CN111568917B - Application of flammulina velutipes polysaccharide extract in preparation of antiviral drugs and extraction method thereof - Google Patents
Application of flammulina velutipes polysaccharide extract in preparation of antiviral drugs and extraction method thereof Download PDFInfo
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Abstract
The invention relates to medical application of a polysaccharide extract, in particular to application of a needle mushroom polysaccharide extract in preparation of an anti-influenza virus medicament and an extraction method thereof. The invention provides an application of a flammulina velutipes polysaccharide extract in preparation of antiviral drugs, foods or health-care products. The invention firstly proposes that the flammulina velutipes polysaccharide extract has a definite anti-influenza virus effect, and the in vitro anti-influenza virus pharmacodynamic study is carried out on the extracted flammulina velutipes polysaccharide extract, and the result shows that the flammulina velutipes polysaccharide extract can obviously inhibit cytopathy caused by influenza virus infection; animal experiments (experiments of H1N1 virus infected mice) are carried out on the extracted needle mushroom polysaccharide extract, and the results prove that the needle mushroom polysaccharide extract can effectively relieve respiratory symptoms caused by H1N1 virus infection, and the application of the needle mushroom polysaccharide extract in the preparation of anti-influenza virus medicaments has good prospects.
Description
Technical Field
The invention relates to medical application of a polysaccharide extract, in particular to application of a needle mushroom polysaccharide extract in preparation of an anti-influenza virus medicament and an extraction method thereof.
Background
Influenza is an acute respiratory disease caused by Influenza Virus (Influenza Virus), has the characteristics of strong infectivity, rapid spread, wide epidemic range and the like, seriously threatens the life and health of human beings and poses great economic threat. The human influenza virus is one of influenza viruses and is divided into three types, namely type A (A), type B (B) and type C (C), wherein the influenza A virus has the greatest threat to human beings due to the most variable antigenicity. Influenza a H1N1 became pandemic in 2009 worldwide highlighting the severity of influenza a virus infection.
Vaccination is the most effective method for preventing and controlling influenza transmission, but major antigens of influenza virus, Hemagglutinin (HA) and Neuraminidase (NA), are susceptible to antigenic shift and antigenic drift, resulting in lack of immunity of human beings against newly emerged strains, and further causing outbreak of influenza virus. Therefore, there is a certain lag in preventing influenza by vaccination, and the preventive effect is limited. While the drugs used to treat influenza viruses are mainly amantadine (e.g., amantadine and rimantadine) and neuraminidase inhibitors (e.g., oseltamivir and zanamivir), such drugs have limited clinical use due to the emergence of drug resistance and their inherent toxic side effects.
Therefore, the extraction of the active ingredients of the anti-influenza virus from natural products is of great significance for the development of new anti-influenza virus medicines.
Disclosure of Invention
The invention aims to provide a flammulina velutipes polysaccharide extract capable of resisting influenza viruses, particularly influenza A H1N1, and an extraction method and application thereof.
In a first aspect, the invention provides the use of a flammulina velutipes polysaccharide extract in the preparation of an antiviral medicament, a food, or a health product.
Preferably, the concentration of the flammulina velutipes polysaccharide extract is between 200 and 2000 mu g/mL. The flammulina velutipes polysaccharide extract within this concentration range has a significant antiviral effect with very minimal toxic effects.
Preferably, the virus is an influenza virus.
Further preferably, the virus is an influenza a virus, for example including but not limited to influenza a H1N1 virus.
Preferably, the food product comprises a food product for special medical use, or a functional food product.
The Flammulina velutipes (Flammulina velutipes) is also called as a common mushroom, a hackberry mushroom, a Desmodium villosum and the like, belongs to the phylum mycota, the subphylum basidiomycota, the class Hymenomycetes, the order Agaricales, the family Tricholomataceae and the genus Lysimachia, and is the earliest edible fungus variety for realizing industrial cultivation in China. At present, the research finds that the flammulina velutipes has the effects of protecting liver, enhancing immune regulation, regulating blood sugar, inhibiting tumors, resisting oxidation and the like, but reports on antiviral research of flammulina velutipes polysaccharide extracts are not found.
The inventor proves through experiments that the flammulina velutipes polysaccharide extract has a particularly obvious effect on resisting influenza viruses, particularly type A viruses and has very low toxicity.
Preferably, the flammulina velutipes polysaccharide extract is extracted from flammulina velutipes fruiting bodies by a water extraction and alcohol precipitation method.
Preferably, the flammulina velutipes polysaccharide extract is prepared by the following steps: pulverizing needle mushroom fruiting body, extracting with water, filtering the extractive solution, concentrating the filtrate to obtain concentrated solution, precipitating the concentrated solution with ethanol, and drying the precipitate to obtain needle mushroom polysaccharide extract.
In a preferred embodiment, the flammulina velutipes polysaccharide extract is extracted by: pulverizing needle mushroom fruiting body, soaking in 10-20 times of water, heating for extraction, filtering to obtain filtrate, repeating the above extraction steps for 1-5 times, mixing filtrates, concentrating the filtrate to obtain concentrated solution, adding ethanol into the concentrated solution to make ethanol volume fraction be 70-90%, standing, transferring the obtained precipitate into baking pan, and vacuum drying at 45 deg.C to obtain needle mushroom polysaccharide extract.
In a second aspect, an antiviral drug is provided, which comprises a flammulina velutipes polysaccharide extract and a pharmaceutically acceptable carrier.
In the above medicine, Flammulina velutipes polysaccharide extract is used as active ingredient.
Preferably, the concentration of the flammulina velutipes polysaccharide extract is between 200 and 2000 mu g/mL. The flammulina velutipes polysaccharide extract within this concentration range has a significant antiviral effect with very minimal toxic effects.
Preferably, the virus is an influenza virus.
Further preferably, the virus is an influenza a virus, for example including but not limited to influenza a H1N1 virus.
Preferably, the flammulina velutipes polysaccharide extract is extracted from flammulina velutipes fruiting bodies by a water extraction and alcohol precipitation method.
Preferably, the flammulina velutipes polysaccharide extract is prepared by the following steps: pulverizing needle mushroom fruiting body, extracting with water, filtering the extractive solution, concentrating the filtrate to obtain concentrated solution, precipitating the concentrated solution with ethanol, and drying the precipitate to obtain needle mushroom polysaccharide extract.
In a preferred embodiment, the flammulina velutipes polysaccharide extract is extracted by: pulverizing needle mushroom fruiting body, soaking in 10-20 times of water, heating for extraction, filtering to obtain filtrate, repeating the above extraction steps for 1-5 times, mixing filtrates, concentrating the filtrate to obtain concentrated solution, adding ethanol into the concentrated solution to make ethanol volume fraction be 70-90%, standing, transferring the obtained precipitate into baking pan, and vacuum drying at 45 deg.C to obtain needle mushroom polysaccharide extract.
In a third aspect, an antiviral food or health product is provided, comprising a flammulina velutipes polysaccharide extract.
Preferably, the concentration of the flammulina velutipes polysaccharide extract is between 200 and 2000 mu g/mL. The inventor proves through experiments that the flammulina velutipes polysaccharide extract in the concentration range has remarkable antiviral effect and very slight toxic effect.
In the above food or health food, Flammulina velutipes polysaccharide extract is used as active ingredient.
Preferably, the virus is an influenza virus.
Further preferably, the virus is an influenza a virus, for example including but not limited to influenza a H1N1 virus.
Preferably, the food product comprises a food product for special medical use, or a functional food product.
Preferably, the flammulina velutipes polysaccharide extract is extracted from flammulina velutipes fruiting bodies by a water extraction and alcohol precipitation method.
Preferably, the flammulina velutipes polysaccharide extract is prepared by the following steps: pulverizing needle mushroom fruiting body, extracting with water, filtering the extractive solution, concentrating the filtrate to obtain concentrated solution, precipitating the concentrated solution with ethanol, and drying the precipitate to obtain needle mushroom polysaccharide extract.
In a preferred embodiment, the flammulina velutipes polysaccharide extract is extracted by: pulverizing needle mushroom fruiting body, soaking in 10-20 times of water, heating for extraction, filtering to obtain filtrate, repeating the above extraction steps for 1-5 times, mixing filtrates, concentrating the filtrate to obtain concentrated solution, adding ethanol into the concentrated solution to make ethanol volume fraction be 70-90%, standing, transferring the obtained precipitate into baking pan, and vacuum drying at 45 deg.C to obtain needle mushroom polysaccharide extract.
In a fourth aspect, a method for extracting flammulina velutipes polysaccharide extract is provided, which comprises the following steps: pulverizing needle mushroom fruiting body, extracting with water, filtering the extractive solution, concentrating the filtrate to obtain concentrated solution, precipitating the concentrated solution with ethanol, and drying the precipitate to obtain needle mushroom polysaccharide extract.
In a preferred embodiment, the extraction method of the flammulina velutipes polysaccharide extract comprises the following steps: pulverizing needle mushroom fruiting body, soaking in 10-20 times of water, heating for extraction, filtering to obtain filtrate, repeating the above extraction steps for 1-5 times, mixing filtrates, concentrating the filtrate to obtain concentrated solution, adding ethanol into the concentrated solution to make ethanol volume fraction 70-90%, standing, transferring the obtained precipitate to baking pan, vacuum drying at 43-50 deg.C, preferably at 45 deg.C to obtain needle mushroom polysaccharide extract.
In the extraction process of the flammulina velutipes extract, environment-friendly water and ethanol are used as solvents, and mild means such as heating extraction and drying are adopted for extraction and purification, so that the flammulina velutipes extract has the advantages of simple process, low production cost and avoidance of organic solvent pollution, and the extraction method can retain the activity of the flammulina velutipes extract to the maximum extent; the obtained Flammulina velutipes polysaccharide extract can be used for preparing anti-influenza virus medicines, foods with special medical application, functional foods or health products.
The invention firstly proposes that the flammulina velutipes polysaccharide extract has a definite anti-influenza virus effect, and the in vitro anti-influenza virus pharmacodynamic study is carried out on the extracted flammulina velutipes polysaccharide extract, and the result shows that the flammulina velutipes polysaccharide extract can obviously inhibit cytopathy caused by influenza virus infection; animal experiments (experiments of H1N1 virus infected mice) are carried out on the extracted needle mushroom polysaccharide extract, and the results prove that the needle mushroom polysaccharide extract can effectively relieve respiratory symptoms caused by H1N1 virus infection, and the application of the needle mushroom polysaccharide extract in the preparation of anti-influenza virus medicaments has good prospects.
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The needle mushroom polysaccharide extract, the extraction method, the application and the beneficial effects thereof are explained in detail below with reference to the accompanying drawings and the detailed embodiments.
FIG. 1 is a graph showing the cytotoxicity results of Flammulina velutipes polysaccharide extract on MDCK cells.
FIG. 2 shows the effect of Flammulina velutipes polysaccharide extract on the survival rate of MDCK cell infected influenza virus.
FIG. 3 is a comparison of lung index of Flammulina velutipes polysaccharide extract against H1N1 virus infected mice.
FIG. 4 is a comparison of the appearance of Flammulina velutipes polysaccharide extract to lung tissue of mice infected with H1N1 virus.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results.
The reagents, methods and apparatus employed in the examples of the invention are conventional in the art, unless otherwise indicated.
Example 1 preparation of Flammulina velutipes polysaccharide extract
(1) Pulverizing needle mushroom fruiting body with pulverizer (25000r/min, 5s), sieving with No. 1 pharmacopeia sieve, collecting filtrate, soaking in 15 times of water until completely wet, heating and extracting for 1.5 hr, filtering, and collecting filtrate; adding 10 times of water into the filter residue, heating and extracting for 1h, filtering, collecting the filtrate, mixing the obtained filtrates, concentrating at 50 deg.C under reduced pressure to about 20L, and further concentrating to about 60% Brix to obtain concentrated solution;
(2) adding absolute ethyl alcohol into the concentrated solution obtained in the step (1) to enable the volume fraction of the absolute ethyl alcohol to be 85%, standing overnight, taking the precipitate, transferring the precipitate into a baking pan, and drying in vacuum at 45 ℃ to obtain the flammulina velutipes polysaccharide extract, wherein the yield is 12.8%, and the polysaccharide content in the extract is 10.1%.
Example 2 cytotoxicity Studies of Flammulina velutipes polysaccharide
The flammulina velutipes polysaccharide extract obtained in example 1 was diluted with PBS to prepare a drug stock solution of 3mg/mL, and stored at 4 ℃.
MDCK cells (canine kidney cells) in good growth state were subjected to EDTA-pancreatin digestion and counted at 2X 104Cells were seeded at 100. mu.L/well in 96-well plates at 37 ℃ with 5% CO2The cell culture box was cultured for 24 hours. When the degree of cell confluence was nearly 90%, the cell culture broth was removed and the cells were washed 2 times with 1 × PBS (phosphate buffered saline). The drug stock solution was serially diluted 2-fold with DMEM containing 2% fetal bovine serum at 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL, 800. mu.g/mL, 1000. mu.g/mL, 2000. mu.g/mL, respectively. Adding the medicinal liquid into the cell culture plate at a ratio of 100 μ L/well, adding MEM medium (minimum Eagle' medium) containing 2% fetal bovine serum into blank control well and normal cell control well at a ratio of 100 μ L/well, and culturing at 37 deg.C with 5% CO2The incubator lasts 48 hours.
After 48 hours, the drug-containing medium was aspirated, rinsed 1-2 times with PBS, and 20. mu.L/well of 5mg/mL MTT solution (thiazole blue) (set to zero) was added thereto at 37 ℃ with 5% CO2The incubator was operated for 4 hours. After 4 hours, the culture plate was taken out, 150. mu.L/well of DMSO solution (dimethyl sulfoxide) was added, after the crystals were dissolved, the absorbance value of each well was measured on a microplate reader at a detection wavelength of 490nm, and the cell viability was calculated according to the following formula.
The 50% toxic concentration was calculated as the drug median toxic concentration (CC 50).
The result of the cytotoxicity research is shown in figure 1, and as can be seen from figure 1, the survival rate of the MDCK cells is between 94.47 and 84.05 percent in the concentration range of 100 mu g/mL to 2000 mu g/mL, which indicates that the half toxic concentration of the flammulina velutipes polysaccharide to the MDCK cells is CC50 which is more than 2000 mu g/mL, and indicates that the flammulina velutipes polysaccharide extract has very low toxicity to the MDCK cells.
Example 3 in vitro anti-influenza Virus study of Flammulina velutipes polysaccharide
MDCK cells in good growth state were cultured at about 2X 10 cells per well4The cells were plated at a density of 96-well cell culture plates at 37 ℃ in 5% CO2The cells are cultured in a cell culture box for about 24 hours, and the cell fusion degree is nearly 100 percent. The cell culture was removed, the cells were washed 2 times with 1 × PBS, and the different drug stocks were serially diluted 2-fold with DMEM containing 2% fetal bovine serum. The Flammulina velutipes polysaccharide extract obtained in example 1 was diluted with PBS to obtain a liquid medicine, which was added to the cell culture plate at 50. mu.L/well, and virus control wells and normal cell control wells at 50. mu.L/well were added to DMEM basal medium containing 2% fetal bovine serum at 37 ℃ with 5% CO2And continuing culturing in the cell culture box. At the same time, 25. mu.L of the drug solution and 25. mu.L of the virus solution (drug-containing virus solution) were mixed, and 25. mu.L of MEM without fetal bovine serum and 25. mu.L of the virus solution were mixed to prepare a drug blank. Putting 96-well culture plate and (non) drug-containing virus liquid into a culture medium containing 5% CO2And incubating for 2h in a constant temperature incubator at 37 ℃. After 2h, the 96-well culture plate was removed, and the drug solutions were separately aspirated and rinsed once with PBS solution. Adding 50 μ L of drug-containing virus solution into each well, shaking, and placing 5% CO2Incubated in an incubator at 37 ℃ for 2 h. After 2h, taking out the 96-well culture plate, respectively removing the drug-containing virus solution, rinsing with PBS solution once, respectively adding 100 μ L/well drug solution containing 2 μ g/mL/TPCK pancreatin, shaking, and placing in a container containing 5% CO2Incubated at 37 ℃ for 48 h.
Subjecting MDCK cells grown in pieces in cell bottles to EDTA-pancreatinCount at 2X 104Cells were seeded at 100. mu.L/well in 96-well plates at 37 ℃ with 5% CO2Incubate overnight. After the whole course of administration, 5% CO at 37 deg.C2After incubation in incubator for 48h, the drug-containing medium was aspirated and washed with PBS 1-2 times, 20. mu.L/well of 5mg/mL MTT solution (set to zero) was added, and 5% CO was added at 37 ℃ with2The incubator is used for 4 hours. And after 4h, taking out the culture plate, adding 150 mu L/hole DMSO solution, after the crystal is dissolved, measuring the absorbance value of each hole on an enzyme-labeling instrument, wherein the detection wavelength is 490nm, and calculating the cell survival rate.
The experimental results are shown in FIG. 2, and the survival rate of the virus cells is about 53.72%; after the dried sample is added, the concentration of the flammulina velutipes polysaccharide extract is within the range of 200 mu g/mL-2000 mu g/mL, the cell survival rate is increased, and is between 61% and 74%, and the cell survival rate is obviously different from that of a virus control group (P is less than 0.01); the result shows that the flammulina velutipes polysaccharide extract has obvious protective effect on MDCK cells infected with H1N1 influenza virus when the concentration is between 200 mu g/mL and 2000 mu g/mL.
Example 4 protective Effect of Flammulina velutipes polysaccharide extract on H1N1 influenza Virus-infected mice
(1) Modeling and administration
Balb/c mice were taken, after adaptive feeding for 3 days, and randomly divided into normal control group, model group, ribavirin group (37.5mg/kg), high (H) and low (L) dose groups (900mg/kg for H group and 300mg/kg for L group) of flammulina velutipes polysaccharide extract prepared in example 1 by weight, 6 mice per group, each drug group was continuously gavaged for 28 days, and normal group and model group were given physiological saline of equal volume.
Starting on day 5 from last, under mild isoflurane anesthesia, 2LD was used in each of the other groups except the normal group50The H1N1 virus diluted solution is dripped into a nasal infection mouse for modeling, and a normal group is dripped into the nasal infection mouse with the same volume of sterile physiological saline, 0.05 ml/mouse, and 6 mice in each group. Continuously observing for 5 days from the day of virus infection, and recording diet and essenceThe body weight of the mice was measured daily for mental state, respiration, hair changes, etc.
(2) Taking materials and detecting
The virus infection is started at 9 o' clock later on day 5, and water is not prohibited for 12 h.
Taking 6 mice, dislocating and killing cervical vertebrae, dissecting and taking lung, observing the lung tissue morphology of each group of mice, weighing the total lung weight, and calculating the lung index.
(3) Results
The lung index experiment results are shown in figure 3, and the lung tissue morphology observation results are shown in figure 4.
The results in FIG. 3 and FIG. 4 show that the pulmonary index of the model group is significantly higher than that of the normal group, which indicates that the lung of the model group animals is obviously edematous and hyperemic (/ gorgeous) after receiving the infection of the PR8 strain of the H1N1 influenza virus. Compared with the model group, the two dose groups of the flammulina velutipes polysaccharide extract can obviously reduce the lung index of tested animals, and obviously relieve the symptoms of edema and congestion (/ blood stasis) of lung tissues, which indicates that the flammulina velutipes polysaccharide extract can effectively relieve respiratory symptoms caused by the infection of the H1N1 influenza virus PR8 strain.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.
Claims (1)
1. The application of the flammulina velutipes polysaccharide extract in preparing the medicine for resisting influenza A virus H1N1 is characterized in that the flammulina velutipes polysaccharide extract is prepared by the following method: pulverizing needle mushroom fruiting body, extracting with water, filtering the extractive solution, concentrating the filtrate to obtain concentrated solution, precipitating the concentrated solution with ethanol, and drying the precipitate to obtain needle mushroom polysaccharide extract.
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