CN110183542A - A kind of extracting method of poplar Phellinus polysaccharide - Google Patents
A kind of extracting method of poplar Phellinus polysaccharide Download PDFInfo
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- CN110183542A CN110183542A CN201910342959.6A CN201910342959A CN110183542A CN 110183542 A CN110183542 A CN 110183542A CN 201910342959 A CN201910342959 A CN 201910342959A CN 110183542 A CN110183542 A CN 110183542A
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 95
- 241000123107 Phellinus Species 0.000 title claims abstract description 81
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 78
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 78
- 241000219000 Populus Species 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000006228 supernatant Substances 0.000 claims abstract description 29
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- 229920005989 resin Polymers 0.000 claims abstract description 22
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- 238000005119 centrifugation Methods 0.000 claims abstract description 13
- 238000010257 thawing Methods 0.000 claims abstract description 12
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 11
- 238000004108 freeze drying Methods 0.000 claims abstract description 10
- 238000009835 boiling Methods 0.000 claims abstract description 7
- 239000000047 product Substances 0.000 claims abstract description 6
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 5
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- 238000007710 freezing Methods 0.000 claims description 7
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- 240000000249 Morus alba Species 0.000 claims description 6
- 235000008708 Morus alba Nutrition 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 239000000659 freezing mixture Substances 0.000 claims 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 11
- 206010061218 Inflammation Diseases 0.000 abstract description 10
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- 230000000694 effects Effects 0.000 abstract description 8
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- 238000000746 purification Methods 0.000 abstract description 3
- 238000001816 cooling Methods 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 13
- 239000012535 impurity Substances 0.000 description 11
- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 238000000502 dialysis Methods 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 239000000049 pigment Substances 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 5
- 238000004452 microanalysis Methods 0.000 description 4
- 150000002772 monosaccharides Chemical class 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 229920001503 Glucan Polymers 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000921 elemental analysis Methods 0.000 description 3
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- 210000002540 macrophage Anatomy 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000012424 Freeze-thaw process Methods 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
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- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical group O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
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- 235000002992 Betula pubescens Nutrition 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010008354 Cervix neoplasm Diseases 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- 241000746344 Sanghuangporus vaninii Species 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
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- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- CJOBVZJTOIVNNF-UHFFFAOYSA-N cadmium sulfide Chemical compound [Cd]=S CJOBVZJTOIVNNF-UHFFFAOYSA-N 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
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- 230000008025 crystallization Effects 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
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Classifications
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Sustainable Development (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention provides a kind of extracting methods of poplar Phellinus polysaccharide, including Phellinus fructification is crushed to 1-5 mesh, with physiological saline impregnate 8~for 24 hours, residue is collected after centrifugation；Then Phellinus fructification residue is boiled into 20~40min under 110~130 DEG C of boiling water, residue is collected after centrifugation；Further thaw in residue plus after water cooling jelly 24-36h；In defrosting mixture plus water obtains mixed liquor, then complex enzyme will be added in mixed liquor, and 2~4h is digested at 50-60 DEG C, is centrifugated to obtain supernatant；It will finally dialyse after supernatant resin adsorption, concentration and freeze-drying obtain poplar Phellinus polysaccharide.The extracting method of poplar Phellinus polysaccharide proposed by the present invention; with the apparent activity for inhibiting tumor cell proliferation; and there is good biocompatibility relative to the polysaccharide of chemical method extraction purification; inflammatory reaction will not be brought, poplar Phellinus polysaccharide of the present invention can be used in anti-tumor drug or health care product.
Description
Technical field
The present invention relates to medicine extractive technique fields, in particular to a kind of extracting method of poplar Phellinus polysaccharide.
Background technique
Phellinus is a kind of medicinal fungi of preciousness.Because the bacterium is usually grown on Mulberry plant in China ALFISOL IN CENTRAL, son
Solid color cadmium yellow and gain the name.Phellinus has had history of more than one thousand years in China as traditional Chinese medicine, in history tree works, mulberry
Yellow also known as Sang Chen, mulberry ear, Phellinus mushroom etc..Phellinus can generally parasitize the tree of the broad leaf trees such as mulberry tree, poplar, white birch, peach, willow
On dry, because its raw tree species is different, kind is not also identical, and ingredient is also multifarious, this also results in the mulberry of a large amount of Science Reports
Yellow polysaccharide component is multifarious, and basic reason is exactly to have obscured the source of raw material.Poplar Phellinus (Phellinus
Vaninii Ljup) be Phellinus one kind, be born in poplar.Also referred to as " North Korea's Phellinus " is produced in one band of Changbai mountain, Jilin.City at present
Circulation is maximum on field, the Phellinus that Korean is most easily accepted by, and the strain and the immediate kind of shape cultivated with South Korea have anti-
The functions such as tumour, strengthen immunity, anti-oxidant and protect liver.
Poplar Phellinus fructification is in lignifying, contains a large amount of chitins and cellulose family macromolecular in cell wall, causes poplar
It is difficult to set Phellinus Polyose extraction.In existing result of study, extracting method is mainly water extract-alcohol precipitation, and recovery rate is lower.Before
Research in, the poplar Phellinus fructification after degreasing is subjected to gradually broken wall, is successively extracted two kinds of polysaccharide with hot water and lye,
Wherein the polysaccharide yield of hot water extraction is lower, and bioactivity is also poor；The polysaccharide yield further extracted with lye is higher, still
It is found in follow-up study, alkali-extracted polysaccharide meeting stimulating expression of macrophage generates a large amount of inflammatory factors, may bring inflammatory reaction, right
Its bio-medical causes certain negative effect.
Therefore it provides a kind of extracting method of poplar Phellinus polysaccharide, improves poplar Phellinus polysaccharide yield and obtains with good
The problem of poplar Phellinus polysaccharide of good biocompatibility is those skilled in the art's urgent need to resolve.
Summary of the invention
The present invention is based at least one above-mentioned technical problem, proposes a kind of extracting method of poplar Phellinus polysaccharide,
First after physiological saline and hot water extract removal of impurities repeatedly, takes cryogenic freezing-microwave quick-thawing method and freeze repeatedly
Melt Phellinus fructification cell, be sufficiently destroyed its cell wall structure, improves the extraction efficiency of poplar Phellinus polysaccharide；Secondly macropore is used
The suction-operated of resin takes off albumen and sloughs pigment, and it is small not introduce other chemistry also under the effect that guarantee cleans to greatest extent
Molecule greatly reduces the bring inflammatory reaction of proposed polysaccharide.
In view of this, including the following steps: the invention proposes a kind of extracting method of poplar Phellinus polysaccharide
(1) Phellinus fructification is crushed to 1-5 mesh, with physiological saline impregnate 8~for 24 hours, repeat 1~3 time, be collected after centrifugation
Residue；
(2) Phellinus fructification residue is boiled under 110~130 DEG C of boiling water 20~40min, repeats 1~3 time, is received after centrifugation
Collect residue；
(3) add water in the residue in step (2), thaw after-15-- 10 DEG C of freezing 24-36h；Freeze-thaw process weight
It is 1-3 times multiple；
(4) add water to obtain mixed liquor in the defrosting mixture of step (3), then complex enzyme, 50-60 will be added in mixed liquor
2~4h is digested at DEG C, is centrifugated to obtain supernatant；
(5) supernatant is cleaned 1~3 time with resin adsorption, and rear dialysis, concentration and freeze-drying obtain poplar Phellinus polysaccharide.
A kind of extracting method of poplar Phellinus polysaccharide provided by the invention, substantially physics extraction process, were being extracted
Other chemical small molecules are not introduced in journey, the extraction efficiency of poplar Phellinus polysaccharide is 6.8%, the poplar Phellinus polysaccharide extracted
With good biocompatibility, and there is the apparent activity for inhibiting tumor cell proliferation, inflammatory reaction will not be brought.
Further, the additional amount of step (2) Phellinus fructification residue and boiling water is 60-80g/L.
Further, the additional amount of residue and water is 200g/L in step (3).
Further, defreezing method in step (3) are as follows: microwave thaws rapidly, power 1000-3000W.
Cryogenic freezing-microwave quick-thawing method multigelation Phellinus fructification cell is wherein utilized, it is sufficiently destroyed
Cell wall structure improves the extraction efficiency of poplar Phellinus polysaccharide.
Further, the additional amount of defrosting mixture and water is 60-80g/L in step (4).
Further, complex enzyme includes cellulase and chitinase in step (4)；Its cellulase and chitinase
Mass ratio is 1:1~3.
Further, the additional amount of complex enzyme is 0.2/10000th~the 0.5 of mixed liquor weight in step (4).
Further, resin is HP20 macroreticular resin in step (5)；Wherein the mass ratio of macroreticular resin and supernatant is 1:
20~50.
Albumen wherein is taken off using the suction-operated of HP20 macroreticular resin and sloughs pigment, in the effect that guarantee cleans to greatest extent
It does not introduce other chemical small molecules under fruit also, greatly reduces the bring inflammatory reaction of proposed polysaccharide.
Based on the second aspect of the present invention, a kind of poplar Phellinus polysaccharide is proposed in preparing anti-tumor drug or health care product
Application.
By above technical scheme, there is the poplar Phellinus polysaccharide that the method for the present invention obtains apparent inhibition tumour cell to increase
The activity grown, and there is good biocompatibility relative to the polysaccharide of chemical method extraction purification, inflammation will not be brought anti-
It answers, and poplar Phellinus polysaccharide of the present invention can be used in anti-tumor drug or health care product.It also have the advantage that
(1) poplar Phellinus polysaccharide origin is in specialty drug resource poplar Phellinus abundance, low in cost；
(2) the polysaccharide extracting process environment-friendly and green, essentially physics extraction process, and recovery rate is 6.8%；
(3) freeze-thaw method is used in the polysaccharide extracting process, is melted broken wall using ice crystal speed, is substantially increased extraction efficiency；
(4) polysaccharide has no toxic side effect, and biocompatibility is good.
Detailed description of the invention
Fig. 1 shows poplar Phellinus polysaccharide PV-F infrared spectrogram in the embodiment of the present invention 2.
Fig. 2 shows poplar Phellinus polysaccharide PV-F carbon-13 nmr spectras in the embodiment of the present invention 2.
Fig. 3 shows the suppression that the poplar Phellinus polysaccharide PV-F of various concentration in the embodiment of the present invention 2 grows Hela cell
Rate processed.
Fig. 4 shows the suppression that the poplar Phellinus polysaccharide PV-F of various concentration in the embodiment of the present invention 2 grows HepG2 cell
Rate processed.
Fig. 5 shows the poplar Phellinus polysaccharide PV-F of various concentration in the embodiment of the present invention 2 to macrophage RAW 264.7
The influence of inflammatory factor NO burst size.
Specific embodiment
To better understand the objects, features and advantages of the present invention, With reference to embodiment
The present invention is further described in detail.It should be noted that in the absence of conflict, the embodiment of the present invention and reality
The feature applied in example can be combined with each other.
In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention, still, the present invention may be used also
To be implemented using other than the one described here other modes, therefore, protection scope of the present invention is not by described below
Specific embodiment limitation.
Embodiment 1
Commercially available poplar Phellinus fructification is wherein bought, the place of production is Changbai Mountain area.
A kind of extracting method of poplar Phellinus polysaccharide, includes the following steps:
(1) Phellinus fructification is crushed to 1-5 mesh, with physiological saline impregnate 8~for 24 hours, repeat 1~3 time, be collected after centrifugation
Residue；
(2) Phellinus fructification residue is boiled into 20~40min under 110~130 DEG C of boiling water, wherein Phellinus fructification residue with
The additional amount of boiling water is 60-80g/L；It repeats 1~3 time, residue is collected after centrifugation；
(3) add water in the residue in step (2), wherein the additional amount of residue and water is 200g/L；At-15-- 10 DEG C
Microwave thaws rapidly after freezing 24-36h, power 1000-3000W；Freeze-thaw process repeats 1-3 times；
(4) water is added its ratio be 60-80g/L to obtain that weight is added in mixed liquor in the defrosting mixture of step (3)
For the complex enzyme of mixed liquor weight 02/10000th~0.5,2~4h is digested at 50-60 DEG C, is centrifugated to obtain supernatant；
(5) supernatant is cleaned 1~3 time with HP20 macroporous resin adsorption, and wherein the mass ratio of macroreticular resin and supernatant is
1:20~50, carry out clear water afterwards and secondary distilled water dialysis, concentration and freeze-drying obtain poplar Phellinus polysaccharide.
Embodiment 2
Poplar Huang fructification 100g is taken, 3 mesh are crushed to, is centrifuged after being impregnated 24 hours with physiological saline, residue is collected；Residue is used
1.5L water extracts half an hour at 120 DEG C, and centrifugation is collected residue, is repeated twice；Residue is dissolved in 0.5L deionized water, and mixing is equal
After even at -12 DEG C cryogenic freezing 24 hours, then microwave thaws rapidly to no ice, power 3000W, in triplicate；After
It is continuous that 1.5 deionized waters are added, the complex enzyme of mixed liquor weight 0.5/10000th is added, complex enzyme is cellulase: chitinase=
1:3 digests 4 hours at 50 DEG C, is centrifugated to obtain supernatant；Supernatant removes free protein and color with HP20 macroreticular resin
The mass ratio of element, macroreticular resin and supernatant is 1:20, and removal of impurities need to be repeated 3 times；It is dialysed respectively with clear water and secondary distilled water
(cutoff Mw=8,000), concentration, freeze-drying obtain poplar Phellinus polysaccharide (PV-F).It is 6.8% that weighing, which calculates yield, is led to
Cross elemental microanalysis method detect protein content is about 0.25% (the results are shown in Table 1 for elemental analysis).
The elemental analysis result of 1 poplar Phellinus polysaccharide PV-F of table
Protein content, as shown in Table 1, low percentages of protein can be calculated by nitrogen content * 6.25.
It is utilized respectively NMR, FT-IR, GC-MS technology detects obtained poplar Phellinus polysaccharide, and result is respectively such as
Table 2 and Fig. 1-2.
The monosaccharide component of 2 poplar Phellinus polysaccharide PV-F of table
As shown in table 2, its available monosaccharide component content of makings chromatography is utilized, it can be seen that principal monosaccharides component
For glucose.
As shown in Figure 1, the infrared appearance at 890cm-1 is unimodal, illustrate for beta glucan structure.Nuclear-magnetism result such as Fig. 2 institute
Show, wherein the peak of 103.7 (C1), 73.9 (C2), 86.9 (C3), 68.9 (C4), 76.6-77.3 (C5) and 61.4 (C6) ppm are
The characteristic feature of β-(1 → 3)-D- glucan, and the peak of 70.5 (C6s) ppm is this table caused by being influenced as the branch on main chain
Bright glucan is in C6 Chu You branch.To sum up, PV-F main component is β-(1 → 3)-D- glucan, and (1 → 6) containing part is bonded
Glucose side group.
Obtained poplar Phellinus polysaccharide is detected by scattering measuring, relevant molecular parameters are as shown in table 3.
The monosaccharide component of 3 poplar Phellinus polysaccharide PV-F of table
In wherein as shown in Table 3, obtaining poplar Phellinus polysaccharide weight average molecular weight by scattering measuring is about 550,000, equal Fang Xuan
Turning radius is 40.8nm, passes through Mw and < s2>z1/2α=0.58 is calculated in scatter diagram, shows that PV-F is in aqueous solution
The random-coil conformation relatively unfolded.It is calculated by viscosimetry, and intrinsic viscosity is 345 in aqueous solution, in aqueous solution
Huggins constant k ˊ value is 0.37, between 0.3-0.5, illustrates that it is dissolved well in water.
Embodiment 3
Poplar Huang fructification 100g is taken, 2 mesh are crushed to, is centrifuged after being impregnated 24 hours with physiological saline, residue is collected；Residue is used
1.5L water extracts half an hour at 120 DEG C, and centrifugation is collected residue, is repeated twice；Residue is dissolved in 0.5L deionized water, and mixing is equal
After even, cryogenic freezing 30 hours at -13 DEG C, then microwave thaws rapidly, power 3000W, in triplicate；It continuously adds
The complex enzyme of mixed liquor weight 0.5/10000th is added in 1.5 deionized waters, and complex enzyme is cellulase: chitinase=1:3, and 50
It is digested 4 hours at DEG C, is centrifugated to obtain supernatant；Supernatant removes free protein and pigment with HP20 macroreticular resin, greatly
The mass ratio of hole resin and supernatant is 1:20, and removal of impurities carries out primary；Respectively with clear water and secondary distilled water dialysis (cutoff
Mw=8,000), concentration, freeze-drying obtains poplar Phellinus polysaccharide.It is 7.1% that weighing, which calculates yield, is examined by elemental microanalysis method
Measuring protein content is about 0.65%, low percentages of protein.Identified, poplar made from chemical structure and embodiment 2 is yellow more
Sugared PV-F is identical, and protein impurities are slightly higher.
Embodiment 4
Poplar Huang fructification 100g is taken, 1-5 mesh is crushed to, is centrifuged after being impregnated 24 hours with physiological saline, residue is collected；Residue
Half an hour is extracted at 120 DEG C with 1.5L water, is centrifuged, residue is collected, is repeated twice；Residue is dissolved in 0.5L deionized water, mixing
After uniformly, freezed 24 hours at -15 DEG C, then microwave thaws rapidly, power 3000W；Continuously add 1.5 deionizations
The complex enzyme of mixed liquor weight 0.5/10000th is added in water, and complex enzyme is cellulase: chitinase=1:3, digests 4 at 50 DEG C
Hour, it is centrifugated to obtain supernatant；Supernatant removes free protein and pigment with HP20 macroreticular resin, macroreticular resin with it is upper
The mass ratio of clear liquid is 1:20, and removal of impurities carries out three times；Respectively with clear water and secondary distilled water dialysis (cutoff Mw=8,000),
Concentration, freeze-drying obtain poplar Phellinus polysaccharide.It is 4.9% that weighing, which calculates yield, reality of the yield compared to freeze thawing in triplicate
It is slightly lower to apply example 1, illustrates that freeze-thaw method can greatly promote its recovery rate.By elemental microanalysis method detect protein content is about
0.28%, low percentages of protein is close with embodiment 2.It is identified, poplar Huang polysaccharide PV- made from chemical structure and embodiment 1
F is identical, and protein impurities content is close.
Embodiment 5
Poplar Huang fructification 100g is taken, 3 mesh are crushed to, is centrifuged after being impregnated 24 hours with physiological saline, residue is collected；Residue is used
1.5L water extracts half an hour at 120 DEG C, and centrifugation is collected residue, is repeated twice；Residue is dissolved in 0.5L deionized water, and mixing is equal
It after even, is freezed 24 hours at -12 DEG C, then microwave thaws rapidly, power 1000W, in triplicate；Continuously add 1.5
The complex enzyme of mixed liquor weight 0.5/10000th is added in deionized water, and complex enzyme is cellulase: chitinase=1:3, and 50 DEG C
Lower enzymatic hydrolysis 4 hours, is centrifugated to obtain supernatant；Supernatant removes free protein and pigment, macropore with HP20 macroreticular resin
The mass ratio of resin and supernatant is 1:20, and removal of impurities need to be repeated 3 times；Respectively with clear water and secondary distilled water dialysis (cutoff Mw
=8,000) it, is concentrated, freeze-drying obtains poplar Phellinus polysaccharide (PV-F).It is 3.8% that weighing, which calculates yield, passes through elemental analysis
Method detect protein content is about 0.26%, low percentages of protein is close with embodiment 2.It is identified, chemical structure and reality
It is identical to apply poplar Huang polysaccharide PV-F made from example 2, protein impurities content is close.The present embodiment uses defrosting power for 1000W, is lower than
Embodiment 2, yield illustrate that yield can be greatly promoted by improving thawing rate well below embodiment 2.
Embodiment 6
Poplar Huang fructification 100g is taken, 3 mesh are crushed to, is centrifuged after being impregnated 24 hours with physiological saline, residue is collected；Residue is used
1.5L water extracts half an hour at 120 DEG C, and centrifugation is collected residue, is repeated twice；Residue is dissolved in 0.5L deionized water, and mixing is equal
It is freezed 8 hours at -4 DEG C after even, then microwave thaws rapidly, power 2000W, in triplicate；Continuously add 1.5 go from
The complex enzyme of mixed liquor weight 0.5/10000th is added in sub- water, and complex enzyme is cellulase: chitinase=1:3, enzyme at 50 DEG C
Solution 4 hours, is centrifugated to obtain supernatant；Supernatant removes free protein and pigment, macroreticular resin with HP20 macroreticular resin
Mass ratio with supernatant is 1:20, and removal of impurities need to be repeated 3 times；Respectively with clear water and secondary distilled water dialysis (cutoff Mw=8,
000) it, is concentrated, freeze-drying obtains poplar Phellinus polysaccharide (PV-F).It is 4.6% that weighing, which calculates yield, is examined by elemental microanalysis method
Measuring protein content is about 0.28%, low percentages of protein, close with embodiment 2.It is identified, chemical structure and embodiment
Poplar Huang polysaccharide PV-F made from 2 is identical, and protein impurities content is close.This implements its yield well below embodiment 2, illustrates sufficiently
Yield can be improved in freezing.
Comparative example 1
Poplar Huang fructification 100g is taken, 2 mesh are crushed to, is centrifuged after being impregnated 24 hours with physiological saline, residue is collected；Residue is used
1.5L water extracts half an hour at 120 DEG C, and centrifugation is collected residue, is repeated twice；Residue is dissolved in the 1.25M NaOH solution of 2L
In, it extracts at room temperature twice, 8 hours every time, collects supernatant after adding acetic acid to neutralize；Supernatant Sevag method removing protein repeats
Three times；Again with 30% hydrogen peroxide depigmentaton, respectively with clear water and secondary distilled water dialysis (cutoff Mw=8,000) after concentration,
Concentration, freeze-drying obtain alkali carries poplar Phellinus polysaccharide (PV-B).It is 4.7% that weighing, which calculates yield, and yield is compared to freeze thawing weight
Multiple embodiment 1 three times is slightly lower, illustrates that freeze-thaw method can greatly promote its recovery rate.By Kjeldahl's method detect protein contains
Amount about 1.3%, protein content is slightly higher.Identified, chemical structure is identical as poplar Huang polysaccharide (PV-F) made from embodiment 1,
Protein impurities content is slightly higher.
Embodiment 7
The tumour cell selected is Cervix neoplasms (Hela) and hepatocellular carcinoma H22.Cell culture is trained using DMEM
Base is supported, (FBS) containing 10% fetal calf serum contains dual anti-100U/ml penicillin and 100 μ g/mL streptomysins.Cell is placed in incubator
It cultivates (37 DEG C of temperature, 5%CO2).Cell survival rate is measured using mtt assay.With 6000, every hole cell inoculation after cell resuspension
In 96 well culture plates, (37 DEG C, 5%CO2) are placed in incubator.After being incubated for 24 hours, every hole addition again is cleaned twice with PBS
200 μ L fresh cultures, after the every hole of experimental group adds the sample of 20 μ L various concentrations to be incubated for 48 hours, 20 μ L MTT are added in every hole
(5mg/mL) solution continues to be put in incubator being incubated for 4 hours, abandons supernatant, and the crystallization of 150 μ L DMSO, Dai formazans is added in every hole
After dissolution, with the every hole luminous intensity (OD) of colorimetric detection at automatic microplate reader (Bio-Rad, Model 550, USA) detection 570nm
Value.The survival rate of cell is calculated by (OD570, sample/OD570, control) × 100%, and OD570, sample are to be added
The luminous intensity of each experimental group of sample, OD570, control are the luminous intensity of control group (PBS).With the Yang Huangduo of various concentration
Two plants of sugar other places reason processing is after tumour cell 48 hours, and tumor cell survival is as shown in Figure 3 and Figure 4, and two kinds of polysaccharide are in 2-
The inhibitory effect of the proliferation of tumour cell is linearly enhanced in the concentration range of 100 μ g/mL.Inhibition of the PV-B to HeLa cell
Rate is slightly worse than PV-F, is slightly better than PV-F to the inhibiting rate of HepG2 cell, illustrates the polysaccharide of two methods extraction to tumour cell
The proliferation of strain has certain inhibiting effect.
Embodiment 8
The inflammatory reaction result for the polysaccharide that distinct methods extract is characterized with the burst size of inflammatory factor NO.NO Griess
Kit detects (green skies Bioisystech Co., Ltd, Shanghai).Cell uses RAW264.7, and cultural method, will with embodiment 7
264.7 cell inoculation of RAW and 96 orifice plates are incubated for 24 hours in advance.After PBS cleaning, fresh DMEM culture medium is added, is added
Blank control adds poplar Phellinus polysaccharide PV-F, PV- of various concentration in PBS, positive control LPS (100ng/mL) and embodiment 2
B.Continue that 96 orifice plates are added in the supernatant (50 μ L) and standard items after being incubated for 24 hours, 50 rooms μ L are sequentially added in each hole
Warm Griess Reagent I and 50 μ L Griess Reagent II.Absorbance is measured in 540nm with automatic microplate reader.Standard
Product draw calculating each sample and the NO burst size of positive control after standard curve.As a result as shown in Figure 5, it can be clearly seen that comparison
1 alkali-extracted polysaccharide PV-B of example can obviously cause the inflammatory reaction of macrophage, and the mentioned polysaccharide PV-F of embodiment 1 is in maximum concentration
When 100 μ g/mL, NO content is still approached with blank group in culture medium, will not bring inflammatory reaction, and biocompatibility is good.
In conclusion the invention proposes a kind of extracting method of poplar Phellinus polysaccharide, it is micro- after repeatedly freezing repeatedly
Wave quick-thawing carries out broken wall to poplar Huang fructification, improves poplar Phellinus polysaccharide yield, and obtained poplar Phellinus polysaccharide has bright
The activity of aobvious inhibition tumor cell proliferation, and there is good bio-compatible relative to the polysaccharide of chemical method extraction purification
Property, inflammatory reaction will not be brought, and poplar Phellinus polysaccharide of the present invention can be used in anti-tumor drug or health care product.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of extracting method of poplar Phellinus polysaccharide, which comprises the steps of:
(1) Phellinus fructification is crushed to 1-5 mesh, with physiological saline impregnate 8~for 24 hours, residue is collected after centrifugation；
(2) the Phellinus fructification residue is boiled into 20~40min under 110~130 DEG C of boiling water, residue is collected after centrifugation；
(3) add water in the residue in step (2), thaw after-15-- 10 DEG C of freezing 24-36h；
(4) add water to obtain mixed liquor in the defrosting mixture of step (3), then complex enzyme will be added in mixed liquor, at 50-60 DEG C
2~4h is digested, supernatant is centrifugated to obtain；
(5) it dialyses after supernatant resin adsorption, concentration and freeze-drying obtain poplar Phellinus polysaccharide.
2. a kind of extracting method of poplar Phellinus polysaccharide according to claim 1, which is characterized in that step (2) described mulberry
The additional amount of yellow fructification residue and the boiling water is 60-80g/L.
3. a kind of extracting method of poplar Phellinus polysaccharide according to claim 1, which is characterized in that described in step (3)
The additional amount of residue and the water is 200g/L.
4. a kind of extracting method of poplar Phellinus polysaccharide according to claim 1, which is characterized in that step (3) described solution
Jelly method are as follows: microwave thaws rapidly, power 1000-3000W.
5. a kind of extracting method of poplar Phellinus polysaccharide according to claim 1, which is characterized in that step (4) described solution
The additional amount for freezing mixture and the water is 60-80g/L.
6. a kind of extracting method of poplar Phellinus polysaccharide according to claim 1, which is characterized in that step (4) is described multiple
Synthase includes cellulase and chitinase；Wherein the cellulase and the chitinase mass ratio are 1:1~3.
7. a kind of extracting method of poplar Phellinus polysaccharide according to claim 1, which is characterized in that step (4) is described multiple
The additional amount of synthase is 0.2/10000th~the 0.5 of the mixed liquor weight.
8. a kind of extracting method of poplar Phellinus polysaccharide according to claim 1, which is characterized in that step (5) described tree
Rouge is macroreticular resin；Wherein the mass ratio of the macroreticular resin and the supernatant is 1:20~50.
9. a kind of -8 poplar Phellinus polysaccharide are preparing answering in anti-tumor drug or health care product according to claim 1
With.
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