CN104151395A - Method for preparing cordyceps polysaccharide peptide - Google Patents
Method for preparing cordyceps polysaccharide peptide Download PDFInfo
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- CN104151395A CN104151395A CN201410213401.5A CN201410213401A CN104151395A CN 104151395 A CN104151395 A CN 104151395A CN 201410213401 A CN201410213401 A CN 201410213401A CN 104151395 A CN104151395 A CN 104151395A
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Abstract
The invention relates to a method for preparing a cordyceps polysaccharide peptide, which comprises the following steps: taking a water extract using cordyceps pure powder as a raw material, and concentrating, centrifugalizing, dialyzing and entrapping the water extract so as to obtain a high-molecular-weight polysaccharide peptide; slowly dropwise adding anhydrous ethanol into a cordyceps polysaccharide peptide solution with a concentration of 1% until the volume percent is 1:0.5 (V/V), and sequentially carrying out centrifugalizing, fractional precipitation and drying on the obtained mixture so as to obtain a first-level component cordyceps polysaccharide peptide 1; taking liquid supernatant, dropwise adding anhydrous ethanol until the volume percent is 1:1 (V/V), treating the obtained product as the above method, collecting 33-50% of precipitates, and drying the collected precipitates so as to obtain a second-level component cordyceps 2; further adding ethanol into liquid supernatant until the volume percent is 1:1.5 (V/V), so that a third-level component cordyceps 3 is obtained; and taking the cordyceps 3, adding water into the cordyceps 3 in a volume ratio of 1:10 to carry out micro-hot dissolving, carrying out column separation on the obtained product many times, dialyzing the obtained product for 12-20 h, concentrating, precipitating and centrifugalizing liquid in a bag, collecting a precipitated part, and freeze-drying the collected precipitated part, so that the cordyceps polysaccharide peptide is obtained. According to the invention, the control samples of the cordyceps polysaccharide peptide are self-prepared, and can be applied to the determination of the products.
Description
Technical field
The present invention relates to a kind of polysaccharide peptide, especially a kind of method of preparing Cordyceps Polysaccharide peptide.
Background technology
Cordyceps sinensis is very famous and precious tonic and health care superfine product, be found the effective constituents such as biologically active substance and amino acid, various trace elements and VITAMIN because containing the multiple known Buddhist monk such as cordycepin, cordycepic acid, adenosine, Cordyceps polysaccharide, SOD, having the growth of calm, relieving cough and reducing sputum and inhibition tumor cell, increase body and fitness and disease prevention is had to effect of peculiar immunity, is the three large treasures equally celebrated for their achievements with ginseng, pilose antler.
Peptide is the rudimentary form of protein, almost relates to the whole process of the life such as human hormone, nerve, growth, reproduction, has adjusting body function, the active substance of the activity that sustains life.
Polysaccharide peptide is a kind of glycoprotein, is the combination of polysaccharide and protein.Generally contain very a high proportion of polysaccharide, they are seen as glycosaminoglycan, have very high molecular weight, are made up of the repeating unit of disaccharide.Polysaccharide peptide has very strong biological activity, and preliminary study polysaccharide Toplink enhancing body immunologic function, has antitumor action and antioxygenation.
Cordyceps sinensis is distinctive a kind of natural, wild, the Chinese caterpillar fungus with precious intellecture property in snowy region area, China Qinghai-Tibet Platean, also claims extremely grass.Any artificial culture Cordyceps militaris (L.) Link. or Cordyceps mycelium all cannot be mentioned in the same breath with it, more can not be by titled with " Cordyceps sinensis ".Prepare voluntarily Cordyceps Polysaccharide peptide reference substance (purity is greater than 95%), and be applied to the mensuration of this series products, this research there is not yet record.
Summary of the invention
The object of the invention is to provide a kind of method of preparing Cordyceps Polysaccharide peptide, prepare voluntarily Cordyceps Polysaccharide peptide reference substance (purity is greater than 95%), and be applied to the mensuration of this series products, and by light scattering detector (HPLC-ELSD), the polysaccharide peptide composition in Cordyceps sinensis sample is carried out to qualitative and quantitative analysis.
The present invention adopts following technical scheme: a kind of method of preparing Cordyceps Polysaccharide peptide, and the method comprises the following steps:
Step 1: get the aqueous extract taking the pure powder of Cordyceps sinensis as raw material, concentrated, centrifugal, dialyse, hold back and obtain contain high molecular weight polysaccharide peptide;
Step 2: get the Cordyceps Polysaccharide peptide solution that contains high molecular weight polysaccharide peptide that in step 1, concentration is 1%, in constantly stirring, slowly dripping dehydrated alcohol to volume percent is 1: 0.5 (V/V), put at 3 DEG C～5 DEG C temperature and deposit 12-20h, centrifugal with 10000-12000rpm, use dehydrated alcohol fractionation precipitation, collect 33% ethanol precipitation part, putting refrigerator vacuum-drying, to obtain the 1st grade of composition be Cordyceps Polysaccharide peptide 1;
Step 3: Cordyceps Polysaccharide peptide 1 supernatant liquor of getting in step 2 drips dehydrated alcohol to 1 ︰ 1 (V/V) again, use dehydrated alcohol fractionation precipitation, the precipitation of collection 33～50%, puts at 3 DEG C～5 DEG C temperature and deposits 12-20h, and after vacuum-drying, obtaining the 2nd grade of composition is Cordyceps sinensis 2; Cordyceps sinensis 2 supernatant liquors are continued to add ethanol to 1 ︰ 1.5 (V/V) again, obtain 3rd level composition Cordyceps sinensis 3, put vacuum-drying and obtain Cordyceps Polysaccharide peptide crude product.
Step 4: get Cordyceps Polysaccharide peptide crude product with 1: 10 ratio (V/V) add water low-grade fever dissolve, centrifugal impurity elimination, separate by least 2 posts of polypropylene amine gel, with the Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution containing 50molL-1NaCl, dialysis 12～20h, in bag taking, liquid is concentrated, by three times of dehydrated alcohol precipitations, centrifugal, collecting precipitation part, dry at-16 DEG C～-20 DEG C temperature, obtaining light brown powdery substance is Cordyceps Polysaccharide peptide.
Preparing unimodally through HPLC, and is that Cordyceps Polysaccharide peptide is unimodal by HPLC-PDA/ELSD detected result, and purity is more than 95%.
In above-mentioned steps (4), adopt the centrifugal impurity elimination of 10000-12000rpm.
In above-mentioned steps (4), contain 50molL
-1its pH=5.8-6.0 of Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of NaCl, best results when concentration is 0.2mol/L.
Best results when polypropylene amine gel adopts Bio-Gel series in above-mentioned steps (4).
The above-mentioned description to structure of the present invention and method is known, and compared to the prior art, tool of the present invention has the following advantages: one, the present invention prepares Cordyceps Polysaccharide peptide reference substance (purity is greater than 95%) voluntarily, can be applied to the mensuration of this series products.Its two, the present invention can carry out qualitative and quantitative analysis to the polysaccharide peptide composition in Cordyceps sinensis sample by light scattering detector (HPLC-ELSD).Its three, sample pre-treatments simple and fast of the present invention, time saving and energy saving, is conducive to the accurate qualitative and quantitative of polysaccharide peptide composition in Cordyceps sinensis product.Its four, method of the present invention is easy and simple to handle, the rate of recovery is high, reproducible, result accurately and reliably, is conducive to the identification and analysis of health factor in Cordyceps sinensis product.
Embodiment
Embodiment mono-:
A method of preparing Cordyceps Polysaccharide peptide, the method comprises the following steps:
Step 1: get the aqueous extract taking the pure powder of Cordyceps sinensis as raw material, concentrated, centrifugal, dialyse, hold back and obtain contain high molecular weight polysaccharide peptide;
Step 2: get the Cordyceps Polysaccharide peptide solution that contains high molecular weight polysaccharide peptide that in step 1, concentration is 1%, in constantly stirring, slowly dripping dehydrated alcohol to volume percent is 1: 0.5 (V/V), put at 3 DEG C of temperature and deposit 12h, centrifugal with 10000rpm, use dehydrated alcohol fractionation precipitation, collect 33% ethanol precipitation part, putting refrigerator vacuum-drying, to obtain the 1st grade of composition be Cordyceps Polysaccharide peptide 1;
Step 3: Cordyceps Polysaccharide peptide 1 supernatant liquor of getting in step 2 drips dehydrated alcohol to 1 ︰ 1 (V/V) again, use dehydrated alcohol fractionation precipitation, the precipitation of collection 33～50%, puts at 3 DEG C of temperature and deposits 12h, and after vacuum-drying, obtaining the 2nd grade of composition is Cordyceps sinensis 2; Cordyceps sinensis 2 supernatant liquors are continued to add ethanol to 1 ︰ 1.5 (V/V) again, obtain 3rd level composition Cordyceps sinensis 3, put vacuum-drying and obtain Cordyceps Polysaccharide peptide crude product.
Step 4: get Cordyceps Polysaccharide peptide crude product and dissolve with 1: 10 ratio low-grade fever that adds water, adopt the centrifugal impurity elimination of 10000rpm, separate by least 2 posts of polypropylene amine gel, with containing 50molL
-1sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of NaCl, dialysis 12h, in bag taking, liquid is concentrated, by three times of dehydrated alcohol precipitations, centrifugal, collecting precipitation part, dry at-16 DEG C of temperature, obtaining light brown powdery substance is Cordyceps Polysaccharide peptide.
Preparing unimodally through HPLC, and is that Cordyceps Polysaccharide peptide is unimodal by HPLC-PDA/ELSD detected result, and purity is more than 95%.
In step (4), contain 50molL
-1its pH=5.8 of Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of NaCl, when concentration is 0.2mol/L, effect is better.
Best results when polypropylene amine gel adopts Bio-Gel series in step (4).
The present invention prepares Cordyceps Polysaccharide peptide reference substance (purity is greater than 95%) voluntarily, can be applied to the mensuration of this series products.The present invention can carry out qualitative and quantitative analysis to the polysaccharide peptide composition in Cordyceps sinensis sample by light scattering detector (HPLC-ELSD).Sample pre-treatments simple and fast of the present invention, time saving and energy saving, is conducive to the accurate qualitative and quantitative of polysaccharide peptide composition in Cordyceps sinensis product.Method of the present invention is easy and simple to handle, and the rate of recovery is high, reproducible, and result accurately and reliably, is conducive to the identification and analysis of health factor in Cordyceps sinensis product.
Embodiment bis-:
Step 1: get the aqueous extract taking the pure powder of Cordyceps sinensis as raw material, concentrated, centrifugal, dialyse, hold back and obtain contain high molecular weight polysaccharide peptide;
Step 2: get the Cordyceps Polysaccharide peptide solution that contains high molecular weight polysaccharide peptide that in step 1, concentration is 1%, in constantly stirring, slowly dripping dehydrated alcohol to volume percent is 1: 0.5 (V/V), put at 4 DEG C of temperature and deposit 16h, centrifugal with 11000rpm, use dehydrated alcohol fractionation precipitation, collect 33% ethanol precipitation part, putting refrigerator vacuum-drying, to obtain the 1st grade of composition be Cordyceps Polysaccharide peptide 1;
Step 3: Cordyceps Polysaccharide peptide 1 supernatant liquor of getting in step 2 drips dehydrated alcohol to 1 ︰ 1 (V/V) again, use dehydrated alcohol fractionation precipitation, the precipitation of collection 33～50%, puts at 4 DEG C of temperature and deposits 16h, and after vacuum-drying, obtaining the 2nd grade of composition is Cordyceps sinensis 2; Cordyceps sinensis 2 supernatant liquors are continued to add ethanol to 1 ︰ 1.5 (V/V) again, obtain 3rd level composition Cordyceps sinensis 3, put vacuum-drying and obtain Cordyceps Polysaccharide peptide crude product.
Step 4: get Cordyceps Polysaccharide peptide crude product and dissolve with 1: 10 ratio low-grade fever that adds water, adopt the centrifugal impurity elimination of 11000rpm, separate by 4 posts of polypropylene amine gel, with containing 50molL
-1sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of NaCl, dialysis 16h, in bag taking, liquid is concentrated, by three times of dehydrated alcohol precipitations, centrifugal, collecting precipitation part, dry at-18 DEG C of temperature, obtaining light brown powdery substance is Cordyceps Polysaccharide peptide.
Preparing unimodally through HPLC, and is that Cordyceps Polysaccharide peptide is unimodal by HPLC-PDA/ELSD detected result, and purity is more than 95%.
In step (4), contain 50molL
-1its pH=5.9 of Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of NaCl, when concentration is 0.2mol/L, effect is better.
Best results when polypropylene amine gel adopts Bio-Gel series in step (4).
This Cordyceps Polysaccharide peptide can be applied in mensuration Cordyceps sinensis quality product.
Embodiment tri-:
A method of preparing Cordyceps Polysaccharide peptide, the method comprises the following steps:
Step 1: get the aqueous extract taking the pure powder of Cordyceps sinensis as raw material, concentrated, centrifugal, dialyse, hold back and obtain contain high molecular weight polysaccharide peptide;
Step 2: get the Cordyceps Polysaccharide peptide solution that contains high molecular weight polysaccharide peptide that in step 1, concentration is 1%, in constantly stirring, slowly dripping dehydrated alcohol to volume percent is 1: 0.5 (V/V), put at 5 DEG C of temperature and deposit 20h, centrifugal with 12000rpm, use dehydrated alcohol fractionation precipitation, collect 33% ethanol precipitation part, putting refrigerator vacuum-drying, to obtain the 1st grade of composition be Cordyceps Polysaccharide peptide 1;
Step 3: Cordyceps Polysaccharide peptide 1 supernatant liquor of getting in step 2 drips dehydrated alcohol to 1 ︰ 1 (V/V) again, use dehydrated alcohol fractionation precipitation, the precipitation of collection 33～50%, puts at 5 DEG C of temperature and deposits 20h, and after vacuum-drying, obtaining the 2nd grade of composition is Cordyceps sinensis 2; Cordyceps sinensis 2 supernatant liquors are continued to add ethanol to 1 ︰ 1.5 (V/V) again, obtain 3rd level composition Cordyceps sinensis 3, put vacuum-drying and obtain Cordyceps Polysaccharide peptide crude product.
Step 4: get Cordyceps Polysaccharide peptide crude product and dissolve with 1: 10 ratio low-grade fever that adds water, adopt the centrifugal impurity elimination of 12000rpm, separate by 6 posts of polypropylene amine gel, with containing 50molL
-1sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of NaCl, dialysis 20h, in bag taking, liquid is concentrated, by three times of dehydrated alcohol precipitations, centrifugal, collecting precipitation part, dry at-20 DEG C of temperature, obtaining light brown powdery substance is Cordyceps Polysaccharide peptide.
Preparing unimodally through HPLC, and is that Cordyceps Polysaccharide peptide is unimodal by HPLC-PDA/ELSD detected result, and purity is more than 95%.
In above-mentioned steps (4), contain 50molL
-1its pH=6.0 of Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of NaCl, when concentration is 0.2mol/L, effect is better.
Best results when polypropylene amine gel adopts Bio-Gel series in step (4).
This Cordyceps Polysaccharide peptide can be applied in mensuration Cordyceps sinensis quality product.
Above are only the specific embodiment of the present invention, but design concept of the present invention is not limited to this, allly utilizes this design to carry out the change of unsubstantiality to the present invention, all should belong to the behavior of invading protection domain of the present invention.
Claims (5)
1. a method of preparing Cordyceps Polysaccharide peptide, is characterized in that, the method comprises the following steps:
(1) get the aqueous extract taking the pure powder of Cordyceps sinensis as raw material, concentrated, centrifugal, dialyse, hold back and obtain contain high molecular weight polysaccharide peptide;
(2) get the Cordyceps Polysaccharide peptide solution that contains high molecular weight polysaccharide peptide that in step (1), concentration is 1%, in constantly stirring, slowly dripping dehydrated alcohol to volume percent is 1: 0.5, put at 3 DEG C～5 DEG C temperature and deposit 12-20h, centrifugal with 10000-12000rpm, use dehydrated alcohol fractionation precipitation, collect 33% ethanol precipitation part, putting refrigerator vacuum-drying, to obtain the 1st grade of composition be Cordyceps Polysaccharide peptide 1;
(3) getting Cordyceps Polysaccharide peptide 1 supernatant liquor in (2) step, to drip dehydrated alcohol to volume percent be 1 ︰ 1 again, use dehydrated alcohol fractionation precipitation, the precipitation of collection 33～50%, put at 3 DEG C～5 DEG C temperature and deposit 12-20h, after vacuum-drying, obtaining the 2nd grade of composition is Cordyceps sinensis 2; It is 1 ︰ 1.5 that Cordyceps sinensis 2 supernatant liquors are continued to add ethanol to volume percent again, obtains 3rd level composition Cordyceps sinensis 3, puts vacuum-drying and obtains Cordyceps Polysaccharide peptide crude product;
(4) getting Cordyceps Polysaccharide peptide crude product dissolves with 1: 10 ratio low-grade fever that adds water, centrifugal impurity elimination, separate by least 2 posts of polypropylene amine gel, with the Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution containing 50molL-1NaCl, dialysis 12～20h, in bag taking, liquid is concentrated, by three times of dehydrated alcohol precipitations, centrifugal, collecting precipitation part, dry at-16 DEG C～-20 DEG C temperature, obtaining light brown powdery substance is Cordyceps Polysaccharide peptide.
2. according to a kind of method of preparing Cordyceps Polysaccharide peptide claimed in claim 1, it is characterized in that: preparing unimodally through HPLC, and is that Cordyceps Polysaccharide peptide is unimodal by HPLC-PDA/ELSD detected result, and purity is more than 95%.
3. according to a kind of method of preparing Cordyceps Polysaccharide peptide claimed in claim 1, it is characterized in that: in described step (4), adopt the centrifugal impurity elimination of 10000-12000rpm.
4. according to a kind of method of preparing Cordyceps Polysaccharide peptide claimed in claim 1, it is characterized in that: its pH=5.8-6.0 of Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution in described step (4), best results when concentration is 0.2mol/L.
5. according to a kind of method of preparing Cordyceps Polysaccharide peptide claimed in claim 1, it is characterized in that: best results when polypropylene amine gel adopts Bio-Gel series in described step (4).
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410213401.5A CN104151395A (en) | 2014-05-20 | 2014-05-20 | Method for preparing cordyceps polysaccharide peptide |
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| CN201410213401.5A CN104151395A (en) | 2014-05-20 | 2014-05-20 | Method for preparing cordyceps polysaccharide peptide |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1482144A (en) * | 2003-07-25 | 2004-03-17 | 山东智灵生物工程有限公司 | Method for preparing galactomannanpeptide and product |
| CN102584963A (en) * | 2012-01-19 | 2012-07-18 | 福建农大菌草技术开发公司 | Active Ganoderma lucidum polysaccharide peptide reference substance, and preparation method and application thereof |
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2014
- 2014-05-20 CN CN201410213401.5A patent/CN104151395A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1482144A (en) * | 2003-07-25 | 2004-03-17 | 山东智灵生物工程有限公司 | Method for preparing galactomannanpeptide and product |
| CN102584963A (en) * | 2012-01-19 | 2012-07-18 | 福建农大菌草技术开发公司 | Active Ganoderma lucidum polysaccharide peptide reference substance, and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JUNG-YOUNG OH 等: "Production of polysaccharide -peptide complexes by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala", 《PROCESS BIOCHEMISTRY》 * |
| 陈肖虹 等: "冬虫夏草多糖肽的提取与制备", 《福建中医药》 * |
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Application publication date: 20141119 |
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