CN104045727B - Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus - Google Patents
Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus Download PDFInfo
- Publication number
- CN104045727B CN104045727B CN201410288047.2A CN201410288047A CN104045727B CN 104045727 B CN104045727 B CN 104045727B CN 201410288047 A CN201410288047 A CN 201410288047A CN 104045727 B CN104045727 B CN 104045727B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- thick polysaccharide
- thick
- water
- inonotus obliquus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 130
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 130
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 130
- 238000000034 method Methods 0.000 title claims abstract description 56
- 239000011347 resin Substances 0.000 title claims abstract description 39
- 229920005989 resin Polymers 0.000 title claims abstract description 39
- 241000414067 Inonotus obliquus Species 0.000 title claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 47
- 241000747105 Fuscoporia Species 0.000 claims abstract description 24
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 17
- 239000002250 absorbent Substances 0.000 claims abstract description 15
- 230000002745 absorbent Effects 0.000 claims abstract description 15
- 239000002994 raw material Substances 0.000 claims abstract description 11
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 10
- 238000005238 degreasing Methods 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 25
- 239000004365 Protease Substances 0.000 claims description 17
- 108090000526 Papain Proteins 0.000 claims description 16
- 229940055729 papain Drugs 0.000 claims description 16
- 235000019834 papain Nutrition 0.000 claims description 16
- 239000000049 pigment Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 13
- 238000009835 boiling Methods 0.000 claims description 11
- 239000012153 distilled water Substances 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 8
- 241001558929 Sclerotium <basidiomycota> Species 0.000 claims description 7
- 229920002678 cellulose Polymers 0.000 claims description 7
- 239000001913 cellulose Substances 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- 150000003384 small molecules Chemical class 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 5
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 claims 1
- 238000007670 refining Methods 0.000 abstract description 15
- 230000014759 maintenance of location Effects 0.000 abstract description 4
- 238000004042 decolorization Methods 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 abstract description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 11
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 239000008395 clarifying agent Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Abstract
The invention discloses the method for a kind of AB-8 of utilization low pole resin for the thick polysaccharide of Inonotus obliquus, comprise the steps: the degreasing pretreatment of A. raw material; B. thick polysaccharide is prepared in hot water lixiviate; C. thick polysaccharide enzymolysis; D. macroporous absorbent resin AB-8 processes; E. little molecule is removed in ultrafiltration; F. vacuum freeze drying; Prepare Fuscoporia obliqua polysaccharide. Use this technique finally to obtain the refining polysaccharide of Inonotus obliquus decolouring and that remove most of albumen and small molecular weight impurity, obtain more much higher sugared retention rate, percent of decolourization and albumen removal efficiency high, and applicable industrialized scaleization is produced the refining polysaccharide of Inonotus obliquus.
Description
Technical field
The invention belongs to biological technical field, specifically relate to the method for a kind of AB-8 of utilization low pole resin for the thick polysaccharide of Inonotus obliquus.
Background technology
Inonotus obliquus [Inonotusobliquus(Fr.) Pilat] be a famous Wild Medicinal fungi among the people. Research shows, Fuscoporia obliqua polysaccharide is one of important active component, has the immunological regulation of enhancing, the multiple pharmacologically active such as antitumor, anti-oxidant, hypoglycemic and antiviral, become one of hot fields of domestic and international research. But the research of the separation and purification to Fuscoporia obliqua polysaccharide report is relatively less at present.
Due to the composition complexity of polysaccharide, the molecular weight distribution of polysaccharide crude extract is wider, polysaccharide kind is also a lot, and previous experiments shows, in the thick polysaccharide of Inonotus obliquus after extraction, contain a certain amount of albumen and pigment, affect chemical constitution and the bioactivity research of polysaccharide, be therefore necessary polysaccharide to carry out separation and purification. But removing albumen and pigment in thick polysaccharide, is a great problem in separation of polysaccharides purifying always, traditional deproteinized and method complex operation, the efficiency of discoloring plain are low. Traditional deproteinized is taking Sevage method as main, method complex operation, and efficiency is low, and the organic solvent of employing is toxic thereby affect the biologically active of polysaccharide; It is better that activated carbon method is removed pigment effect, but the active carbon powder in later stage is difficult for removal, and it is fine that hydrogen peroxide method is removed pigment effect because of stronger antioxidation activity, but larger to the activity influence of polysaccharide, is therefore badly in need of exploring a kind of new process for purification.
Macroporous absorbent resin is the polymer adsorbing material that a class does not contain cation exchange groups, there is good macroporous netlike structure and larger specific area, have good adsorption activity, some macroreticular resin has been used in the deproteinized research in plant polyose, has obtained good effect.
Application publication number CN102603906A(application number 201110461635.8) Chinese patent literature a kind of preparation method of the Fuscoporia obliqua polysaccharide aqueous solution is disclosed: a kind of preparation method of the Fuscoporia obliqua polysaccharide aqueous solution, comprise the following steps: trichloroacetic acid takes off albumen: in Inonotus obliquus extract, add trichloroacetic acid, leave standstill, then Separation of Solid and Liquid, gets and separates the solid matter obtaining; Activated carbon decolorizing: to step; Gained solid matter by volume adds the water of 50～100 times, then adds activated carbon decolorizing, and then Separation of Solid and Liquid is got gained supernatant; Clarify with natural clarifying agent: gained supernatant adds natural clarifying agent clarification.
Macroreticular resin there is not yet bibliographical information in order to remove the thick polysaccharide of Inonotus obliquus.
Summary of the invention
The object of this invention is to provide the technique of the thick polysaccharide of a kind of refining Inonotus obliquus; core technology of the present invention is: applying biological enzyme and anion exchange absorbing resin substitute traditional Sevage method, activated carbon method and hydrogen peroxide method and remove albumen and pigment, is applicable to scale industrialization and prepares the refining polysaccharide of Inonotus obliquus.
Technical scheme of the present invention is as follows:
Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus, it comprises the steps: the degreasing pretreatment of A. raw material; B. thick polysaccharide is prepared in hot water lixiviate; C. thick polysaccharide enzymolysis; D. macroporous absorbent resin AB-8 processes; E. little molecule is removed in ultrafiltration; F. vacuum freeze drying; Prepare Fuscoporia obliqua polysaccharide.
Foregoing method, preferred scheme is, steps A. the degreasing pretreatment of raw material refers to, gets dry inonotus obliquus sclerotium, pulverizes, it is preferred that the 60-95% ethanolic solution that is placed in 4-8 times of volume soaks 16-24h(, the 70-90% ethanolic solution that is placed in 6-7 times of volume soaks 18-20h, is more preferably, and 80% ethanolic solution that is placed in 6.5 times of volumes soaks 20h), to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
Foregoing method, preferred scheme is, the lixiviate of step B. hot water is prepared thick polysaccharide and is referred to, after pretreated residue is air-dry, adds 10-20 times of distilled water (v/w), extracting in boiling water 1-3h, 80-120 order filtered through gauze (preferred, add 13-18 times of distilled water (v/w), extracting in boiling water 1.5-2.5h, 85-100 order filtered through gauze, be more preferably, add 15 times of distilled water (v/w), extracting in boiling water 2h, 100 order filtered through gauze), filter residue repeats to extract 1-3 time, and merging filtrate is concentrated, centrifugal, spraying is dried to obtain thick polysaccharide.
Foregoing method, preferred scheme is that the thick polysaccharide enzymolysis of step C. refers to, thick polysaccharide is dissolved in water, add papain, 35-45 DEG C of reaction 20-40min(is preferred, 36-42 DEG C of reaction 25-35min, be more preferably 40 DEG C of reaction 30min), obtain the enzymolysis liquid of thick polysaccharide. Be more preferably, after thick polysaccharide is dissolved in water, be formulated as 0.5-2.0%(w/w) the aqueous solution of the aqueous solution (preferred, to be formulated as 0.6-1.8%(w/w), be more preferably, be formulated as 1.5%(w/w) the aqueous solution). Be more preferably the 3-5%(w/w that papain addition is thick polysaccharide solution) (preferred, the 3.5-4.5%(w/w that addition is thick polysaccharide solution), be more preferably the 4.0%(w/w that addition is thick polysaccharide solution)).
Foregoing method, preferred scheme is, step D. macroporous absorbent resin AB-8 processes and refers to, the enzymolysis liquid of thick polysaccharide adds in the AB-8 resin chromatographic column that pretreatment is good, according to the thick polysaccharide enzymolysis liquid=1.2-2.0 of AB-8 tree fat ︰ ︰ 1(w/v), flow control is preferred at 1.5-2.2ml/min(, according to the thick polysaccharide enzymolysis liquid=1.5-1.8 of AB-8 tree fat ︰ ︰ 1(w/v), flow control is at 1.5-2ml/min, be more preferably, according to the AB-8 tree thick polysaccharide of fat ︰ enzymolysis liquid=1.6 ︰ 1(w/v), flow control is at 1.8ml/min), collect efflux. Be more preferably, described pretreatment refers to, macroporous absorbent resin AB-8 soaked in absolute ethyl alcohol 24h, and be eluted to colourlessly with absolute ethyl alcohol, then wash with water to without alcohol taste.
Foregoing method, preferably scheme is, step e. ultrafiltration is removed little molecule and is referred to, adopts the hollow cellulose film that milipore filter molecular weight is 10,000 to carry out ultrafiltration, removes the small-molecule substance below 10,000.
Foregoing method, preferably scheme is, step F. vacuum freeze drying refers to, first Fuscoporia obliqua polysaccharide sample is positioned over to 3h at-20 DEG C, then proceed at-80 DEG C more than freezing 6h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
The invention discloses the refining polysaccharide of a kind of Inonotus obliquus and preparation technology thereof, preparation technology is as follows: 1, the pretreatment of raw material; 2, the stripping of Fuscoporia obliqua polysaccharide; 3, protease hydrolytic; 4, resin anion (R.A.) processing; 5, ultrafiltration, then dry. Use this technique finally to obtain the refining polysaccharide of Inonotus obliquus decolouring and that remove most of albumen and small molecular weight impurity, obtain more much higher sugared retention rate, percent of decolourization and albumen removal efficiency high, and applicable industrialized scaleization is produced the refining polysaccharide of Inonotus obliquus.
Detailed description of the invention
Below in conjunction with embodiment, technical solution of the present invention is encyclopaedized, but protection domain is not by this restriction. Raw materials usedly in embodiment all can obtain from market.
Embodiment 1Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus, comprise the steps:
A. the degreasing pretreatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 60% ethanolic solution that is placed in 4 times of volumes soaks 16h, to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. thick polysaccharide is prepared in hot water lixiviate: after pretreated residue is air-dry, add 10 times of distilled water (v/w), and extracting in boiling water 1h, 80 order filtered through gauze, filtrate is concentrated, centrifugal, and spraying is dried to obtain thick polysaccharide.
C. thick polysaccharide enzymolysis: thick polysaccharide is dissolved in water, is formulated as 0.5%(w/w) the aqueous solution, add papain, the 3%(w/w that papain addition is thick polysaccharide solution), 35 DEG C of reaction 20min, obtain the enzymolysis liquid of thick polysaccharide.
D. macroporous absorbent resin AB-8 processes and refers to, the enzymolysis liquid of thick polysaccharide adds pretreatment (macroporous absorbent resin AB-8 soaked in absolute ethyl alcohol 24h, and be eluted to colourless with absolute ethyl alcohol, then wash with water to without alcohol taste) in good AB-8 resin chromatographic column, according to the AB-8 tree thick polysaccharide of fat ︰ enzymolysis liquid=1.2 ︰ 1(w/v), flow control, at 1.5ml/min, is collected efflux.
E. little molecule is removed in ultrafiltration: adopt the hollow cellulose film that milipore filter molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000, obtain Fuscoporia obliqua polysaccharide sample.
F. vacuum freeze drying: first Fuscoporia obliqua polysaccharide sample is positioned over to 3h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
Embodiment 2Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus, comprise the steps:
A. the degreasing pretreatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 95% ethanolic solution that is placed in 8 times of volumes soaks 24h, to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. thick polysaccharide is prepared in hot water lixiviate: after pretreated residue is air-dry, add 20 times of distilled water (v/w), and extracting in boiling water 3h, 120 order filtered through gauze, filter residue repeats to extract 2 times, and merging filtrate is concentrated, centrifugal, and spraying is dried to obtain thick polysaccharide.
C. thick polysaccharide enzymolysis: thick polysaccharide is dissolved in water, is formulated as 2.0%(w/w) the aqueous solution, add papain, the 5%(w/w that papain addition is thick polysaccharide solution), 45 DEG C of reaction 40min, obtain the enzymolysis liquid of thick polysaccharide.
D. macroporous absorbent resin AB-8 processes: macroporous absorbent resin AB-8 soaked in absolute ethyl alcohol 24h, and be eluted to colourlessly with absolute ethyl alcohol, then wash with water to without alcohol taste.
The enzymolysis liquid of thick polysaccharide adds in the AB-8 resin chromatographic column that pretreatment is good, according to the AB-8 tree thick polysaccharide of fat ︰ enzymolysis liquid=2.0 ︰ 1(w/v), flow control, at 2.2ml/min, is collected efflux.
E. little molecule is removed in ultrafiltration: adopt the hollow cellulose film that milipore filter molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000.
F. vacuum freeze drying: first Fuscoporia obliqua polysaccharide sample is positioned over to 3h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
Embodiment 3Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus, it comprises the steps:
A. the degreasing pretreatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 90% ethanolic solution that is placed in 7 times of volumes soaks 20h, to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. thick polysaccharide is prepared in hot water lixiviate: after pretreated residue is air-dry, add 18 times of distilled water (v/w), and extracting in boiling water 2.5h, 100 order filtered through gauze, filter residue repeats to extract 3 times, and merging filtrate is concentrated, centrifugal, and spraying is dried to obtain thick polysaccharide.
C. thick polysaccharide enzymolysis: thick polysaccharide is dissolved in water, is formulated as 1.8%(w/w) the aqueous solution, add papain, the 4.5%(w/w that papain addition is thick polysaccharide solution), 42 DEG C of reaction 35min, obtain the enzymolysis liquid of thick polysaccharide.
D. macroporous absorbent resin AB-8 processes: the enzymolysis liquid of thick polysaccharide adds in the AB-8 resin chromatographic column that pretreatment (pretreatment mode and embodiment 1-2 are same) is good, according to the AB-8 tree thick polysaccharide of fat ︰ enzymolysis liquid=1.8 ︰ 1(w/v), flow control, at 2ml/min, is collected efflux.
E. little molecule is removed in ultrafiltration: adopt the hollow cellulose film that milipore filter molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000.
F. vacuum freeze drying: first Fuscoporia obliqua polysaccharide sample is positioned over to 3h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
Embodiment 4Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus, comprise the steps:
A. the degreasing pretreatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 70% ethanolic solution that is placed in 6 times of volumes soaks 18h, to remove degrease, single (widow) sugar of part and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. thick polysaccharide is prepared in hot water lixiviate: after pretreated residue is air-dry, add 13 times of distilled water (v/w), and extracting in boiling water 1.5h, 85 order filtered through gauze, extract 1 time, and filtrate is concentrated, centrifugal, and spraying is dried to obtain thick polysaccharide.
C. thick polysaccharide enzymolysis: thick polysaccharide is dissolved in water, is formulated as 0.5%(w/w) the aqueous solution, add papain, the 3.5%(w/w that papain addition is thick polysaccharide solution), 36 DEG C of reaction 25min, obtain the enzymolysis liquid of thick polysaccharide.
D. macroporous absorbent resin AB-8 processes: the enzymolysis liquid of thick polysaccharide adds in the AB-8 resin chromatographic column that pretreatment is good, according to the AB-8 tree thick polysaccharide of fat ︰ enzymolysis liquid=1.5 ︰ 1(w/v), flow control, at 1.5ml/min, is collected efflux.
E. little molecule is removed in ultrafiltration: adopt the hollow cellulose film that milipore filter molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000.
F. vacuum freeze drying: first Fuscoporia obliqua polysaccharide sample is positioned over to 3h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
Embodiment 5Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus, it comprises the steps:
A. the degreasing pretreatment of raw material: get dry inonotus obliquus sclerotium, pulverize, 80% ethanolic solution that is placed in 6.5 times of volumes soaks 20h, to remove degrease, part oligosaccharides and fat-soluble pigment, the shady and cool place of residue is air-dry.
B. thick polysaccharide is prepared in hot water lixiviate: after pretreated residue is air-dry, add 15 times of distilled water (v/w), and extracting in boiling water 2h, 100 order filtered through gauze, filter residue repeats to extract 2 times, and merging filtrate is concentrated, centrifugal, and spraying is dried to obtain thick polysaccharide.
C. thick polysaccharide enzymolysis: thick polysaccharide is dissolved in water, is formulated as 1.5%(w/w) the aqueous solution, add papain, the 4.0%(w/w that papain addition is thick polysaccharide solution), 40 DEG C of reaction 30min, obtain the enzymolysis liquid of thick polysaccharide.
D. macroporous absorbent resin AB-8 processes: the enzymolysis liquid of thick polysaccharide adds pretreatment, and (described pretreatment refers to, macroporous absorbent resin AB-8 soaked in absolute ethyl alcohol 24h, and be eluted to colourless with absolute ethyl alcohol, then wash with water to without alcohol taste) in good AB-8 resin chromatographic column, according to the AB-8 tree thick polysaccharide of fat ︰ enzymolysis liquid=1.6 ︰ 1(w/v), flow control, at 1.8ml/min, is collected efflux.
E. little molecule is removed in ultrafiltration: adopt the hollow cellulose film that milipore filter molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000.
F. vacuum freeze drying: first Fuscoporia obliqua polysaccharide sample is positioned over to 3h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6h, then proceed to vacuum freeze drier dry, obtain refining Fuscoporia obliqua polysaccharide.
Test exampleThe mensuration of embodiment 5 gained Fuscoporia obliqua polysaccharide polyoses contents and protein content, and the calculating of albumen removal efficiency, pigment removal efficiency and polysaccharide retention rate. The wherein content (%) of content (the %)-reduced sugar of polyoses content (%)=total reducing sugar.
Total sugar content is measured the phenolsulfuric acid method that adopts; Content of reducing sugar is measured the DNS method that adopts; Determining the protein quantity adopts Bradford method.
In formula: A0, A11 is respectively the front and absorbance of rear solution at wavelength 450nm place of decolouring of decolouring.
In formula: C0、C11 is respectively the front and absorbance of rear solution at wavelength 450nm place of decolouring of decolouring.
In formula: M0, M11 are respectively the polyoses content before and after processing.
Be the result comparison of the refining thick polysaccharide of Inonotus obliquus of Sevage method of the present invention, activated carbon method and hydrogen peroxide method, this shows that macroreticular resin AB-8 has the ability of removing albumen and pigment concurrently below. See the following form:
The present invention adopts the method for the refining thick polysaccharide of Inonotus obliquus of AB-8 resin anion (R.A.), not only can remove albumen, also have the ability of removing pigment concurrently, therefore the purity of polysaccharide of preparation is higher, compare with the thick polysaccharide before processing, pigment removal efficiency is 90.1%, albumen removal efficiency is 85.1%, polysaccharide retention rate 72.1%.
Finally it should be noted that, embodiment is the detailed description of the invention of optimum of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement. Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (13)
1. utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus, it is characterized in that, comprise the steps:
A. the degreasing pretreatment of raw material: get dry inonotus obliquus sclerotium, pulverize, the 60-95% ethanolic solution that is placed in 4-8 times of volume soaks 16-24h, to remove degrease, part monose and fat-soluble pigment, the shady and cool place of residue is air-dry;
B. thick polysaccharide is prepared in hot water lixiviate: after pretreated residue is air-dry, add 10-20 times of distilled water (v/w), and extracting in boiling water 1-3h, 80-120 order filtered through gauze, filter residue repeats to extract 1-3 time, and merging filtrate is concentrated, centrifugal, and spraying is dried to obtain thick polysaccharide;
C. thick polysaccharide enzymolysis: be formulated as 0.5-2.0%(w/w after thick polysaccharide is dissolved in water) the aqueous solution, add papain, the 3-5%(w/w that papain addition is thick polysaccharide solution), 35-45 DEG C of reaction 20-40min, obtains the enzymolysis liquid of thick polysaccharide;
D. macroporous absorbent resin AB-8 processes: the enzymolysis liquid of thick polysaccharide adds in the AB-8 resin chromatographic column that pretreatment is good, according to the thick polysaccharide enzymolysis liquid=1.2-2.0 of AB-8 tree fat ︰ ︰ 1(w/v), flow control, at 1.5-2.2ml/min, is collected efflux; Described pretreatment refers to, macroporous absorbent resin AB-8 soaked in absolute ethyl alcohol 24h, and be eluted to colourlessly with absolute ethyl alcohol, then wash with water to without alcohol taste;
E. little molecule is removed in ultrafiltration: adopt the hollow cellulose film that milipore filter molecular weight is 10,000 to carry out ultrafiltration, remove the small-molecule substance below 10,000, obtain Fuscoporia obliqua polysaccharide sample;
F. vacuum freeze drying; Prepare Fuscoporia obliqua polysaccharide; Described vacuum freeze drying refers to, first Fuscoporia obliqua polysaccharide sample is positioned over to 3h at-20 DEG C, then proceeds at-80 DEG C more than freezing 6h, then proceeds to vacuum freeze drier dry.
2. method according to claim 1, is characterized in that, steps A: the 70-90% ethanolic solution that is placed in 6-7 times of volume soaks 18-20h.
3. method according to claim 2, is characterized in that, steps A: 80% ethanolic solution that is placed in 6.5 times of volumes soaks 20h.
4. method according to claim 1, is characterized in that, step B: add 13-18 times of distilled water (v/w), extracting in boiling water 1.5-2.5h, 85-100 order filtered through gauze.
5. method according to claim 4, is characterized in that, step B: add 15 times of distilled water (v/w), extracting in boiling water 2h, 100 order filtered through gauze.
6. method according to claim 1, is characterized in that, step C:36-42 DEG C of reaction 25-35min.
7. method according to claim 6, is characterized in that, step C:40 DEG C of reaction 30min.
8. method according to claim 1, is characterized in that, step C: after thick polysaccharide is dissolved in water, be formulated as 0.6-1.8%(w/w) the aqueous solution.
9. method according to claim 8, is characterized in that, step C: after thick polysaccharide is dissolved in water, be formulated as 1.5%(w/w) the aqueous solution.
10. method according to claim 1, is characterized in that, step C: the 3.5-4.5%(w/w that papain addition is thick polysaccharide solution).
11. methods according to claim 10, is characterized in that step C: the 4.0%(w/w that papain addition is thick polysaccharide solution).
12. methods according to claim 1, is characterized in that step D: according to the thick polysaccharide enzymolysis liquid=1.5-1.8 of AB-8 tree fat ︰ ︰ 1(w/v), flow control, at 1.5-2ml/min, is collected efflux.
13. methods according to claim 12, is characterized in that step D: according to the AB-8 tree thick polysaccharide of fat ︰ enzymolysis liquid=1.6 ︰ 1(w/v), flow control, at 1.8ml/min, is collected efflux.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410288047.2A CN104045727B (en) | 2014-06-25 | 2014-06-25 | Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410288047.2A CN104045727B (en) | 2014-06-25 | 2014-06-25 | Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104045727A CN104045727A (en) | 2014-09-17 |
| CN104045727B true CN104045727B (en) | 2016-05-25 |
Family
ID=51499164
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410288047.2A Active CN104045727B (en) | 2014-06-25 | 2014-06-25 | Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104045727B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104844720B (en) * | 2015-06-19 | 2016-11-09 | 贵州师范学院 | A kind of separating and extracting process of hundred tail gracilis polysaccharides |
| CN106420500B (en) * | 2016-11-30 | 2019-07-12 | 广东芭薇生物科技股份有限公司 | A kind of composition of the extract containing Inonotus obliquus and its application |
| CN109748982B (en) * | 2019-01-30 | 2021-06-04 | 吉林医药学院 | Preparation method and application of inonotus obliquus polysaccharide |
| CN112521523B (en) * | 2020-12-15 | 2023-03-24 | 山西康欣药业有限公司 | Method for extracting and purifying inonotus obliquus polysaccharide |
| CN114009647A (en) * | 2021-11-18 | 2022-02-08 | 九愿健康产业科技(苏州)有限公司 | Preparation method of inonotus obliquus granule beverage |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102038720A (en) * | 2010-12-17 | 2011-05-04 | 中国农业大学 | Fuscoporia obliqua active ingredients capable of lowering blood sugar and preparation method and application of fuscoporia obliqua active ingredients |
| CN102603906A (en) * | 2011-12-30 | 2012-07-25 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of inonotus obliquus polysaccharide aqueous solution |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100340663B1 (en) * | 2000-12-16 | 2002-06-20 | 정종상 | A method of preparing the antitumor and immuno-activity polysaccharide from the artificial cultivation of inonotus obliquus, and its use |
-
2014
- 2014-06-25 CN CN201410288047.2A patent/CN104045727B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102038720A (en) * | 2010-12-17 | 2011-05-04 | 中国农业大学 | Fuscoporia obliqua active ingredients capable of lowering blood sugar and preparation method and application of fuscoporia obliqua active ingredients |
| CN102603906A (en) * | 2011-12-30 | 2012-07-25 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of inonotus obliquus polysaccharide aqueous solution |
Non-Patent Citations (1)
| Title |
|---|
| 两种不同来源桦褐孔菌多糖的抗氧化与免疫活性的研究;武永德;《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》;20120315(第3期);第26-27页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104045727A (en) | 2014-09-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104045727B (en) | Utilize the method for AB-8 low pole resin for the thick polysaccharide of Inonotus obliquus | |
| CN104045724B (en) | The method that Fuscoporia obliqua polysaccharide is prepared in extraction from Inonqqus obliquus | |
| CN104045728B (en) | Adopt the method for D101 resin for the thick polysaccharide of Inonotus obliquus | |
| CN106381319A (en) | High-efficiency extraction and separation method of grape seed proanthocyanidin oligomer | |
| CN101200487B (en) | Method for preparing asiatic centella total saponins by using macroporous adsorption resin | |
| CN103519180B (en) | Preparation method of maca extract | |
| CN104045726B (en) | Utilize the method for the refining thick polysaccharide of Inonotus obliquus of macroporous absorbent resin DA201 | |
| CN102491938A (en) | Purification method of deoxynojirimycin | |
| AU2020101569A4 (en) | A Method of Extracting Xanthotoxol from the Root of Angelica Dahurica | |
| CN105175566B (en) | Polyamide column and macroporous resin column connection post method remove the method for protein and pigment in Radix Panacis Quinquefolii polysaccharide extract | |
| CN104045725B (en) | D301G resin anion(R.A) is adopted to refine the method for Phaeopoms obliquus Crude polysaccharides | |
| CN101229335B (en) | Enzyme method for preparing smilax scobinicaulis total saponin extract | |
| CN101108871A (en) | Technique for extracting cycli phosphate adenosine from chinese date | |
| CN102180985B (en) | Method for extracting and purifying mugwort polysaccharides | |
| CN104031159B (en) | 732 resin cation (R.C.)s are adopted to refine the method for Phaeopoms obliquus Crude polysaccharides | |
| CN107522761A (en) | A kind of method and its hypoglycemic purposes for isolating and purifying the O mulberry cloth disaccharide glycosides of delphinidin 3 | |
| CN104231011B (en) | Preparation method of verbascoside | |
| CN102351928B (en) | Method for extracting iridoid active ingredients from plantain seeds | |
| CN110054706A (en) | The method for the fucoidin concentration extracted in one main laminaria | |
| CN104945530A (en) | Method for refining Korean pine pinecone polysaccharide from macroreticular resin | |
| CN110054704B (en) | Method for refining mesona chinensis benth polysaccharide by combining ammonium sulfate and CTAB (cetyl trimethyl ammonium bromide) precipitation with macroporous resin | |
| CN111150765B (en) | Fingered citron flavone extract and separation and purification method thereof | |
| CN106336440A (en) | Method for extracting and separating out oleanolic acid from olive leaves | |
| CN108210554B (en) | Method for separating and purifying alcohol-soluble total flavonoids from liquorice | |
| CN101953880B (en) | Method for preparing pueraria flavonid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| CB03 | Change of inventor or designer information |
Inventor after: Ji Yanbin Inventor after: Li Tao Inventor after: Zhou Zhichao Inventor after: Zhang Bo Inventor before: Chen Wenling |
|
| COR | Change of bibliographic data | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160425 Address after: 300000 No. 220, Dongting Road, Tanggu District, Tianjin, Tianjin ten, N1006 laboratory building, international biological medicine Joint Research Institute Applicant after: Three source biotechnology (Tianjin) Co., Ltd. Address before: 250014 Ji'nan Province, Lixia District, Shandong Road, No. 26, No. 1 home, home, building, culture and culture, No. 1201 Applicant before: Chen Wenling |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information |
Inventor after: Kang Jun Inventor after: Ji Yanbin Inventor after: Li Tao Inventor after: Zhou Zhichao Inventor after: Zhang Bo Inventor before: Ji Yanbin Inventor before: Li Tao Inventor before: Zhou Zhichao Inventor before: Zhang Bo |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20190605 Address after: 300 000 building 807, A3, Hailande Industrial Park, Caizhi Road, Xiqing District, Tianjin Patentee after: Li Yongping Address before: 300000 N1006, 10th floor, Experimental Building of Tianjin International Biomedical Research Institute, 220 Dongting Road, Tanggu District, Tianjin Patentee before: Three source biotechnology (Tianjin) Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200120 Address after: 102600 Unit 403, Unit 1, 15th Floor, Changfengyuan, Huangcun Town, Daxing District, Beijing Patentee after: Du Lihong Address before: 300 000 building 807, A3, Hailande Industrial Park, Caizhi Road, Xiqing District, Tianjin Patentee before: Li Yongping |
|
| TR01 | Transfer of patent right | ||
| CP02 | Change in the address of a patent holder |
Address after: 241000 economic and Technological Development Zone Phase II, Nanling County, Wuhu City, Anhui Province Patentee after: Du Lihong Address before: 102600 Unit 403, Unit 1, 15th Floor, Changfengyuan, Huangcun Town, Daxing District, Beijing Patentee before: Du Lihong |
|
| CP02 | Change in the address of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201015 Address after: No.4, 2 / F, Zhonglou unit, No.399, Qunli Fourth Avenue, Daoli District, Harbin City, Heilongjiang Province, 150010 Patentee after: Harbin Shenhua Huikang biological Co., Ltd Address before: 241000 economic and Technological Development Zone Phase II, Nanling County, Wuhu City, Anhui Province Patentee before: Du Lihong |
|
| TR01 | Transfer of patent right |