti-RSV sargassum fusiforme polysaccharide according to claim 3, wherein the method comprises the following steps: in the step (1), the feed liquid ratio is 1: 100-150.

5. The method for preparing the anti-RSV sargassum fusiforme polysaccharide according to claim 3, wherein the method comprises the following steps: in the step (2), the feed liquid ratio is 1: 15-30.

6. The method for preparing the anti-RSV sargassum fusiforme polysaccharide according to claim 3, wherein the method comprises the following steps: the addition amount of the absolute ethyl alcohol in the step (2) is 30-40 times of the mass of the sargassum fusiforme crude polysaccharide.

7. The method for preparing the anti-RSV sargassum fusiforme polysaccharide according to claim 3, wherein the method comprises the following steps:

the packing materials of the medium-pressure preparation liquid chromatograph in the step (4) are selected as follows: DE-52.