CN110256591B - Cordyceps sinensis polysaccharide extract and preparation method and application thereof - Google Patents
Cordyceps sinensis polysaccharide extract and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
- A61K36/066—Clavicipitaceae
- A61K36/068—Cordyceps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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Abstract
The invention belongs to the field of extraction of traditional Chinese medicinal materials, and provides a cordyceps sinensis polysaccharide extract, a preparation method and application thereof, in particular to a preparation method of the cordyceps sinensis polysaccharide extract, the cordyceps sinensis polysaccharide extract with high antitumor activity prepared by the preparation method, and application of the cordyceps sinensis polysaccharide extract in preparation of antitumor drugs. The preparation method comprises the steps of precipitating crude polysaccharide with ethanol, deproteinizing with Sevag reagent, and decolorizing with macroporous resin to obtain deproteinized and decolorized Cordyceps polysaccharide extract.
Description
Technical Field
The invention belongs to the field of extraction of traditional Chinese medicinal materials, relates to preparation of a polysaccharide extract of the traditional Chinese medicinal materials, and particularly relates to preparation and application of a polysaccharide extract of cordyceps sinensis.
Background
The cordyceps sinensis is a traditional and rare Chinese medicinal material in China, belongs to ascomyceta (Ascomycota) Chaetomium (Sordariomycetes), Hypocrea (Hypocrea) and nematoda (Ophiocery picitaceae), has a long medicinal history in China, and has the effects of tonifying lung and kidney, and stopping bleeding and reducing phlegm. Modern chemical and pharmacological research shows that the cordyceps sinensis contains various chemical components such as nucleoside, sugar, sterol, protein and the like, and has pharmacological effects of regulating immunity, relieving lung diseases, resisting tumors and the like.
At the early stage, the anti-tumor effect of Cordyceps sinensis is reported, for example, the inhibition rate of Cordyceps sinensis polysaccharide to mouse sarcoma S180 is 28% -33% when the concentration of Cordyceps sinensis polysaccharide is 100mg/10g, as described in the literature pharmacological action of Cordyceps sinensis polysaccharide (Chinese herbal medicine, 1985, 16(7): 18-23); the literature, "Effect of cordyceps sinensis on the promotion and differentiation of human leukemia U937 cells" [ life sciences,60(25):2349-2359] reports that the inhibition rate of cordyceps sinensis polysaccharide on human leukemia U937cells is 78% -83% at a concentration of 10 μ g/mL; some researches have been made on the antitumor activity of the crude extract of Cordyceps sinensis, for example, patent "an antitumor activity of fresh Cordyceps sinensis extract and its preparation method and application" (application No.: CN201611221881.5) discloses that the suppression rate of breast cancer cell MDA-MB-453 by fresh Cordyceps sinensis water extract at a concentration of 0.25-3 mg/mL is 39.0-86.7%.
The polysaccharide is widely present in animal cell membranes, plant and microbial cell walls, is a natural high molecular compound, is separated from fruiting bodies, mycelia or fermentation liquor of fungi, and has various biological activities of resisting viruses, resisting coagulation, resisting tumors, regulating immunity, reducing blood fat and the like. However, the extraction of the fungal polysaccharide has many problems due to the influence of protein and pigment removal in the extraction process of the fungal polysaccharide, and the characteristics of poor extraction purity and low stability, such as high polarity, difficult volatilization, thermal instability, high molecular weight and the like of the polysaccharide, such that the extraction of the fungal polysaccharide has many problems. At present, there is a great demand for cordyceps sinensis polysaccharide extracts having high antitumor activity.
Disclosure of Invention
The invention provides a preparation method of a cordyceps sinensis polysaccharide extract, provides the cordyceps sinensis polysaccharide extract with high anti-tumor activity prepared by the preparation method, and also provides application of the cordyceps sinensis polysaccharide extract in preparing anti-tumor medicines. The cordyceps polysaccharide extract is a polysaccharide extract which is deproteinized and decolored, and has good inhibitory activity on cancer cells, particularly on leukemia K-562 cells.
In a first aspect, the present invention provides a method for preparing a cordyceps sinensis polysaccharide extract, comprising the following steps:
(1) extracting Cordyceps with water under heating to obtain extract, and eluting with water to obtain water extract;
(2) adding ethanol into the water extract obtained in the step (1) for ethanol precipitation to obtain ethanol precipitation crude polysaccharide;
(3) dissolving the alcohol-precipitated crude polysaccharide obtained in the step (2) in water to obtain a crude polysaccharide solution, and then adding a sevag reagent according to the volume ratio of the crude polysaccharide solution to the sevag reagent of 3:1-5:1 for deproteinization to obtain deproteinized crude polysaccharide; and
(4) and (4) decoloring the deproteinized crude polysaccharide obtained in the step (3) by using macroporous resin.
Preferably, the method comprises the steps of:
(1) extracting Cordyceps with water at 80-100 deg.C under heating to obtain extract, and eluting with water to obtain water extract;
(2) adding ethanol into the water extract obtained in the step (1) for ethanol precipitation to obtain ethanol precipitation crude polysaccharide;
(3) dissolving the alcohol-precipitated crude polysaccharide obtained in the step (2) in water according to a material-liquid ratio of 1: 18-1: 22(g/mL) to obtain a crude polysaccharide solution, and then adding a sevag reagent according to a volume ratio of the crude polysaccharide solution to the sevag reagent of 3:1-5:1 to perform deproteinization to obtain deproteinized crude polysaccharide; and
(4) and (4) decoloring the deproteinized crude polysaccharide obtained in the step (3) by using macroporous resin.
Preferably, in the step (1), the heating extraction is reflux extraction, the material-liquid ratio of the heating extraction is 1:8-1:12(kg/L), and the extraction is carried out for 2-4 hours; preferably, the residue after heating extraction is continuously subjected to reflux extraction for 2-3 times according to the material-liquid ratio of 1:8-1:12(kg/L), each time for 2-4h, and the extracting solutions are combined.
Preferably, after the extract is obtained in step (1), the extract is adsorbed on a macroporous resin and eluted with water to obtain an aqueous extract.
Preferably, the aqueous extract in the step (2) is an aqueous solution of the aqueous extract with a material-to-liquid ratio of 1:3-1:6 (g/mL); in the step (2), the ethanol is absolute ethanol, and the volume ratio of the absolute ethanol to the aqueous solution of the water extract is 3:1-5: 1.
Preferably, the aqueous extract in the step (2) is an aqueous solution of the aqueous extract with a material-to-liquid ratio of 1:5 (g/mL); in the step (2), the ethanol is absolute ethanol, and the volume ratio of the absolute ethanol to the aqueous solution of the water extract is 4: 1.
Preferably, the alcohol precipitation in the step (2) is carried out at room temperature, and the alcohol precipitation time is more than 8 hours.
Preferably, the volume ratio of the crude polysaccharide solution to the sevag reagent in the step (3) is 4: 1.
Preferably, step (3) is repeated before step (4) until the centrifuged aqueous layer and the organic phase have no protein layer, and the deproteinized aqueous layer solution is obtained as the deproteinized crude polysaccharide, specifically, the following steps are repeated: adding a sevag reagent into the crude polysaccharide solution, centrifuging after fully shaking, taking a water phase layer, and repeatedly performing deproteinization operation until no protein layer exists between the centrifuged water phase layer and the organic phase layer to obtain a deproteinized water phase layer solution, namely the deproteinized crude polysaccharide; preferably, the deproteinized aqueous phase layer solution is further added with 4-5 times volume of absolute ethyl alcohol, and after alcohol precipitation, the crude polysaccharide after deproteinization is obtained by centrifugation.
Preferably, in the step (3), 4 to 5 times volume of absolute ethanol is slowly added to the deproteinized aqueous phase solution while stirring, and the mixture is precipitated with ethanol at 4 ℃ overnight and then centrifuged at 5000rpm for 10 minutes to obtain deproteinized crude polysaccharide (DPCSP).
Preferably, in the step (4), the macroporous resin is AB-8 macroporous resin, D-941 macroporous resin, D-113 macroporous resin or D-101 macroporous resin; more preferably, in said step (5), the macroporous resin is AB-8 macroporous resin.
Preferably, the step (4) is: adding the deproteinized crude polysaccharide obtained in the step (3) to an AB-8 macroporous resin column, eluting with water to obtain an eluent, concentrating, and dialyzing with 2kDa dialysis bag pure water to obtain a solution in a dialysis bag after dialysis; preferably, the solution in the dialysis bag is further freeze-dried to obtain crude de-pigmented polysaccharide (DCCSP).
Preferably, the invention provides a preparation method of the cordyceps sinensis polysaccharide extract, which comprises the following steps:
(1) extracting pulverized Cordyceps with water under reflux for 2-4 hr at a ratio of material to liquid of 1:8-1:12 (kg/L); preferably, the residue after reflux extraction is continuously subjected to reflux extraction for 2-3 times according to the material-liquid ratio of 1:8-1:12(kg/L), the extraction time is 2-4h each time, and the extracting solutions are combined;
concentrating the extractive solution to obtain extract, adsorbing with HP-20 macroporous resin, eluting with water, concentrating the eluate, and freeze drying to obtain water extract;
(2) dissolving the water extract obtained in the step (1) in water according to a material-to-liquid ratio of 1:3-1:6(g/mL) to obtain a water solution of the water extract; then adding absolute ethyl alcohol for alcohol precipitation to obtain alcohol precipitation crude polysaccharide, wherein the volume ratio of the absolute ethyl alcohol to the aqueous solution of the water extract is 3:1-5: 1;
(3) adding the alcohol-precipitated crude polysaccharide obtained in the step (2) into water according to a material-liquid ratio of 1: 18-1: 22(g/mL) to obtain a crude polysaccharide solution, and then adding a sevag reagent according to a volume ratio of the crude polysaccharide solution to the sevag reagent of 4:1 to perform deproteinization, wherein the specific operation is as follows: adding a sevag reagent into the crude polysaccharide solution, centrifuging after fully shaking, taking a water phase layer, and repeatedly performing deproteinization operation until no protein layer exists between the centrifuged water phase layer and the organic phase layer to obtain a deproteinized water phase layer solution, namely the deproteinized crude polysaccharide; preferably, the deproteinized aqueous phase layer solution is further added with 4-5 times volume of absolute ethyl alcohol, and the crude polysaccharide after deproteinization is obtained by alcohol precipitation and centrifugation; and
(4) adding the deproteinized crude polysaccharide obtained in the step (3) to an AB-8 macroporous resin column, eluting with water to obtain an eluent, concentrating, and dialyzing with 2kDa dialysis bag pure water to obtain a dialyzed solution in a dialysis bag; preferably, the solution in the dialysis bag is further freeze-dried to obtain crude de-pigmented polysaccharide (DCCSP).
Preferably, the invention provides a preparation method of the cordyceps sinensis polysaccharide extract, which comprises the following steps:
(1) extracting pulverized Cordyceps with water under reflux for 2-4 hr at a ratio of 1:8-1:12(kg/L), extracting the residue under reflux for 2-3 times at a ratio of 1:8-1:12(kg/L), each time for 2-4 hr, and mixing extractive solutions;
concentrating the extractive solution to obtain extract, adsorbing with HP-20 macroporous resin, eluting with water, concentrating the eluate, and freeze drying to obtain water extract;
(2) dissolving the water extract obtained in the step (1) in water according to the material-liquid ratio of 1:5(g/mL) to obtain a water extract aqueous solution; then adding absolute ethyl alcohol for alcohol precipitation to obtain alcohol precipitation crude polysaccharide, wherein the volume ratio of the absolute ethyl alcohol to the aqueous solution of the water extract is 4: 1;
(3) adding the alcohol precipitated crude polysaccharide obtained in the step (2) into water according to the material-liquid ratio of 1:20(g/mL) to obtain a crude polysaccharide solution, and then adding a sevag reagent according to the volume ratio of the crude polysaccharide solution to the sevag reagent of 4:1 to remove protein, wherein the specific operation is as follows: adding sevag reagent into the crude polysaccharide solution, fully oscillating, centrifuging, taking a water phase layer, and repeatedly performing deproteinization operation until the centrifuged water phase layer and the organic phase layer have no protein layer to obtain a deproteinized water phase layer solution; adding 4-5 times volume of anhydrous ethanol into the deproteinized aqueous phase layer solution, precipitating with ethanol at 4 deg.C, and centrifuging at 5000rpm for 10min to obtain deproteinized crude polysaccharide; and
(4) and (3) adding the deproteinized crude polysaccharide obtained in the step (3) to an AB-8 macroporous resin column, eluting with water to obtain an eluent, concentrating, dialyzing with 2kDa dialysis bag pure water to obtain a solution in a dialysis bag, and further freezing and drying the solution in the dialysis bag to obtain crude decolorant polysaccharide (DCCSP).
In a second aspect, the invention provides a cordyceps sinensis polysaccharide extract prepared by the method of the invention.
In a third aspect, the invention provides a cordyceps sinensis polysaccharide extract, which has a total sugar content of 85-90% and comprises low molecular weight polysaccharides and high molecular weight polysaccharides, wherein the low molecular weight polysaccharides are polysaccharides with an average molecular weight of 6.5-7.0 kDa and account for about 75% of the total sugar by mass, and the high molecular weight polysaccharides have an average molecular weight of more than 670kDa and account for about 25% of the total sugar by mass.
Preferably, the average molecular weight of the low molecular weight polysaccharides in the cordyceps sinensis polysaccharide extract is 6.86 kDa.
Preferably, the cordyceps sinensis polysaccharide extract shows three peaks in total at a retention time of 9.610 minutes, 12.225 minutes and 17.705 minutes by gel chromatography, and the peaks respectively correspond to a molecular weight of 670kDa and account for 3.328%; 21.849 is accounted for by molecular weight of more than 670 kDa; and 74.829% with a molecular weight of 6.86 kDa.
In a fourth aspect, the invention provides the cordyceps sinensis polysaccharide extract prepared by the method of the invention or the application of the cordyceps sinensis polysaccharide extract in preparing anti-tumor medicines.
Preferably, the tumor is breast cancer, lymphoma, leukemia, melanoma, colon cancer and lung cancer.
Preferably, the tumors are breast cancer MDA-MB-453, lymphoma Raji, leukemia K-562 and melanoma SK-MEL-28.
The "ratio of the material to the liquid" in the invention refers to the ratio of the amount of the material to the amount of the solvent. The ratio of the material to the liquid in the extraction in the step (1) of the invention is 1:8-1:12(kg/L), which means that the ratio of the cordyceps sinensis to the extraction solvent water is 1:8-1:12(kg/L), i.e. when 1kg of cordyceps sinensis is extracted with water, the volume of the water is 8L-12L. If the water extract is an aqueous water extract solution with a material-to-liquid ratio of 1:3-1:6(g/mL), the water extract solution is obtained by mixing the concentrated water extract without the solvent and the solvent water in a ratio of 1:3-1:6(g/mL), namely, when 1g of the water extract is dissolved into an aqueous solution by using water, the volume of the water is 3mL-18 mL; the sevag reagent is a solution prepared by mixing chloroform and n-butanol according to a volume ratio of 4:1(mL: mL).
The term "about" as used herein means an approximate value or range, e.g., "about 75% by mass" as used herein means that the mass of low molecular weight polysaccharide is about 75% of the total polysaccharide extract mass, i.e., within a range of 75 ± 5%, all falling within the range defined by "about 75%", e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%/78%. Ranges of 79%, 80% all fall within the range of "about 75%"; preferably, all ranges within 75 ± 3% fall within the range defined by "about 75%". Likewise, "about 25% by mass" means that the mass of the high molecular weight polysaccharide accounts for about 25% of the mass of the total polysaccharide extract, i.e., within a range of 25. + -. 5% both falling within the range defined by "about 75%"; preferably, all ranges within 25 ± 3% fall within the range defined by "about 25%".
The preparation method of the cordyceps sinensis polysaccharide extract is simple and convenient to operate, and the obtained polysaccharide extract has high total sugar content and high activity.
The D113 cation exchange resin is a macroporous weakly acidic acrylic cation exchange resin containing a carboxylic acid functional group (-COOH) in its crosslinked acrylic backbone. The resin is mainly used for industrial water treatment, and can also be used for industrial wastewater treatment, metal recovery, and biochemical medicine separation and purification.
D941 anion exchange resin is macroporous methyl acrylate copolymer cross-linked polymer, and is polyamine alkalescent ion exchange resin obtained through aminolysis of polyethylene polyamine, and is mainly used for refining and decolorizing in food industry such as saccharide, and decolorizing and refining natural medicines such as stevioside, ginsenoside, notoginsenoside, antibiotic, etc.
The D101 resin is a spherical, non-polar polymeric adsorbent. The resin is a crosslinked styrene-divinylbenzene copolymer. The resin has special selectivity for saponin and flavone based on proper pore size and specific surface. The resin is suitable for extracting saponin, flavonoid and some organic substances from aqueous solution.
The AB-8 resin is a spherical polystyrene type weak polar adsorption resin (note: the resin has a certain ester group on the surface and is originally marked as weak polar, but the adsorption mechanism is still hydrophobic and is now determined as nonpolar). The resin is a crosslinked polymer, which is different from the hydrophobic adsorbent of the early stage, and has a weak hydrophilic group attached to the skeleton structure thereof; unlike common ion exchange resins, the ion exchange resins have only non-ionizing functional groups in their structures. The resin has large specific surface area and pore size, is suitable for adsorbing various traditional Chinese medicine components with certain hydrophobicity, and has large adsorption capacity, easy elution and good adsorption kinetics performance. Is stable to heat, organic solvents and acid and alkali under common use conditions, so the service life is longer. It has no adsorption to protein, saccharide, inorganic acid, alkali, salt and small molecule hydrophilic organic matter, so that it can separate Chinese medicine components from these matters. Most suitable for water-soluble and weakly polar substance extraction, separation and purification.
Drawings
FIG. 1 is a gel chromatogram of the obtained Cordyceps sinensis polysaccharide extract DCCSP.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that various changes and modifications based on the inventive concept herein will occur to those skilled in the art and are intended to be included within the scope of the present invention. The reagents used are not indicated by the manufacturer, and are all conventional products commercially available.
Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
In the embodiment of the invention, the dry cordyceps medicinal material is sourced from Yichang Shanchengsheng Shuihu cordyceps limited company.
Examples
1. Reagent and consumable
Table 1:
2. apparatus and device
Table 2:
example 1
Extracting cordyceps sinensis polysaccharide:
(1) alcohol precipitation
Pulverizing 15Kg of dried Cordyceps, extracting with hot water under reflux, and extracting with 120L of water for 2 hr each time for 3 times. Mixing extractive solutions, concentrating, adsorbing with HP-20 macroporous resin, eluting with water, concentrating eluate, and freeze drying to obtain Cordyceps water extract solid.
Placing 300g of the cordyceps sinensis aqueous extract solid in a 5L beaker, adding 1500mL of water, dissolving in a water bath at 60 ℃, and cooling to obtain a cordyceps sinensis aqueous extract solution with a material-to-liquid ratio of 1:5 (g/mL). Subpackaging the cordyceps aqueous extract solution into 2 measuring cups of 5L in equal amount, respectively adding absolute ethyl alcohol with 4 times of volume, sealing by using a preservative film, and carrying out alcohol precipitation at room temperature overnight. Then, centrifuging for 30 minutes at a centrifugal force of 3000g, combining alcohol precipitates, heating in a water bath at 80 ℃ to volatilize ethanol, and obtaining 46.5g of crude polysaccharide precipitated by alcohol.
(2) Sevag deproteinization
Dissolving the alcohol precipitated crude polysaccharide in 1L of water, dissolving in a water bath at 80 ℃, subpackaging the solution in a centrifuge cup, adding Sevag reagent (chloroform: n-butyl alcohol is 4:1, V/V) according to a ratio of 4:1 (sample: Sevag reagent, V/V), oscillating for 30 minutes, centrifuging at 4000rpm for 10 minutes, dividing the solution into three layers after centrifugation, sequentially comprising an organic layer, a protein layer and an aqueous phase layer from bottom to top, taking the aqueous phase layer, and repeating the deproteinizing step for 11 times until no obvious protein layer exists between the aqueous phase and the organic phase. Adding 4 times volume of anhydrous ethanol slowly into the deproteinized aqueous phase layer solution while stirring, precipitating with ethanol at 4 deg.C overnight, and centrifuging at 5000rpm for 10min to obtain deproteinized crude polysaccharide (DPCSP).
(3) Depigmentation
Weighing about 2g of macroporous resin D113, D941, D101 and AB-8 which are pretreated and subjected to suction filtration respectively in a beaker, adding 10mL of polysaccharide solution (obtained by dissolving the deproteinized crude polysaccharide obtained in the step (2) in 600mL of water) respectively, oscillating for 3 hours at 120 times/min, centrifuging, taking 5mL of supernatant into a colorimetric tube, and selecting the macroporous resin with the lightest color of the supernatant after adsorption for subsequent tests. The color depth is D113> D941> D101> AB-8.
Therefore, AB-8 macroporous resin was used in this example. Dissolving the deproteinized crude polysaccharide obtained in the step (2) in 600mL of water, adding the polysaccharide solution onto an AB-8 macroporous resin column (5.0 x 55cm), eluting with water until the eluent is not colored with phenol-sulfuric acid, combining the eluates, concentrating, dialyzing the concentrate with 2k Da dialysis bag pure water to obtain a dialyzed solution in a dialysis bag, and freeze-drying the solution in the dialysis bag to obtain crude destaining polysaccharide (DCCSP) powder. The total sugar content of DCCSP determined by phenol-sulfuric acid method was 87.42%.
Example 2
Crude depigmenting polysaccharide (DCCSP) antitumor test:
(1) cell culture: the leukemia K-562 cells, the breast cancer MDA-MB-453 cells, the lymphoma cancer Raji cells and the colorectal cancer COLO205 cells are placed in a DMEM culture medium, the melanoma SK-MEL-28 cells are placed in an RPMI-1640(Roswell Park medical Institute-1640) culture medium, and then all the cells are placed in a 5% carbon dioxide incubator at 37 ℃ for culture. Cells were observed under an inverted microscope 1 time daily and the medium was changed every 2 days. And carrying out cell passage when the cells in the culture bottle grow to 85-95% of fusion. Discarding the old culture solution, repeatedly washing with PBS for 2 times, adding 2mL of 0.25% pancreatin digestive juice (except HL-60), rounding cells, floating, adding 4mL of culture solution to stop digestion, transferring to a 15mL sterile centrifuge tube, centrifuging for 4 minutes at 1000 r/min, discarding supernatant, and transferring to a new sterile culture bottle at a ratio of 1:4 for culture.
(2) Preparing the medicine: the crude de-pigmented polysaccharide (DCCSP) powder prepared in example 1 was weighed out into the culture medium to prepare 6mg/mL solutions, diluted 3-fold to a series of gradient concentrations with the initial final concentration as the initial final concentration, set up 9 concentration points in total, and then filtered through a 0.22 μm sterile filter to sterilize.
(3) And (3) activity detection: cells in the logarithmic growth phase are collected, counted, resuspended in complete medium, adjusted to the appropriate concentration, and seeded into 96-well plates. At 37 ℃ 5% CO2After incubation under conditions for 24 hours. Crude depigmenting polysaccharide (DCCSP) was added at 100. mu.L/well. Then, the mixture was left at 37 ℃ with 5% CO2Incubate for 72 hours in an incubator. Adding 10% of CCK-8 (fine)Cell counting kit-8), placing the reagent in an incubator at 37 ℃ for incubation for 1-2 hours, detecting the absorbance (A) value of each hole at 450nm of an enzyme-labeling instrument, and respectively calculating the IC50 value (mg/mL) of each drug at 72 hours by data processing software. The results are shown in the following table:
table 3: IC50 value of drug to tumor cell
| Cells | DCCSP(mg/mL) |
| RAJI | 0.1993 |
| K-562 | 0.754 |
| COLO205 | 1.586 |
| MDA-MB-453 | 0.013 |
| SK-MEL-28 | 1.167 |
Example 3
Method for measuring molecular weight distribution of decolored crude polysaccharide (DCCSP)
(1) Solution preparation:
20mM ammonium acetate: ammonium acetate 1.54g is weighed, dissolved by adding 1000mL of water, filtered and degassed by ultrasound.
Series dextran control solutions: appropriate dextran controls of different molecular weights (1, 5, 12, 25, 50, 80, 150, 410, 670kDa) were prepared as 1mg/mL solutions and passed through 0.45 μm membranes.
DCCSP solution: an appropriate amount of DCCSP was taken to prepare a 2mg/mL solution, which was passed through a 0.45 μm membrane.
(2) Gel chromatography analysis: a chromatographic column: TSKgel G5000PWXL (7.8 mm. times.30 cm, 10 μm) and guard column TSKgel guard column PWXL (6.0 mm. times.4 cm); mobile phase: 20mM ammonium acetate, isocratic elution; flow rate: 0.6 mL/min; column temperature: room temperature; sample introduction amount: 20 mu L of the solution; evaporation temperature: 30 ℃; temperature of the drift tube: 30 ℃; carrier gas: air, flow rate 1.60 SLM; gain value: 1.0.
(3) data processing: and (5) carrying out linear regression analysis on the retention time by using the logarithmic value of the molecular weight of the glucan to obtain a molecular weight correction curve. And (4) according to the retention time of each chromatographic peak in the test solution, calculating the corresponding molecular weight by using a correction curve. And calculating the proportion of each component in the analyzed chromatographic peak by an area normalization method.
(4) As a result: performing regression analysis by using the logarithm value of the series of glucan molecular weights as an ordinate and the retention time as an abscissa to obtain a regression equation of-0.4218 x +11.304, R20.9762. As can be seen from FIG. 1, DCCSP consists of three main components, and the molecular weights of peaks 1 and 2 are both greater than 670kDa and the molecular weight of peak 3 is 6.86kDa, as calculated by the regression equation. The ratios of peaks 1, 2 and 3 were 3.328%, 21.849% and 74.829% by normalization.
TABLE 4 polysaccharide DCCSP retention time (min) and molecular weight (Da)
| Peak name | Retention time | Molecular weight | Is in percentage by weight |
| 1 | 9.610 | >670kDa | 3.328 |
| 2 | 12.225 | >670kDa | 21.849 |
| 3 | 17.705 | 6.86kDa | 74.829 |
Claims (11)
1. A Cordyceps sinensis polysaccharide extract, wherein the total sugar content in the extract is 85% -90%, the extract comprises low molecular weight polysaccharide and high molecular weight polysaccharide, the low molecular weight polysaccharide is polysaccharide with an average molecular weight of 6.5-7.0 kDa, and accounts for 75% +/-5% of the total sugar by mass, the high molecular weight polysaccharide has an average molecular weight of more than 670kDa, and accounts for 25% +/-5% of the total sugar by mass;
the cordyceps sinensis polysaccharide extract is prepared by the following steps:
(1) extracting Cordyceps with water at 80-100 deg.C under heating to obtain extractive solution, adsorbing with HP-20 macroporous resin, and eluting with water to obtain water extract; wherein the heating extraction is reflux extraction, the material-liquid ratio of the heating extraction is 1:8-1:12 kg/L, and the extraction is carried out for 2-4 hours;
(2) adding ethanol into the water extract obtained in the step (1) for ethanol precipitation to obtain ethanol precipitation crude polysaccharide; wherein the water extract is an aqueous solution of water extract with the material-liquid ratio of 1:3-1:6 g/mL; the ethanol is absolute ethanol, and the volume ratio of the absolute ethanol to the aqueous solution of the water extract is 3:1-5: 1;
(3) dissolving the alcohol-precipitated crude polysaccharide obtained in the step (2) in water according to the material-liquid ratio of 1: 18-1: 22 g/mL to obtain a crude polysaccharide solution, and then adding a Sevag reagent according to the volume ratio of the crude polysaccharide solution to the Sevag reagent of 3:1-5:1 to remove protein to obtain deproteinized crude polysaccharide; and
(4) and (3) decoloring the deproteinized crude polysaccharide obtained in the step (3) by using AB-8 macroporous resin, eluting by using water to obtain eluent, concentrating, dialyzing by using 2kDa dialysis bag pure water to obtain a dialyzed solution in a dialysis bag, and freeze-drying the dialyzed solution in the dialysis bag to obtain the cordyceps sinensis polysaccharide extract.
2. The polysaccharide extract of Cordyceps sinensis according to claim 1, wherein the low molecular weight polysaccharide has an average molecular weight of 6.86 kDa.
3. The polysaccharide extract of cordyceps sinensis according to claim 1, which shows three peaks in total at a retention time of 9.610 minutes, 12.225 minutes and 17.705 minutes by gel chromatography analysis, and respectively corresponds to a molecular weight of 670kDa of 3.328%; 21.849% of molecular weight more than 670 kDa; and 74.829% with a molecular weight of 6.86 kDa.
4. The method for preparing the cordyceps sinensis polysaccharide extract according to any one of claims 1 to 3, comprising the following steps:
(1) extracting Cordyceps with water at 80-100 deg.C under heating to obtain extractive solution, adsorbing with HP-20 macroporous resin, and eluting with water to obtain water extract; wherein the heating extraction is reflux extraction, the material-liquid ratio of the heating extraction is 1:8-1:12 kg/L, and the extraction is carried out for 2-4 hours;
(2) adding ethanol into the water extract obtained in the step (1) for ethanol precipitation to obtain ethanol precipitation crude polysaccharide; wherein the water extract is an aqueous solution of water extract with the material-liquid ratio of 1:3-1:6 g/mL; the ethanol is absolute ethanol, and the volume ratio of the absolute ethanol to the aqueous solution of the water extract is 3:1-5: 1;
(3) dissolving the alcohol-precipitated crude polysaccharide obtained in the step (2) in water according to the material-liquid ratio of 1: 18-1: 22 g/mL to obtain a crude polysaccharide solution, and then adding a Sevag reagent according to the volume ratio of the crude polysaccharide solution to the Sevag reagent of 3:1-5:1 to remove protein to obtain deproteinized crude polysaccharide; and
(4) and (3) decoloring the deproteinized crude polysaccharide obtained in the step (3) by using AB-8 macroporous resin, eluting by using water to obtain eluent, concentrating, dialyzing by using 2kDa dialysis bag pure water to obtain a dialyzed solution in a dialysis bag, and freeze-drying the dialyzed solution in the dialysis bag to obtain the cordyceps sinensis polysaccharide extract.
5. The method according to claim 4, wherein in the step (1), the residue after heating extraction is continuously refluxed and extracted for 2-3 times according to the material-to-liquid ratio of 1:8-1:12 kg/L, each time for 2-4 hours, and the extracting solutions are combined.
6. The method according to claim 4, wherein the aqueous extract in the step (2) is an aqueous extract solution with a feed-to-liquid ratio of 1:5 g/mL; in the step (2), the ethanol is absolute ethanol, and the volume ratio of the absolute ethanol to the aqueous solution of the water extract is 4: 1.
7. The method of claim 4, wherein the alcohol precipitation in step (2) is performed at room temperature and the alcohol precipitation time is greater than 8 hours.
8. The process of claim 4, wherein the volume ratio of the crude polysaccharide solution to the Sevag reagent in step (3) is 4: 1.
9. The method according to claim 4, wherein step (3) is repeated before step (4) until the centrifuged aqueous layer and the organic phase have no protein layer, and the deproteinized aqueous layer solution is obtained as the deproteinized crude polysaccharide.
10. The method according to claim 4, wherein the deproteinized aqueous layer solution is further added with 4-5 times volume of anhydrous ethanol, precipitated with ethanol and centrifuged to obtain deproteinized crude polysaccharide.
11. Use of the polysaccharide extract of cordyceps sinensis according to any one of claims 1 to 3 or prepared by the method according to any one of claims 4 to 10 in the preparation of a medicament for treating tumors; wherein the tumor is selected from one or more of blood cancer, breast cancer, lymphoma cancer and colorectal cancer.
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