CN117224493A - Preparation method of ginseng polysaccharide sulfated nano-composite - Google Patents
Preparation method of ginseng polysaccharide sulfated nano-composite Download PDFInfo
- Publication number
- CN117224493A CN117224493A CN202311219736.3A CN202311219736A CN117224493A CN 117224493 A CN117224493 A CN 117224493A CN 202311219736 A CN202311219736 A CN 202311219736A CN 117224493 A CN117224493 A CN 117224493A
- Authority
- CN
- China
- Prior art keywords
- ginseng polysaccharide
- solution
- sulfated
- ginseng
- preparing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000208340 Araliaceae Species 0.000 title claims abstract description 90
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 title claims abstract description 90
- 235000003140 Panax quinquefolius Nutrition 0.000 title claims abstract description 90
- 235000008434 ginseng Nutrition 0.000 title claims abstract description 90
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 80
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 80
- 150000004676 glycans Chemical class 0.000 title claims abstract description 76
- 239000002114 nanocomposite Substances 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 239000000203 mixture Substances 0.000 claims abstract description 13
- 238000003756 stirring Methods 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 238000009835 boiling Methods 0.000 claims abstract description 5
- 239000012141 concentrate Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 61
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 238000000502 dialysis Methods 0.000 claims description 15
- 238000002791 soaking Methods 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 239000011521 glass Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 239000005457 ice water Substances 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 239000002244 precipitate Substances 0.000 claims description 8
- 101001096557 Dickeya dadantii (strain 3937) Rhamnogalacturonate lyase Proteins 0.000 claims description 7
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 7
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 7
- 229960000583 acetic acid Drugs 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 239000012362 glacial acetic acid Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 5
- 238000005520 cutting process Methods 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000010298 pulverizing process Methods 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- UDYFLDICVHJSOY-UHFFFAOYSA-N sulfur trioxide pyridine complex Chemical compound O=S(=O)=O.C1=CC=NC=C1 UDYFLDICVHJSOY-UHFFFAOYSA-N 0.000 claims description 4
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000011033 desalting Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 241000712461 unidentified influenza virus Species 0.000 abstract description 13
- 241000700605 Viruses Species 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 8
- 206010022000 influenza Diseases 0.000 abstract description 3
- 230000035755 proliferation Effects 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 238000012404 In vitro experiment Methods 0.000 abstract description 2
- 238000005904 alkaline hydrolysis reaction Methods 0.000 abstract description 2
- 239000002131 composite material Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 13
- -1 polysaccharide sulfate Chemical class 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000003928 nasal cavity Anatomy 0.000 description 2
- 239000007922 nasal spray Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000005723 virus inoculator Substances 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
Landscapes
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a preparation method of a sulfated nano-composite of ginseng polysaccharide, and relates to the technical field of biomedicine. The method comprises the following steps: step one: preparing ginseng polysaccharide solution; step two: boiling and extracting the ginseng polysaccharide solution to obtain ginseng polysaccharide concentrated solution; step three: adding complex enzyme preparation into ginseng polysaccharide concentrate, stirring and mixing by a stirrer, performing water bath enzymolysis at 45-50deg.C for 2-2.5 hr, and centrifuging the mixture in a centrifuge to obtain supernatant. According to the invention, the panaxan is prepared by combining weak acidolysis, weak alkaline hydrolysis and composite enzymolysis, and the panaxan sulfated nano-composite is synthesized by a chemical method, and in vitro experiments prove that the panaxan sulfated nano-composite can inhibit the proliferation of influenza viruses, and compared with panaxan, the panaxan sulfated nano-composite has improved anti-influenza virus effect.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method of a sulfated nanocomposite of ginseng polysaccharide.
Background
Influenza virus can enter the body through skin mucosa, combine with ribosome of host cell, continue to assemble, replicate enzymes and proteins, and further replicate to form new virus. The development of anti-influenza virus products is mainly started from two aspects, namely, the prevention of the combination of viruses and host cells; and secondly, preventing the production of new viruses in host cells.
The ginseng polysaccharide can inhibit invasion of virus from digestive system, has effects of preventing and adjuvant treating pneumonia caused by influenza virus, enhancing immunity, reducing blood lipid, and improving intestinal bacteria.
In the prior art, ginseng polysaccharide is mostly prepared into inhalants such as oral preparations or nasal sprays and the like, so that the inhalants enter nasal cavities, air pipes and lungs to prevent viruses from infecting body tissues, and the inhalants are used for preventing and treating influenza viruses, and the performance of the ginseng polysaccharide is rarely improved, so that the effect of the ginseng polysaccharide on external application is common, and therefore, the preparation method of the ginseng polysaccharide sulfated nano-composite is provided.
Disclosure of Invention
The invention aims to provide a preparation method of ginseng polysaccharide sulfated nano-composites, which aims to solve the problems in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions: the preparation method of the ginseng polysaccharide sulfated nano-composite comprises the following steps:
step one: preparing ginseng polysaccharide solution;
step two: boiling and extracting the ginseng polysaccharide solution to obtain ginseng polysaccharide concentrated solution;
step three: adding complex enzyme preparation into ginseng polysaccharide concentrate, stirring and mixing by a stirrer, performing water bath enzymolysis at 45-50deg.C for 2-2.5h, and centrifuging the mixture in a centrifuge to obtain supernatant;
step four: dialyzing the supernatant by a dialysis bag, concentrating the dialysate, adding 90-95% ethanol solution into the concentrated solution until the final concentration of ethanol is 78-82%, placing into a constant temperature box, standing for 20-24h at 3-5deg.C, and centrifuging by a centrifuge to obtain precipitate;
step five: placing the precipitate into a container, washing with 80-85% ethanol for 2-3 times, washing with 90-95% ethanol for 2-3 times, adding distilled water for dissolution, placing the mixed solution into a drying box, drying at 50-60deg.C, and pulverizing to obtain Ginseng radix polysaccharide powder;
step six: preparing a reaction solution;
step seven: placing 50g of ginseng polysaccharide powder into a beaker containing a reaction solution, placing the beaker into a constant temperature box, stirring at a constant temperature of 50-55 ℃ for 2.5-3h, cooling to room temperature through an ice water bath, and then adding 2-2.5mol/L NaOH solution until the pH value of the mixed solution is regulated to 7;
step eight: and (3) carrying out dialysis and desalting for 68-72h through a dialysis bag, collecting liquid in the bag, and freeze-drying to finally obtain the ginseng polysaccharide sulfated nano-composite.
Further, in the first step, the preparation method of the ginseng polysaccharide solution comprises the following steps:
s1, taking 1kg of ginseng, cleaning, adding the ginseng into a transparent glass barrel containing 10L of water for soaking for 10-12 hours, cutting the ginseng into 1cm sections, and soaking again for 5-8 hours;
s2, adding glacial acetic acid into a transparent glass barrel, adjusting the pH value of the solution to 6.0, and placing the transparent glass barrel in a constant temperature box for soaking for 2 hours at 37 ℃;
s3, adjusting the pH of the solution to 7.6 by using 0.2mol/L sodium bicarbonate, placing the solution in an incubator again, soaking the solution for 1h at 35 ℃, and adding glacial acetic acid to adjust the pH to be neutral;
and S4, filtering the mixed solution in the step S3 through a filter to obtain the ginseng polysaccharide solution.
Further, in the second step, the ginseng polysaccharide solution extract is heated to 100 ℃, and boiled and extracted for 2-3 times until the extract is concentrated to 1L.
Further, in the third step, the complex enzyme preparation is composed of the following substances: the endo alpha-1, 5 arabinase, beta glucosidase and rhamnogalacturonase are sequentially 1 (1.8-2) (0.4-0.5) in mass ratio, and the compound enzyme preparation is obtained by mixing endo alpha-1, 5 arabinase, beta glucosidase and rhamnogalacturonase.
Further, in the third step, the addition amount of the complex enzyme preparation is 0.8-1.1g.
Further, in the fourth step, the concentration temperature of the dialysate is 55-65 ℃ until the dialysate is concentrated to 450-500ml.
In the sixth step, the preparation method of the reaction solution comprises the following steps: 150mL of pyridine is measured, the mixture is added into a beaker, 30g of sulfur trioxide-pyridine is added into the mixture while stirring in an ice-water bath, the mixture is stirred for 10min, the beaker containing the reaction solution is placed into a water bath at 90 ℃, the heating is stopped after 1h, and the water bath is cooled to room temperature, so that the reaction solution is obtained.
Further, the molecular weight of the dialysis bag in the fourth step and the eighth step is 3500Da.
Compared with the prior art, the invention has the beneficial effects that:
according to the preparation method of the ginseng polysaccharide sulfate nano-composite, the ginseng polysaccharide is prepared by combining weak acidolysis, weak alkaline hydrolysis and composite enzymolysis, and the ginseng polysaccharide sulfate nano-composite is synthesized by a chemical method, and in vitro experiments prove that the ginseng polysaccharide sulfate nano-composite can inhibit the proliferation of influenza viruses, and compared with the ginseng polysaccharide, the ginseng polysaccharide sulfate nano-composite has improved anti-influenza virus effect.
Drawings
FIG. 1 shows the inhibition of influenza virus by ginseng polysaccharide at different concentrations;
FIG. 2 shows the effect of ginseng polysaccharide (A) and ginseng polysaccharide sulfated nanocomposite (B) on influenza virus proliferation.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the prior art, ginseng polysaccharide is mostly prepared into inhalants such as oral preparations or nasal sprays and the like, so that the inhalants enter nasal cavities, air pipes and lungs to prevent viruses from infecting body tissues, and the inhalants are used for preventing and treating influenza viruses, and the performance of the ginseng polysaccharide is generally improved, so that the effect of the ginseng polysaccharide on external application is generally improved, and therefore, the preparation method of the ginseng polysaccharide sulfated nano-composite is provided, which comprises the following steps:
step one: preparing ginseng polysaccharide solution;
step two: heating the ginseng polysaccharide solution extract to 100 ℃, and boiling and extracting for 2-3 times until the extract is concentrated to 1L to obtain ginseng polysaccharide concentrate;
step three: adding 0.8-1.1g of complex enzyme preparation into ginseng polysaccharide concentrated solution, stirring and mixing by a stirrer, carrying out water bath enzymolysis for 2-2.5h at 45-50 ℃, and then placing the mixed solution into a centrifuge for mixture centrifugation to obtain supernatant;
step four: dialyzing the supernatant by a dialysis bag, concentrating the dialysate, specifically, concentrating the dialysate at 55-65 ℃ until the concentration of the dialysate reaches 450-500ml, adding 90-95% ethanol solution into the concentrated solution after concentration until the final concentration of ethanol reaches 78-82%, then placing the solution into a constant temperature box, standing for 20-24h at 3-5 ℃, and finally centrifuging by a centrifuge to obtain a precipitate;
step five: placing the precipitate into a container, washing with 80-85% ethanol for 2-3 times, washing with 90-95% ethanol for 2-3 times, adding distilled water for dissolution, placing the mixed solution into a drying box, drying at 50-60deg.C, and pulverizing to obtain Ginseng radix polysaccharide powder;
step six: preparing a reaction solution;
step seven: placing 50g of ginseng polysaccharide powder into a beaker containing a reaction solution, placing the beaker into a constant temperature box, stirring at a constant temperature of 50-55 ℃ for 2.5-3h, cooling to room temperature through an ice water bath, and then adding 2-2.5mol/L NaOH solution until the pH value of the mixed solution is regulated to 7;
step eight: and (3) carrying out dialysis and desalting for 68-72h through a dialysis bag, collecting liquid in the bag, and freeze-drying to finally obtain the ginseng polysaccharide sulfated nano-composite.
The preparation of the ginseng polysaccharide solution needs to be known, and in the first step, the preparation method of the ginseng polysaccharide solution comprises the following steps:
s1, taking 1kg of ginseng, cleaning, adding the ginseng into a transparent glass barrel containing 10L of water for soaking for 10-12 hours, cutting the ginseng into 1cm sections, and soaking again for 5-8 hours;
s2, adding glacial acetic acid into a transparent glass barrel, adjusting the pH value of the solution to 6.0, and placing the transparent glass barrel in a constant temperature box for soaking for 2 hours at 37 ℃;
s3, adjusting the pH of the solution to 7.6 by using 0.2mol/L sodium bicarbonate, placing the solution in an incubator again, soaking the solution for 1h at 35 ℃, and adding glacial acetic acid to adjust the pH to be neutral;
and S4, filtering the mixed solution in the step S3 through a filter to obtain the ginseng polysaccharide solution.
Preparation of the Complex enzyme preparation it is to be appreciated that in step three, the complex enzyme preparation is composed of: the endo alpha-1, 5 arabinase, beta glucosidase and rhamnogalacturonase are sequentially 1 (1.8-2) (0.4-0.5) in mass ratio, and the compound enzyme preparation is obtained by mixing endo alpha-1, 5 arabinase, beta glucosidase and rhamnogalacturonase.
In the sixth step, the preparation method of the reaction solution is as follows: 150mL of pyridine is measured, the mixture is added into a beaker, 30g of sulfur trioxide-pyridine is added into the mixture while stirring in an ice-water bath, the mixture is stirred for 10min, the beaker containing the reaction solution is placed into a water bath at 90 ℃, the heating is stopped after 1h, and the water bath is cooled to room temperature, so that the reaction solution is obtained.
In this protocol, the dialysis bag molecular weight in step four and step eight is 3500Da.
Specific:
the preparation method of the ginseng polysaccharide comprises the following steps: washing 1kg of ginseng, adding 10L of water, soaking for 12 hours, cutting into 1cm sections, soaking again for 6 hours, boiling and extracting for 2 times at 100 ℃ until the concentration is 1L, adding 1g of complex enzyme preparation (prepared by a laboratory, the mass ratio of endo alpha-1, 5 arabinase, beta-glucosidase and rhamnogalacturonase is 1:2:0.5 in sequence) into the concentrated solution, mixing, carrying out enzymolysis for 2 hours in a water bath at 50 ℃, centrifuging the reaction mixture at normal temperature, taking the supernatant, dialyzing the supernatant by a dialysis bag, concentrating the dialyzate, specifically, concentrating the dialyzate at 60 ℃ until the concentration temperature of the dialyzate is 500ml, adding 95% ethanol solution into the supernatant until the final concentration of ethanol is 80%, standing for 24 hours at 4 ℃, and finally centrifuging to obtain a precipitate; washing the precipitate with 80% ethanol for 3 times, washing with 95% ethanol for 2 times, oven drying at 60deg.C, and pulverizing to obtain Ginseng radix polysaccharide powder;
the preparation method of the ginseng polysaccharide sulfated nano-composite comprises the following steps: 150mL of pyridine is measured, the pyridine is added into a beaker, 30g of sulfur trioxide-pyridine is added while stirring in an ice water bath, stirring is carried out for 10min, the beaker is placed into a water bath at 90 ℃, 50g of ginseng polysaccharide powder is added, stirring is carried out for 3h at a constant temperature of 55 ℃, the ice water bath is cooled to room temperature, 2mol/L NaOH solution is added, pH is regulated to 7, dialysis is carried out for 72h through a 3500Da dialysis bag, liquid in the bag is collected, freeze drying is carried out, and the sulfated nano-composite of ginseng polysaccharide is obtained, and the degree of substitution of sulfate of the sulfated nano-composite of ginseng polysaccharide is measured to be 1.65.
Examples:
canine kidney cells (MDCK) cells stored in liquid nitrogen were removed and resuscitated. After three successive passes, well growing MDCK cells were trypsinized and counted, inoculated in 96 well plates with a cell mass of 1 x 10≡5 per well, and cultured overnight in a 5% CO2 constant temperature cell incubator at 37 ℃. The influenza virus H1N1-UI182 strain is a mouse adapted strain of 2009 influenza A virus H1N1 (A/Changchun/01/2009 (H1N 1)) (DOI: 10.3390/v 8040115). First, the H1N1-UI182 strain stored at-80℃was removed and slowly thawed on ice. Subsequently, a virus dilution with a content of 100TCID50 was inoculated into a 96-well plate, and the cell culture medium was replaced after adsorption for 1-2 hours in a cell culture box. After 5h of virus inoculation, the culture solution was removed and 100. Mu.L of a 100-fold dilution of ginseng polysaccharide and ginseng polysaccharide sulfated nanocomposite solution (0. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, 400. Mu.g/mL, 800. Mu.g/mL, 1000. Mu.g/mL) was rapidly added, and a blank cell control well and a virus control well were additionally established for 6 replicates. The inoculated cell plates were placed in a 5% CO2 incubator at 37℃for 48h. Finally, the CCK8 method is used for detecting the cell activity, observing the change of cell morphology, and calculating the half maximum effective inhibition concentration (EC 50) of the drug on influenza virus.
The calculation formula is as follows:
inhibition = (drug treatment OD-virus control OD)/(cell control OD-virus control OD)) ×100%.
Experimental results: the ginseng polysaccharide and the ginseng polysaccharide sulfated nano-composite have excellent antiviral activity, and can obviously inhibit the replication of influenza virus H1N1-UI182 strain in MDCK cells. The half maximal effective inhibitory concentration (EC 50) of Ginseng Polysaccharide (GPS) against H1N1-UI182 strain was 257.5 μg/mL (FIG. 2A); whereas the half maximal effective inhibitory concentration (EC 50) of the sulfated nanocomposite of ginseng polysaccharide (pGPS) against H1N1-UI182 strain was 108.5. Mu.g/mL (FIG. 2B).
The ginseng polysaccharide and the ginseng polysaccharide sulfated nano-composite can obviously inhibit the replication of influenza viruses in vitro, and the sulfated ginseng polysaccharide sulfated nano-composite has better effect of inhibiting the proliferation of the influenza viruses.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made hereto without departing from the spirit and scope of the invention as defined by the appended embodiments and equivalents thereof.
Claims (8)
1. The preparation method of the sulfated nano-composite of ginseng polysaccharide is characterized by comprising the following steps:
step one: preparing ginseng polysaccharide solution;
step two: boiling and extracting the ginseng polysaccharide solution to obtain ginseng polysaccharide concentrated solution;
step three: adding complex enzyme preparation into ginseng polysaccharide concentrate, stirring and mixing by a stirrer, performing water bath enzymolysis at 45-50deg.C for 2-2.5h, and centrifuging the mixture in a centrifuge to obtain supernatant;
step four: dialyzing the supernatant by a dialysis bag, concentrating the dialysate, adding 90-95% ethanol solution into the concentrated solution until the final concentration of ethanol is 78-82%, placing into a constant temperature box, standing for 20-24h at 3-5deg.C, and centrifuging by a centrifuge to obtain precipitate;
step five: placing the precipitate into a container, washing with 80-85% ethanol for 2-3 times, washing with 90-95% ethanol for 2-3 times, adding distilled water for dissolution, placing the mixed solution into a drying box, drying at 50-60deg.C, and pulverizing to obtain Ginseng radix polysaccharide powder;
step six: preparing a reaction solution;
step seven: placing 50g of ginseng polysaccharide powder into a beaker containing a reaction solution, placing the beaker into a constant temperature box, stirring at a constant temperature of 50-55 ℃ for 2.5-3h, cooling to room temperature through an ice water bath, and then adding 2-2.5mol/L NaOH solution until the pH value of the mixed solution is regulated to 7;
step eight: and (3) carrying out dialysis and desalting for 68-72h through a dialysis bag, collecting liquid in the bag, and freeze-drying to finally obtain the ginseng polysaccharide sulfated nano-composite.
2. The method for preparing a sulfated nanocomposite of ginseng polysaccharide according to claim 1, wherein in the first step, the method for preparing a ginseng polysaccharide solution comprises:
s1, taking 1kg of ginseng, cleaning, adding the ginseng into a transparent glass barrel containing 10L of water for soaking for 10-12 hours, cutting the ginseng into 1cm sections, and soaking again for 5-8 hours;
s2, adding glacial acetic acid into a transparent glass barrel, adjusting the pH value of the solution to 6.0, and placing the transparent glass barrel in a constant temperature box for soaking for 2 hours at 37 ℃;
s3, adjusting the pH of the solution to 7.6 by using 0.2mol/L sodium bicarbonate, placing the solution in an incubator again, soaking the solution for 1h at 35 ℃, and adding glacial acetic acid to adjust the pH to be neutral;
and S4, filtering the mixed solution in the step S3 through a filter to obtain the ginseng polysaccharide solution.
3. The method for preparing sulfated nanocomposites according to claim 1, wherein in the second step, the ginseng polysaccharide solution extract is heated to 100 ℃ and boiled for 2-3 times until the extract is concentrated to 1L.
4. The method for preparing sulfated nanocomposites of ginseng polysaccharide according to claim 1, wherein in the third step, the complex enzyme preparation is composed of: the endo alpha-1, 5 arabinase, beta glucosidase and rhamnogalacturonase are sequentially 1 (1.8-2) (0.4-0.5) in mass ratio, and the compound enzyme preparation is obtained by mixing endo alpha-1, 5 arabinase, beta glucosidase and rhamnogalacturonase.
5. The method for preparing a sulfated nanocomposite of ginseng polysaccharide according to claim 4, wherein the complex enzyme preparation is added in an amount of 0.8-1.1g in the third step.
6. The method for preparing sulfated nanocomposites of ginseng polysaccharide according to claim 1, wherein in the fourth step, the concentration temperature of the dialysate is 55-65 ℃ until the dialysate is concentrated to 450-500ml.
7. The method for preparing sulfated nanocomposite of ginseng polysaccharide according to claim 1, wherein in the sixth step, the preparation method of the reaction solution comprises: 150mL of pyridine is measured, the mixture is added into a beaker, 30g of sulfur trioxide-pyridine is added into the mixture while stirring in an ice-water bath, the mixture is stirred for 10min, the beaker containing the reaction solution is placed into a water bath at 90 ℃, the heating is stopped after 1h, and the water bath is cooled to room temperature, so that the reaction solution is obtained.
8. The method for preparing a sulfated nanocomposite of ginseng polysaccharide according to claim 1, wherein the molecular weight of the dialysis bag in the fourth and eighth steps is 3500Da.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311219736.3A CN117224493A (en) | 2023-09-21 | 2023-09-21 | Preparation method of ginseng polysaccharide sulfated nano-composite |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311219736.3A CN117224493A (en) | 2023-09-21 | 2023-09-21 | Preparation method of ginseng polysaccharide sulfated nano-composite |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117224493A true CN117224493A (en) | 2023-12-15 |
Family
ID=89085691
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311219736.3A Pending CN117224493A (en) | 2023-09-21 | 2023-09-21 | Preparation method of ginseng polysaccharide sulfated nano-composite |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117224493A (en) |
-
2023
- 2023-09-21 CN CN202311219736.3A patent/CN117224493A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111658664B (en) | Application of sea cucumber polysaccharide in resisting novel coronavirus | |
| CN111588732A (en) | Application of fucoidin in resisting novel coronavirus | |
| US8614070B2 (en) | Process for the co-production of chitin, its derivatives and polymers containing glucose, mannose and/or galactose, by the fermentation of the yeast Pichia pastoris | |
| CN110028533A (en) | A kind of method and application of the refining amino glucosamine salt hydrochlorate from microbial fermentation solution | |
| CN102807630B (en) | Low-molecular-weight chitosan and glucosamine co-production technology | |
| CN114032273B (en) | Multifunctional American ginseng hydrolytic peptide and preparation method and application thereof | |
| CN103012609A (en) | Method for extracting fucosan sulphate from sea cucumber cooking waste liquor | |
| FI63762B (en) | FOERFARANDE FOER FRAMSTAELLNING AV SOM EXPANDERINGSMEDEL AV BLDPLASMA LAEMPLIG HYDROXIETYLSTAERKELSE | |
| CN117224493A (en) | Preparation method of ginseng polysaccharide sulfated nano-composite | |
| CN109628347A (en) | One plant of luminous bacillus FC615 and its cultural method and application | |
| WO2009022221A2 (en) | Composition and method to stimulate growth and defense against pathogens in plants | |
| CN1895665A (en) | Scorpionfish-ink polysaccharide and its preparation | |
| EP0491114A1 (en) | A process for preparing new non-covalent polysaccharide-protein associations having pharmacological activity | |
| CN116284468A (en) | Tremella aurantialba acidic polysaccharide, preparation method thereof and application thereof in improving ulcerative colitis | |
| WO2021253643A1 (en) | Application of sulphated polysaccharides against novel coronavirus | |
| CN102078335A (en) | Preparation method of latic acid hirudo liquid | |
| CN113350381B (en) | Method for increasing protein ratio of pearl hydrolysate and treatment solution | |
| CN101891837A (en) | Carboxymethylation bifidobacterium exopolysaccharide, preparation method thereof and application thereof | |
| CN112870217B (en) | Application of manno-glucuronic acid poly (oligo) saccharide and sulfated derivative thereof in preparation of drugs for treating osteoporosis | |
| CN107163163A (en) | A kind of processing method of chondroitin sulfate product | |
| CN107164340B (en) | Method for extracting superoxide dismutase from fresh pig blood | |
| CN114732827B (en) | Application of sulfated polysaccharides from different marine organisms and pharmaceutical composition thereof | |
| CN107629145B (en) | Method for extracting hyaluronic acid from wood frog skin | |
| CN114316081B (en) | A sulfated polysaccharide of Botrytis longipedicularis with SARS-CoV-2 inhibiting activity, and its preparation method and application | |
| CN110878129A (en) | Glucosamine heparin salt and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |