CN106309470B

CN106309470B - Application of sargassum fusiforme polysaccharide

Info

Publication number: CN106309470B
Application number: CN201610763357.4A
Authority: CN
Inventors: 闫滨; 周革非; 邢荣莲; 高敏; 岳路路
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-08-30
Filing date: 2016-08-30
Publication date: 2021-05-18
Anticipated expiration: 2036-08-30
Also published as: CN106309470A

Abstract

The invention discloses application of sargassum fusiforme polysaccharide, wherein the sargassum fusiforme polysaccharide has antiviral activity, and the application of the sargassum fusiforme polysaccharide refers to application of the sargassum fusiforme polysaccharide with the antiviral activity in preparation of medicines for resisting RSV or EV71 viruses. The invention discovers that the sargassum fusiforme polysaccharide has obvious inhibiting effect on RSV and EV71 viruses for the first time, the drug toxicity inhibiting index (TI) of the sargassum fusiforme polysaccharide can reach about 1000, and discloses that the sargassum fusiforme polysaccharide is applied to preparing antiviral drugs and can inhibit RSV and EV71 viruses, so that the sargassum fusiforme polysaccharide can eliminate phlegm and soften hard masses, induce diuresis to alleviate edema, discharge heat and remove dampness, and has potential application value in the field of treating RSV and EV71 virus infectious diseases.

Description

Application of sargassum fusiforme polysaccharide

Technical Field

The invention relates to the technical field of medicines, in particular to application of sargassum fusiforme polysaccharide in preparation of medicines for inhibiting Respiratory Syncytial Virus (RSV) and enterovirus 71 (Human enterovirus 71, EV 71).

Background

The Cyrtymenia Sparsa is dried thallus of Cyrtymenia Sparsa (Sargassaceae, Sargassum fusiforme (Harv.) Setchell) belonging to Sargassaceae (Sargassaceae) genus Sargassum. Cyrtymenia Sparsa is called as "seaweed of small leaf", also called as seaweed, antler tip, sea barley, etc. Mainly produced in the territorial waters of Liaoning, Shandong, Fujian, Zhejiang and Guangdong, etc., and is mostly wild, collected in summer and autumn, and the dried algae are used as the medicine. Listed under the seaweed item of the department of herbage in compendium of materia Medica, records: the salty Sargassum fusiforme can moisten down and the cold can discharge heat and drink water. Therefore, it can eliminate goiter, tumor and tuberculosis, and eliminate edema, beriberi, retained fluid and damp-heat of phlegm qi, so that pathogenic qi can flow out from urine. It is listed as a Chinese herb in Shen nong Ben Cao Jing, which means that it is indicated for "goiter and tumor with accumulation of qi, cervical sclerosing and pain, carbuncle, swelling, abdominal mass and qi, thunder and tinnitus in the upper and lower abdomen, and twelve edema in the lower. This herb is bitter, salty, cold and nontoxic in flavor. It enters liver, stomach and kidney meridians. Has effects in eliminating phlegm, softening hard masses, inducing diuresis to alleviate edema, clearing away heat, and eliminating dampness. And (3) character identification: the whole body is shrunk to form a curled mass, the black brown color is obtained, the surface is frosted, the length is 15-40 cm, and the texture is crisp and fragile. The meat is sticky, smooth and flexible after being soaked, the main shaft is cylindrical, the surface is rough, the main shaft has short branches and branches which are alternate, no thorn-shaped bulges exist, the leaf-shaped bulges are linear, clubbed and slightly flat, and sometimes the front end of the main shaft is expanded and hollow to form bubbles or shield-shaped. A spindle-shaped or spherical air bag is arranged between the armpits, the length of the air bag is 5-10 cm, the air bag is clustered, and the bag handle is longer. The pessary is cylindrical or oblong. Fishy smell, salty taste.

The Sargassum fusiforme polysaccharide (SPF) is a water-soluble polysaccharide extracted from the whole alga Hizikia fusiforme, and the main components of the Sargassum fusiforme alga mainly comprise algin and fucoidan sulfate. Modern pharmacological studies show that the sargassum fusiforme polysaccharide has the effects of resisting tumors, reducing blood sugar and blood fat, enhancing the immunity of the organism, delaying senility and the like. Various pharmacological activities of sargassum fusiforme are closely related to polysaccharide structure thereof. The SPF is an Acidic polysaccharide mixture with similar composition structure, good water solubility and high viscosity, mainly comprises alginic acid (APA), brown sugar (FPS) and brown starch (Laminaran), and the analysis of the content of the polysaccharide in the sargassum fusiforme is an important basis for the research, development and utilization of the efficacy of the sargassum fusiforme.

RSV is the most important viral pathogen for viral lower respiratory tract infections in infants and young children worldwide. In recent years, RSV infection has been found to be an important pathogen in immunosuppressed adults and the elderly. Most RSV infections are upper respiratory tract infections, but many infants can develop pneumonia and lower respiratory tract infections.

EV71 is one of the major pathogens of human hand-foot-and-mouth disease (HFMD), a group of acute viral diseases that have become public health problems worldwide.

Disclosure of Invention

The invention aims to provide application of sargassum fusiforme polysaccharide.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the application of the sargassum fusiforme polysaccharide has antiviral activity, and the application of the sargassum fusiforme polysaccharide in preparing the medicines for resisting RSV or EV71 virus.

Preferably, the preparation method of the sargassum fusiforme polysaccharide is a water extraction and alcohol precipitation method.

More preferably, the water extraction and alcohol precipitation method comprises the specific steps of mixing sargassum fusiforme powder (dried at 45 ℃ for 2 hours) with distilled water, boiling, liquid exchange and extraction, concentrating the extracted filtrate, adding 5% trichloroacetic acid, centrifuging to remove protein, dialyzing the obtained solution, freezing, adding 95% ethanol to precipitate polysaccharide, centrifuging to obtain precipitate, and vacuum drying to obtain sargassum fusiforme polysaccharide powder.

More preferably, the mass ratio of the sargassum fusiforme powder to the distilled water is 1: 30.

More preferably, the number of times of liquid exchange extraction is 3.

More preferably, the centrifugation speed is 5000r/min, and the centrifugation time is 20-30 min.

More preferably, the dialysis time is 2.5 to 3.5 days.

The invention has the beneficial effects that:

the invention discovers that the sargassum fusiforme polysaccharide has obvious inhibiting effect on RSV and EV71 viruses for the first time, the drug toxicity inhibiting index (TI) of the sargassum fusiforme polysaccharide can reach about 1000, and discloses that the sargassum fusiforme polysaccharide is applied to preparing antiviral drugs and can inhibit RSV and EV71 viruses, so that the sargassum fusiforme polysaccharide can eliminate phlegm and soften hard masses, induce diuresis to alleviate edema, discharge heat and remove dampness, and has potential application value in the field of treating RSV and EV71 virus infectious diseases.

Drawings

Figure 1 is a microscope picture of RSV virus control: MA104 cells showed complete CPE schematic (200 ×);

fig. 2 is an EV71 virus control microscope picture: full CPE appearance in RD cells; (200 ×);

fig. 3 is a microscope picture (200 ×) of MA104 cells under protection;

fig. 4 is a microscope picture (200 ×) of protected RD cells.

Detailed Description

The invention will be further explained with reference to the drawings.

The effect of crude polysaccharide of sargassum fusiforme on virus is studied:

(1) preparing crude extract of sargassum fusiforme polysaccharide:

taking sargassum fusiforme powder: distilled water 1:30 g/g, boiling in water for 4.5-6h, and extracting for 3 times in the middle of liquid change. Collecting filtrate, concentrating by rotary evaporation to 1/4, adding 5% trichloroacetic acid to adjust pH to 5.0-6.5, centrifuging at 5000r/min for 20min to remove protein in crude polysaccharide solution, dialyzing the obtained solution in a dialysis bag (about 3 d), freezing the solution overnight, adding 4 times volume of 95% ethanol to precipitate polysaccharide, centrifuging to obtain precipitate, and vacuum drying to obtain crude polysaccharide powder. The above extracts were dissolved in cell maintenance solution (1640 containing 2% newborn bovine serum, product of GIBICO) respectively for use.

(2) Cell and virus strains:

are provided by the institute for basic medicine of Shandong provincial academy of medical sciences: MA104 (rhesus monkey embryonic kidney cells, RSV sensitive cells as shown in fig. 3), RD (rhabdomyosarcoma cells, EV71 sensitive cells as shown in fig. 4); EV71 (isolated from infectious disease Hospital hand-foot-and-mouth disease patients in Jinan City, and confirmed to be EV71 by full sequence determination), RSV (Long strain, introduced in viral institute of preventive medicine and sciences in China in 10 months of 2000).

(3) Cytotoxicity assay:

referring to the literature of Wang Xiaoyan, Zhang Meiying, Wang Yafeng and the like, the research of in vitro anti-coxsackie virus B3, herpes simplex virus type 1 and hepatitis B virus of betel nut extract, Shizhen Chinese medicine, 2008; 19(12): 2954-2955, spotting cells on a 96-well plate to form a monolayer, diluting a drug-containing cell maintenance solution 2-fold from the 1 st well to 12 concentrations, plating the diluted solution on the 96-well plate, repeating the steps for 3 wells at each dilution, and incubating the diluted solution at 37 ℃ with 5% CO₂Culturing under the condition, observing CPE under a microscope, and continuously observing drug toxicity. CPE greater than 50% was considered toxic. Then staining with neutral red, setting the measurement wavelength to 540nm, measuring OD value, the cell survival rate is the result of comparing the OD value of the experimental group and the OD value of the cell control group, and calculating by Reed-Muench method to obtain the half poisoning concentration (50% of toxin concentration, TC)₅₀)。

TC₅₀Either [ Antilog (log higher than 50% of the value of the lesion rate drug dilution-pd)]×C_{First hole drug concentration}。

(4) And (3) toxicity inhibition test:

referring to the documents of Wang Xiaoyan, Zhang Meiying, Wang Yafeng, etc., the extract of Arecae semen can resist Coxsackie virus B3, herpes simplex virus 1 and hepatitis B virus in vitro2008, Shizhen national medicine; 19(12): 2954 to 2955, cells spotted on a 96-well plate are grown as a monolayer, and a drug-containing cell maintenance solution is diluted 2-fold from the 1 st well to 12 concentrations, and the diluted solution is seeded on the 96-well plate (50. mu.L/well), and 3 wells are repeated for each dilution, and a cell control and a virus control are provided. In addition to the cell controls, 50. mu.L of virus at 100TCID50 was added to each well. 35 ℃ and 5% CO₂Incubations were performed under conditions, and the incubation was terminated after microscopic observation for cytopathic effects continued until virus control CPE reached 90%. The absorbance A was measured by a microplate reader, and the dilution of CPE 50% was regarded as the half Effective Concentration (EC) of the drug₅₀)。

EC₅₀Either [ Antilog (log higher than 50% viability drug dilution-pd)]×C_{First hole drug concentration}. According to the half Toxic Concentration (TC) of the tested medicine₅₀) And half Effective Concentration (EC) of the drug₅₀) The two are compared to obtain a toxicity inhibiting index (TI), and if the TI is more than 4, the medicine is judged to be effective.

(5) As a result:

firstly, the concentration TC of the drug in the median poisoning₅₀The determination of (1): the cells of the control group are observed under a microscope to have good shapes and compact adherent growth. Breaking the medicinal cells, displaying toxic form, measuring OD value, and calculating TC of the medicine according to formula₅₀. TC of sargassum fusiforme polysaccharide₅₀At MA104 of 2^-1.3(ii) a At RD of 2^-1.6。

② the drug antiviral test: observation under a microscope shows that the cells of the control group have good morphology and compact adherent; the RSV and EV71 virus control groups both have more than 90% of CPE in about 24h, and the RSV has the main characteristics of necrosis and breakage in MA104 (shown in figure 1); EV71 is characterized primarily by increased refractive index, rounding off, and sloughing of CPE in RD (as shown in fig. 2).

The groups of cells in the antiviral test are subjected to a drug dilution gradient (one time each dilution, 12 times in total), and CPE appears according to the gradient rule:

rsv test groups: RSV-MA104 cells were completely protected before dilution 5, followed by CPE, and CPE appeared to be progressively more pronounced as drug concentration decreased.

Ev71 test group: EV71-RD cells were completely protected before dilution 9, followed by CPE, and CPE appeared to be progressively more pronounced as drug concentration decreased.

Staining a 96-well plate to be tested with 1% neutral red, measuring absorbance A at 540nm with an enzyme-labeling instrument, subtracting A value of a virus control group from A value of each group, and comparing A value of each experimental group with A value of a cell control group to obtain EC₅₀，EC₅₀And TC₅₀In comparison to TI, as shown in table 1:

rsv test groups: inhibiting RSV from CPE, EC in MA104 cells₅₀Is 2^-11.2μ g/mL, TI 955.43.

Ev71 test group: inhibition of EV71 development of CPE, EC in RD cells₅₀Is 2^-11.8μ g/mL, TI 1176.27.

For ease of observation and comparison, the details are given in Table 1.

TABLE 1 Hizikia fusiforme polysaccharide toxicity inhibition test results

From the table above, the crude extract of sargassum fusiforme polysaccharide has obvious inhibition effect on both RSV and EV71 viruses, which indicates that sargassum fusiforme polysaccharide not only can eliminate phlegm and soften hard masses, induce diuresis and reduce edema, and discharge heat and remove dampness, but also has potential application value in the field of treating RSV and EV71 virus infectious diseases. The invention provides experimental basis for clinical application of sargassum fusiforme polysaccharide in treating virus infectious diseases, provides certain guiding significance for developing anti-RSV and EV71 virus medicines, and has important reference value.

Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive faculty, based on the technical solutions of the present invention.

Claims

1. The application of the sargassum fusiforme polysaccharide is characterized in that the sargassum fusiforme polysaccharide has antiviral activity, and the application of the sargassum fusiforme polysaccharide with the antiviral activity refers to the application of the sargassum fusiforme polysaccharide in preparing an anti-EV 71 virus medicament;

the preparation method of sargassum fusiforme polysaccharide comprises water extraction and alcohol precipitation;

the water extraction and alcohol precipitation method comprises the specific steps of mixing sargassum fusiforme powder with distilled water, boiling with water, changing the solution for extraction, concentrating the extracted filtrate, adding 5% trichloroacetic acid, centrifuging to remove protein, dialyzing the obtained solution, freezing, adding 95% ethanol to precipitate polysaccharide, centrifuging to obtain precipitate, and drying in vacuum to obtain sargassum fusiforme polysaccharide powder;

wherein the mass ratio of the sargassum fusiforme powder to the distilled water is 1: 30; the liquid changing and extracting times are 3; the centrifugation speed is 5000r/min, and the centrifugation time is 20-30 min; the dialysis time is 2.5-3.5 days.