CN110256591A - A kind of Chinese caterpillar fungus polysaccharide extract and its preparation method and application - Google Patents
A kind of Chinese caterpillar fungus polysaccharide extract and its preparation method and application 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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- 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
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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, 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
Abstract
The invention belongs to Chinese medicines to extract field, provide a kind of Chinese caterpillar fungus polysaccharide extract and its preparation method and application, specifically, provide a kind of preparation method of Chinese caterpillar fungus polysaccharide extract, and the Chinese caterpillar fungus polysaccharide extract having compared with high anti-tumor activity prepared through such preparation method, such Chinese caterpillar fungus polysaccharide extract is additionally provided in preparation for the application in anti-tumor drug.The preparation method includes using ethyl alcohol alcohol precipitation Thick many candies, de- albumen, and the step of with macroreticular resin depigmentation is carried out with Sevag reagent, to obtain the Chinese caterpillar fungus polysaccharide extract through taking off albumen and depigmentation.
Description
Technical field
The invention belongs to Chinese medicines to extract field, be related to the preparation of Chinese medicine polyoses extract, more particularly to the worm summer in winter
The preparation and its application of grass polysaccharide extract.
Background technique
Cordyceps sinensis is the traditional rare traditional Chinese medicine in China, is under the jurisdiction of Ascomycota (Ascomycota) excrement shell Gammaproteobacteria
(Sordariomycetes), Hypocreales (Hypocreales), nematode grass section (Ophiocordycipitaceae), in China
With long medicinal history, have effects that invigorating the lung and the kidney, hemostasis and phlegm.Modern chemistry and pharmacological research show the worm summer in winter
Grass has containing multiclass chemical components such as nucleosides, carbohydrate, sterol, protein and adjusts immunity, alleviate pulmonary disease, is antitumor
Equal pharmacological actions.
Also have early period reported literature cordyceps sinensis have antitumor action, as document " pharmacological action of Cordyceps sinensis polysaccharide " [in
Herbal medicine, 1985,16 (7): 18-23] to record Cordyceps sinensis polysaccharide concentration be 28% to S180 sarcoma inhibiting rate when being 100mg/10g
~33%；Document " Effect of cordyceps sinensis on the proliferation and
differentiation of human leukemic U937cells》[life sciences,60(25):2349-2359]
Reporting when Chinese caterpillar fungus polysaccharide concentration is 10 μ g/mL is 78%~83% to human leukemia U937 cell inhibitory rate；Also there is portion
Point research is the anti-tumor activity for cordyceps sinensis crude extract, as " a kind of fresh cordyceps sinensis of anti-tumor activity mentions patent
Take object and its preparation method and application " (application number: CN201611221881.5) disclose fresh cordyceps sinensis water extract dense
Degree is 39.0%~86.7% to the inhibiting rate of breast cancer cell MDA-MB-453 when being 0.25~3mg/mL.
Polysaccharide is widely present in animal cell membrane, plant and microorganism wall, is a kind of natural polymer,
Fungi polysaccharide is that one kind is separated from the fructification of fungi, mycelium or fermentation liquid, some fungi polysaccharides have disease-resistant
The multiple biological activities such as poison, anticoagulation, antitumor, immunological regulation, reducing blood lipid.However, due in fungi polysaccharide extraction process by
To the influence of albumen, pigment removal, moreover, that there is also DNA purities is poor, stability is not high, as polysaccharide itself it is highly polar, wave
Raise difficult questions, thermal instability, high molecular weight the features such as so that there are many problems for the extraction of fungi polysaccharide.Currently, for have compared with
There is very big demands for the Chinese caterpillar fungus polysaccharide extract of high anti-tumor activity.
Summary of the invention
The present invention provides a kind of preparation methods of Chinese caterpillar fungus polysaccharide extract, provide through such preparation method system
The standby Chinese caterpillar fungus polysaccharide extract having compared with high anti-tumor activity additionally provides such Chinese caterpillar fungus polysaccharide extract and exists
Preparation is for the application in anti-tumor drug.Chinese caterpillar fungus polysaccharide extract of the invention is through de- albumen and depigmentation
Polyoses extract has preferable inhibitory activity to cancer cell, especially has good inhibitory activity to leukemia K-562 cell.
In a first aspect, the method includes such as the present invention provides a kind of preparation method of Chinese caterpillar fungus polysaccharide extract
Lower step:
(1) cordyceps sinensis is obtained into extract with water heating extracting, and acquisition water extract is eluted with water；
(2) ethyl alcohol progress alcohol precipitation is added in the water extract obtained into step (1) and obtains alcohol precipitation Thick many candies；
(3) by step (2) obtain alcohol precipitation Thick many candies it is soluble in water obtain Thick many candies solution, later according to Thick many candies solution
Volume ratio with sevag reagent is that 3:1~5:1 addition sevag reagent carries out de- albumen, the Thick many candies after obtaining de- albumen；With
And
(4) Thick many candies after the de- albumen of step (3) acquisition are subjected to depigmentation with macroreticular resin.
Preferably, described method includes following steps:
(1) by cordyceps sinensis, with water, the heating extraction at 80 DEG C -100 DEG C obtains extract, and acquisition water is eluted with water and mentions
Object；
(2) ethyl alcohol progress alcohol precipitation is added in the water extract obtained into step (1) and obtains alcohol precipitation Thick many candies；
(3) the alcohol precipitation Thick many candies that step (2) obtain are obtained slightly according to solid-liquid ratio 1:18~1:22 (g/mL) is soluble in water
Polysaccharide solution is later that 3:1~5:1 addition sevag reagent carries out de- egg according to the volume ratio of Thick many candies solution and sevag reagent
White, after obtaining de- albumen Thick many candies；And
(4) Thick many candies after the de- albumen of step (3) acquisition are subjected to depigmentation with macroreticular resin.
Preferably, in the step (1), heating extraction is refluxing extraction, and the solid-liquid ratio of heating extraction is 1:8-1:12
(kg/L), it extracts 2-4 hours；Preferably, the residue after the heating extraction continues according to solid-liquid ratio to be 1:8-1:12 (kg/L)
It carries out refluxing extraction 2-3 times, extracts 2-4h, combined extract every time.
Preferably, it obtains after extract in step (1) by extract macroporous resin adsorption and acquisition water is eluted with water
Extract.
Preferably, water extract is water extract aqueous solution that solid-liquid ratio is 1:3-1:6 (g/mL) in the step (2)；It is described
Ethyl alcohol is dehydrated alcohol in step (2), and the volume ratio of the dehydrated alcohol and water extract aqueous solution is 3:1-5:1.
Preferably, water extract is water extract aqueous solution that solid-liquid ratio is 1:5 (g/mL) in the step (2)；The step
(2) ethyl alcohol is dehydrated alcohol in, and the volume ratio of the dehydrated alcohol and water extract aqueous solution is 4:1.
Preferably, alcohol precipitation carries out at room temperature in the step (2), and the alcohol precipitation time is greater than 8 hours.
Preferably, the volume ratio of Thick many candies solution and sevag reagent is 4:1 in the step (3).
Preferably, repeat the aqueous layer and organic alternate no albumen of step (3) after centrifugation before step (4)
Layer, the aqueous layer solution after obtaining de- albumen is as the Thick many candies after de- albumen, specifically, repeating following steps: Xiang Suoshu
Sevag reagent is added in Thick many candies solution, is centrifuged after fulling shake, aqueous layer simultaneously repeats de- albumen operation until centrifugation
Aqueous layer and organic alternate no albumin layer afterwards, the aqueous layer solution after obtaining de- albumen are the Thick many candies after de- albumen；It is excellent
The dehydrated alcohol of 4-5 times of volume is further added in the aqueous layer solution after the de- albumen, is centrifuged to obtain de- egg after alcohol precipitation for selection of land
Thick many candies after white.
Preferably, in the step (3), it is slowly added to 4-5 times while stirring into the aqueous layer solution after de- albumen
The dehydrated alcohol of volume, at 4 DEG C, alcohol precipitation is overnight, and then 5000rpm is centrifuged 10 minutes, obtains de- albumen Thick many candies (DPCSP).
Preferably, in the step (4), macroreticular resin is AB-8 macroreticular resin, D-941 macroreticular resin, D-113 macropore
Resin or D-101 macroreticular resin；It is highly preferred that macroreticular resin is AB-8 macroreticular resin in the step (5).
Preferably, the step (4) are as follows: the Thick many candies after the de- albumen obtained in step (3) are added to AB-8 macropore tree
On rouge column, eluent is eluted with water to obtain, is concentrated, and is dialysed with 2kDa bag filter pure water, solution in the bag filter after being dialysed；
Preferably, solution is further freeze-dried and obtains depigmentation Thick many candies (DCCSP) in the bag filter.
Preferably, the present invention provides a kind of preparation method of Chinese caterpillar fungus polysaccharide extract, include the following steps:
(1) by the cordyceps sinensis of crushing with water refluxing extraction 2-4 hours, the solid-liquid ratio of extraction is 1:8-1:12 (kg/L)；
Preferably, the residue after the refluxing extraction continues to be 1:8-1:12 (kg/L) progress refluxing extraction 2-3 times according to solid-liquid ratio, often
Secondary extraction 2-4h, combined extract；
Extracting solution is concentrated to get extract, with HP-20 macroporous resin adsorption and is eluted with water, eluent concentration, freezing
Water extract is obtained after drying；
(2) water extract obtained in step (1) is obtained into water extract according to solid-liquid ratio 1:3~1:6 (g/mL) is soluble in water
Aqueous solution；Then dehydrated alcohol progress alcohol precipitation is added and obtains alcohol precipitation Thick many candies, wherein the dehydrated alcohol and water extract aqueous solution
Volume ratio be 3:1-5:1；
(3) the alcohol precipitation Thick many candies that step (2) obtain are added to the water to obtain slightly according to solid-liquid ratio 1:18~1:22 (g/mL)
Polysaccharide solution is added sevag reagent according to the volume ratio 4:1 of Thick many candies solution and sevag reagent later and carries out de- albumen, specifically
Operation are as follows: sevag reagent is added in Xiang Suoshu Thick many candies solution, is centrifuged after fulling shake, aqueous layer simultaneously repeats de- egg
Aqueous layer and organic alternate no albumin layer of the white operation after centrifugation, the aqueous layer solution after obtaining de- albumen are to take off albumen
Thick many candies afterwards；Preferably, the dehydrated alcohol of 4-5 times of volume, alcohol are further added in the aqueous layer solution after the de- albumen
It is heavy to be then centrifuged for obtaining the Thick many candies after de- albumen；And
(4) Thick many candies after the de- albumen of step (3) acquisition are added in AB-8 macroporous resin column, are eluted with water to obtain elution
Liquid, concentration, and solution in the bag filter after being dialysed with 2kDa bag filter pure water；Preferably, molten in the bag filter
Liquid, which is further freeze-dried, obtains depigmentation Thick many candies (DCCSP).
Preferably, the present invention provides a kind of preparation method of Chinese caterpillar fungus polysaccharide extract, include the following steps:
(1) by the cordyceps sinensis of crushing with water refluxing extraction 2-4 hours, the solid-liquid ratio of extraction is 1:8-1:12 (kg/L),
Residue after the refluxing extraction continues to be that 1:8-1:12 (kg/L) is carried out refluxing extraction 2-3 times according to solid-liquid ratio, extracts every time
2-4h, combined extract；
Extracting solution is concentrated to get extract, with HP-20 macroporous resin adsorption and is eluted with water, eluent concentration, freezing
Water extract is obtained after drying；
(2) by the water extract obtained in step (1), according to solid-liquid ratio 1:5 (g/mL), soluble in water to obtain water extract water-soluble
Liquid；Then dehydrated alcohol progress alcohol precipitation is added and obtains alcohol precipitation Thick many candies, wherein the body of the dehydrated alcohol and water extract aqueous solution
Product is than being 4:1；
(3) alcohol precipitation Thick many candies that step (2) obtain are added to the water to obtain Thick many candies according to solid-liquid ratio 1:20 (g/mL) molten
Liquid is added sevag reagent according to the volume ratio 4:1 of Thick many candies solution and sevag reagent later and carries out de- albumen, concrete operations
Are as follows: it is centrifuged after being fullyd shake after addition sevag reagent into Thick many candies solution, it is straight that aqueous layer simultaneously repeats de- albumen operation
Aqueous layer and organic alternate no albumin layer after to centrifugation, the aqueous layer solution after obtaining de- albumen；Water after the de- albumen
The dehydrated alcohol of 4-5 times of volume is further added in phase layer solution, then 5000rpm centrifugation 10min obtains de- egg in 4 DEG C of alcohol precipitations
Thick many candies after white；And
(4) Thick many candies after the de- albumen of step (3) acquisition are added in AB-8 macroporous resin column, are eluted with water to obtain elution
Liquid, concentration, and dialyse solution in the bag filter after obtaining with 2kDa bag filter pure water, it is described after bag filter in solution it is further
Freeze-drying obtains depigmentation Thick many candies (DCCSP).
Second aspect, the present invention provides a kind of Chinese caterpillar fungus polysaccharide extractions being prepared by the method for the invention
Object.
The third aspect, the present invention provides a kind of Chinese caterpillar fungus polysaccharide extract, total sugar content 85%-90%, packets
Containing low-molecular-weight polysaccharide and high molecular weight polysaccharide, the low-molecular-weight polysaccharide is that average molecular weight is the more of 6.5kDa-7.0kDa
Sugar, accounts for about the 75% of total reducing sugar in mass, and the average molecular weight of the high molecular weight polysaccharide is greater than 670kDa, accounts in mass
About the 25% of total reducing sugar.
Preferably, the average molecular weight of low-molecular-weight polysaccharide is 6.86kDa in the Chinese caterpillar fungus polysaccharide extract.
Preferably, the Chinese caterpillar fungus polysaccharide extract through gel chromatography analysis shows that at retention time 9.610 minutes,
Three peaks are shown altogether within 12.225 minutes and 17.705 minutes, respectively correspond molecular weight greater than 670kDa and account for 3.328%；Molecular weight is big
21.849 are accounted in 670kDa；And molecular weight is that 6.86kDa accounts for 74.829%.
Fourth aspect, the present invention provides the Chinese caterpillar fungus polysaccharide extract prepared by method of the invention or institutes of the present invention
The Chinese caterpillar fungus polysaccharide extract stated is in preparation for the application in anti-tumor drug.
Preferably, the tumour is breast cancer, lymph cancer, leukemia, melanoma, colon cancer and lung cancer.
Preferably, the tumour is breast cancer MDA-MB-453, lymph cancer Raji, leukemia K-562 and melanoma SK-
MEL-28。
Heretofore described " solid-liquid ratio " refers to the ratio of inventory Yu solvent for use amount.In step of the present invention (1) extraction
Solid-liquid ratio is 1:8-1:12 (kg/L), indicates that the ratio of cordyceps sinensis and Extraction solvent water is 1:8-1:12 (kg/L), that is, indicates
When being extracted to 1kg cordyceps sinensis with water, the volume of water used is 8L-12L.It in addition is 1:3- if water extract is solid-liquid ratio
The water extract aqueous solution of 1:6 (g/mL), then it represents that the ratio of not solvent-laden water extract and aqueous solvent is 1:3-1:6 after concentration
(g/mL) the water extract aqueous solution being mixed to get, that is, indicate when to 1g water extract water dissolution aqueous solution is made when, water used
Volume is 3mL-18mL；Sevag reagent of the present invention is after chloroform is mixed with n-butanol according to volume ratio 4:1 (mL:mL)
Solution.
Heretofore described " about " indicates an approximate number value or range, for example " accounts in mass big in the present invention
About 75% " expression low-molecular-weight polysaccharide quality accounts for total polysaccharide extractive mass ratio about 75%, i.e., in 75 ± 5% ranges all
Belong to the range that " about 75% " defines, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%/78%., 79%,
80% range belongs to the range of " about 75% "；Preferably, belong to what " about 75% " defined in 75 ± 3% ranges
Range.Equally, " accounting for about 25% in mass " indicates that high molecular weight polysaccharide quality accounts for total polysaccharide extractive mass ratio about
25%, i.e., the range that " about 75% " defines is belonged in 25 ± 5% ranges；Preferably, it is belonged in 25 ± 3% ranges
The range that " about 25% " defines.
The preparation method of Chinese caterpillar fungus polysaccharide extract of the invention is simple, convenient, and the polyoses extract of acquisition is total
Sugared content is high, and activity is high.
D113 cation exchange resin is a kind of macroporous type acidulous acrylic acid cation exchanger resin, in its crosslinking third
Contain carboxylic acid function's base (- COOH) in olefin(e) acid skeleton.The resin is mainly used for Treatment of Industrial Water, can be used at industrial wastewater
Reason, the separation and purification of metal recovery, biochemical drug.
The D941 anion exchange resin resin is the methyl acrylate copoly cross-linked polymer polymer of macroporous structure, is led to
It crosses polyethylene polyamine and carries out the polyamines base weak-base ion exchange resin that aminolysis obtains, be mainly used in the food industry such as carbohydrate
Refinery decolorization, the decolorizing and refining of the natural drugs such as stevioside, ginsenoside, notoginsenoside, antibiotic.
D101 resin is a kind of spherical, non-polar polymer adsorbent.The resin is a cross-linked styrene-divinylbenzene
Copolymer.Based on suitable aperture and specific surface, which has special selectivity to saponin and flavones.The resin is suitable for from water
Saponin, flavonoids and certain organic matters are extracted in solution.
AB-8 resin is that one kind is spherical, (annotation: the resin surface has certain ester to polystyrene type low pole absorption resin
Base, original are designated as low pole；But adsorption mechanism is still hydrophobicity, is now set to nonpolarity).The resin is a kind of cross-linked polymer, it
It is different from the hydrophobic adsorption agent of early stage, it attached slightly water-wet group in its skeleton structure；Again not with general ion exchange resin
Together, only non-ionic function base in its structure.The specific surface area of the resin and aperture are larger, and being suitable for adsorbing all kinds of has
Certain hydrophobic traditional Chinese medicine ingredients, adsorbance is larger, and elution is easy, and adsorption dynamics adsorption kinetics is functional.To heat, organic solvent and one
As acid, alkali under use condition stablize, therefore service life is longer.It is hydrophilic to albumen, carbohydrate, inorganic acid, alkali, salt, small molecule
Property organic matter does not adsorb, thus can separate general traditional Chinese medicine ingredients with these substances.Optimum is water-soluble, has low pole object
The extracting and developing of matter, purifying.
Detailed description of the invention
Fig. 1 is the gel chromatography figure of the Chinese caterpillar fungus polysaccharide extract DCCSP obtained.
Specific embodiment
As described below is the preferred embodiment of the present invention, and what the present invention was protected is not limited to following preferred implementation side
Formula.It should be pointed out that for those skilled in the art on the basis of the inventive concept, several deformations for making and
It improves, belongs to protection scope of the present invention.Manufacturer person is not specified in agents useful for same, and being can be by the routine of commercially available acquisition
Product.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in
Within protection scope of the present invention.
Dry cordyceps sinensis crude drug source is in Yichang mountain city Shui Dou cordyceps sinensis Co., Ltd in the embodiment of the present invention.
Embodiment
1. reagent and consumptive material
Table 1:
2. instrument and equipment
Table 2:
Embodiment 1
The extraction of Chinese caterpillar fungus polysaccharide:
(1) alcohol precipitation
By the dry cordyceps sinensis pulverizing medicinal materials of 15Kg, hot water return is extracted, is extracted altogether 3 times with 120L water every time, is extracted every time
2h.Combined extract, concentration, extracting solution HP-20 macroporous resin adsorption is eluted with water, and eluent concentration, freeze-drying obtain
Obtain cordyceps summer grass water extract solid.
300g cordyceps sinensis water extract solid is placed in 5L beaker, 1500mL water is added, 60 DEG C of water-bath dissolutions are cooling,
Obtain the cordyceps sinensis water extract solution that solid-liquid ratio is 1:5 (g/mL).Cordyceps sinensis water extract solution even is dispensed into 2 5L measuring cups, point
Not Jia Ru 4 times of volume dehydrated alcohols, sealed with preservative film, at room temperature alcohol precipitation stay overnight.Then, divided with 3000g centrifugal force 30
Clock merges alcohol hypostasis, and 80 DEG C of heating water baths volatilize ethyl alcohol, obtains alcohol precipitation Thick many candies 46.5g.
(2) Sevag takes off albumen
Alcohol precipitation Thick many candies are dissolved in 1L water, solution is dispensed into Centrifuge Cup by 80 DEG C of water-baths dissolution, by 4:1 (sample:
Sevag reagent, V/V) Sevag reagent (chloroform: n-butanol=4:1, V/V) is added, it vibrates 30 minutes, 4000rpm is centrifuged 10 points
Clock, solution is divided into three layers after centrifugation, is followed successively by organic layer, albumin layer and aqueous layer from the bottom up, and aqueous layer repeats this
De- albumen step 11 time, until water phase and organic alternate without obvious albumin layer.Slowly add into the aqueous layer solution after de- albumen
Enter the dehydrated alcohol of 4 times of volumes, it is stirring while adding, it is then stayed overnight in 4 DEG C of alcohol precipitations, 5000rpm is centrifuged 10 minutes, obtains de- albumen
Thick many candies (DPCSP).
(3) depigmentation
Weigh respectively pretreatment and filtered macroreticular resin D113, D941, D101 and AB-8 about 2g in beaker, respectively plus
Enter 10mL polysaccharide solution (the de- albumen Thick many candies that above-mentioned steps (2) obtain are dissolved in gained in 600mL water), 120 beats/min of vibrations
It swings 3 hours, takes 5mL supernatant in colorimetric cylinder after centrifugation, after the most shallow macroreticular resin of supernatant fluid color carries out after selection absorption
Continuous test.Color depth is D113 > D941 > D101 > AB-8.
Therefore, AB-8 macroreticular resin is used in the present embodiment.The de- albumen Thick many candies that step (2) obtain are dissolved in
In 600mL water, polysaccharide solution is added on AB-8 macroporous resin column (5.0 × 55cm), is eluted with water to eluent and phenol sulphur
Acid do not develop the color, merge eluent, concentration, concentrate dialysed with 2k Da bag filter pure water after bag filter in solution,
Solution in the bag filter is freeze-dried, depigmentation Thick many candies (DCCSP) powder is obtained.The total reducing sugar of Phenol sulfuric acid procedure measurement DCCSP
Content is 87.42%.
Embodiment 2
Depigmentation Thick many candies (DCCSP) anti-tumor experiment:
(1) cell culture: by leukemia K-562 cell, breast cancer MDA-MB-453 cell, lymthoma cancer Raji cell and knot
Carcinoma of the rectum COLO205 cell is placed in DMEM culture medium, and melanoma SK-MEL-28 cell is placed in RPMI-1640
In (Roswell Park Memorial Institute-1640) culture medium, it is then placed in 37 DEG C, the training of 5% carbon dioxide
It supports and is cultivated in case.Daily inverted microscope is observed cell 1 time, and it is primary to change culture solution every 2 days.Cell in culture bottle is grown into
Cell passage is carried out when 85%~95% fusion.Old culture solution is discarded, PBS repeated flushing 2 times, 0.25% pancreatin digestive juice is added
After (except HL-60) 2mL, cell Bian Yuan ﹑ float, 4mL culture solution is added and terminates digestion, transfers to 15mL sterile centrifugation tube
In, it is centrifuged 4 minutes within 1000r/ minutes, abandons supernatant, be transferred in new sterile culture flask and cultivated with 1:4 ratio.
(2) drug is prepared: being accurately weighed depigmentation Thick many candies (DCCSP) powder that embodiment 1 is prepared respectively and is extremely trained
It supports in base, is configured to the solution of 6mg/mL, with the concentration for initial final concentration, be diluted to a series of gradient concentrations according to 3 times, altogether
If 9 concentration points, then through 0.22 μm of sterile filters filtration sterilization.
(3) Activity determination: collecting logarithmic growth phase cell, counts, and with complete medium again suspension cell, adjusts cell
Concentration is inoculated with 96 orifice plates to suitable concentration.At 37 DEG C, 5%CO2Under the conditions of be incubated for 24 hours after.It is separately added by 100 holes μ L/
Depigmentation Thick many candies (DCCSP).Then, 37 DEG C are placed in, 5%CO2In incubator, it is incubated for 72 hours.It is (thin that 10%CCK-8 is added
Born of the same parents' counting reagent kit -8) reagent, it is placed in 37 DEG C of incubators and is incubated for 1-2 hours, the absorbance in each hole is detected at microplate reader 450nm
(A) value, data processing software calculate separately IC50 value (mg/mL) of each drug at 72 hours.As a result as shown in the table:
Table 3: IC50 value of the drug to tumour cell
| Cell | DCCSP(mg/mL) |
| RAJI | 0.1993 |
| K-562 | 0.754 |
| COLO205 | 1.586 |
| MDA-MB-453 | 0.013 |
| SK-MEL-28 | 1.167 |
Embodiment 3
Depigmentation Thick many candies (DCCSP) molecular weight distribution determination method
(1) prepared by solution:
20mM ammonium acetate: weighing ammonium acetate 1.54g, and water 1000mL is added to dissolve, filtering, ultrasonic degassing.
Serial glucan reference substance solution: appropriate different molecular weight (1,5,12,25,50,80,150,410,670kDa) is taken
Glucan reference substance be configured to the solution of 1mg/mL, cross 0.45 μm of film.
DCCSP solution: taking appropriate DCCSP to be configured to 2mg/mL solution, crosses 0.45 μm of film.
(2) gel chromatography is analyzed: chromatographic column: TSKgel G5000PWXL (7.8mm × 30cm, 10 μm) and guard column
TSKgel guardcolumn PWXL(6.0mm×4cm)；Mobile phase: 20mM ammonium acetate, isocratic elution；Flow velocity: 0.6mL/
min；Column temperature: room temperature；Sample volume: 20 μ L；Evaporating temperature: 30 DEG C；Drift tube temperature: 30 DEG C；Carrier gas: air, flow velocity
1.60SLM；Yield value: 1.0.
(3) data processing: linear regression analysis is carried out to retention time with the logarithm of dextran molecule amount, obtains molecular weight
Calibration curve.According to the retention time of chromatographic peak each in test solution, corresponding molecular weight is acquired by calibration curve.With area
Normalization method calculates each component ratio shared by analyzed chromatographic peak.
(4) result: using the logarithm of serial dextran molecule amount as ordinate, retention time is that abscissa carries out recurrence point
Analysis, obtains regression equation y=-0.4218x+11.304, R2=0.9762.As seen from Figure 1, DCCSP is by three major part groups
At can be calculated by regression equation, peak 1,2 molecular weight are all larger than 670kDa, and 3 molecular weight of peak is 6.86kDa.Pass through normalization method
The ratio for obtaining appearance 1,2 and 3 is 3.328%, 21.849% and 74.829%.
Table 4- polysaccharide DCCSP retention time (min) and molecular weight (Da)
| Peak title | Retention time | Molecular weight | Accounting % |
| 1 | 9.610 | >670kDa | 3.328 |
| 2 | 12.225 | >670kDa | 21.849 |
| 3 | 17.705 | 6.86kDa | 74.829 |
Claims (14)
1. a kind of preparation method of Chinese caterpillar fungus polysaccharide extract, includes the following steps:
(1) cordyceps sinensis is obtained into extract with water heating extracting, and acquisition water extract is eluted with water；
(2) ethyl alcohol progress alcohol precipitation is added in the water extract obtained into step (1) and obtains alcohol precipitation Thick many candies；
(3) by step (2) obtain alcohol precipitation Thick many candies it is soluble in water obtain Thick many candies solution, later according to Thick many candies solution with
The volume ratio of sevag reagent is that 3:1~5:1 addition sevag reagent carries out de- albumen, the Thick many candies after obtaining de- albumen；And
(4) Thick many candies after the de- albumen of step (3) acquisition are subjected to depigmentation with macroreticular resin.
2. a kind of preparation method of Chinese caterpillar fungus polysaccharide extract, includes the following steps:
(1) by cordyceps sinensis, with water, the heating extraction at 80 DEG C -100 DEG C obtains extract, and acquisition water extract is eluted with water；
(2) ethyl alcohol progress alcohol precipitation is added in the water extract obtained into step (1) and obtains alcohol precipitation Thick many candies；
(3) the alcohol precipitation Thick many candies that step (2) obtain are obtained into Thick many candies according to solid-liquid ratio 1:18~1:22 (g/mL) is soluble in water
Solution is later that 3:1~5:1 addition sevag reagent carries out de- albumen according to the volume ratio of Thick many candies solution and sevag reagent,
Thick many candies after obtaining de- albumen；And
(4) Thick many candies after the de- albumen of step (3) acquisition are subjected to depigmentation with macroreticular resin.
3. method according to claim 1 or 2, in the step (1), heating extraction is refluxing extraction, heating extraction
Solid-liquid ratio is 1:8-1:12 (kg/L), is extracted 2-4 hours；Preferably, the residue after the heating extraction continues according to solid-liquid ratio
For 1:8-1:12 (kg/L) progress refluxing extraction 2-3 times, extract 2-4 hours every time, combined extract；Preferably, in step (1)
It obtains extract macroporous resin adsorption after extract and acquisition water extract is eluted with water.
It is 1:3-1:6 (g/mL) that 4. method according to claim 1 or 2, in the step (2), water extract, which is solid-liquid ratio,
Water extract aqueous solution；Ethyl alcohol is dehydrated alcohol in the step (2), and the volume ratio of the dehydrated alcohol and water extract aqueous solution is
3:1-5:1；Preferably,
Water extract is the water extract aqueous solution that solid-liquid ratio is 1:5 (g/mL) in the step (2)；Ethyl alcohol is in the step (2)
The volume ratio of dehydrated alcohol, the dehydrated alcohol and water extract aqueous solution is 4:1.
5. method according to claim 1 or 2, alcohol precipitation carries out at room temperature in the step (2), and the alcohol precipitation time is greater than 8
Hour.
6. method according to claim 1 or 2, Thick many candies solution and the volume ratio of sevag reagent are in the step (3)
4:1。
7. method according to claim 1 or 2 repeats the water phase of step (3) after centrifugation before step (4)
Layer and organic alternate no albumin layer, the aqueous layer solution after obtaining de- albumen is as the Thick many candies after de- albumen；
Preferably, the dehydrated alcohol of 4-5 times of volume is further added in the aqueous layer solution after the de- albumen, is centrifuged after alcohol precipitation
Thick many candies after albumen must be taken off.
8. method according to claim 1 or 2, in the step (4), macroreticular resin is AB-8 macroreticular resin, D-941
Macroreticular resin, D-113 macroreticular resin or D-101 macroreticular resin；It is preferred that AB-8 macroreticular resin.
9. method according to claim 1 or 2, wherein step (4) are as follows: thick more after the de- albumen for obtaining step (3)
Sugar is added in AB-8 macroporous resin column, is eluted with water to obtain eluent, concentration, and is dialysed with 2kDa bag filter pure water
Solution in bag filter afterwards；Preferably, solution is freeze-dried in the bag filter obtains Chinese caterpillar fungus polysaccharide extract
(DCCSP)。
10. the Chinese caterpillar fungus polysaccharide extract prepared by any one of claim 1-9 the method.
11. a kind of Chinese caterpillar fungus polysaccharide extract, total sugar content 85%-90% include low-molecular-weight polysaccharide and macromolecule
Polysaccharide is measured, the low-molecular-weight polysaccharide is the polysaccharide that average molecular weight is 6.5kDa-7.0kDa, accounts for total reducing sugar in mass about
75%, the average molecular weight of the high molecular weight polysaccharide is greater than 670kDa, accounts for about the 25% of total reducing sugar in mass.
12. Chinese caterpillar fungus polysaccharide extract according to claim 11, the average molecular weight of the low-molecular-weight polysaccharide are
6.86kDa。
13. Chinese caterpillar fungus polysaccharide extract according to claim 11, through gel chromatography analysis shows that dividing in retention time 9.610
Clock shows three peaks in 12.225 minutes and 17.705 minutes altogether, respectively corresponds molecular weight greater than 670kDa and accounts for 3.328%；Molecule
Amount is greater than 670kDa and accounts for 21.849%；And molecular weight is that 6.86kDa accounts for 74.829%.
14. the Chinese caterpillar fungus polysaccharide extract or claim 10- that are prepared by any one of claim 1-9 the method
13 described in any item Chinese caterpillar fungus polysaccharide extracts are in preparation for the application in anti-tumor drug.
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