CN106946998A - Natural antioxidant grifola frondosa polysaccharide CCPP-1 and preparation method and application thereof - Google Patents
Natural antioxidant grifola frondosa polysaccharide CCPP-1 and preparation method and application thereof Download PDFInfo
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- loudspeaker
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- 101100438623 Caenorhabditis elegans ccpp-1 gene Proteins 0.000 title claims abstract description 53
- 150000004676 glycans Chemical class 0.000 title claims abstract description 33
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 33
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 33
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 10
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 240000001080 Grifola frondosa Species 0.000 title abstract 3
- 235000007710 Grifola frondosa Nutrition 0.000 title abstract 3
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 claims abstract description 19
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims abstract description 10
- 235000013305 food Nutrition 0.000 claims abstract description 9
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 6
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims abstract description 5
- 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 claims abstract description 5
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims abstract description 5
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims abstract description 5
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 5
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000002537 cosmetic Substances 0.000 claims abstract description 5
- 239000008103 glucose Substances 0.000 claims abstract description 5
- 239000000203 mixture Substances 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 241000358321 Craterellus cornucopioides Species 0.000 claims description 16
- 239000012153 distilled water Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 13
- 230000002000 scavenging effect Effects 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 235000009508 confectionery Nutrition 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 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 description 6
- 238000010828 elution Methods 0.000 claims description 6
- -1 hydroxyl radical free radical Chemical class 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 239000013049 sediment Substances 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 5
- 238000007710 freezing Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000013019 agitation Methods 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000004148 unit process Methods 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 6
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 abstract description 4
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 abstract description 4
- 229940097043 glucuronic acid Drugs 0.000 abstract description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 2
- 150000002772 monosaccharides Chemical class 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 235000006708 antioxidants Nutrition 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 241000233866 Fungi Species 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 230000007760 free radical scavenging Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 3
- 230000003064 anti-oxidating effect Effects 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 150000005045 1,10-phenanthrolines Chemical class 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241001489099 Cantharellus Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003214 pyranose derivatives Chemical group 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
Classifications
-
- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Polymers & Plastics (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Sustainable Development (AREA)
- Birds (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
the invention discloses a natural antioxidant grifola frondosa polysaccharide CCPP-1 and a preparation method and application thereof, wherein the molecular weight of the grifola frondosa polysaccharide CCPP-1 is 1.85 × 106Da, whose monosaccharide composition consists of glucose in molar ratio: rhamnose: mannose: xylose: glucuronic acid 0.47: 0.05: 1: 0.86: 0.006. CCPP-1 extracted by the invention has obvious effect of removing DPPH and hydroxyl free radicals, when the concentration is 400ug/ml, the removal rate is equivalent to VC, and the CCPP-1 can be used as a natural high-efficiency antioxidant and applied to the fields of food, medicine, cosmetics and the like.
Description
Technical field
The present invention relates to grey loudspeaker granulose CCPP-1 of a kind of natural and its production and use, belong to food
Product manufacture field.
Background technology
Fungi polysaccharide is by its fructification or the mycelium produced by fermentation, zymotic fluid isolated one
Plant active polysaccharide.In recent years, fungi polysaccharide is because its extensive bioactivity and the effect naturally having no side effect are by more and more
Concern and research.Polysaccharide has important biological or activity in immunological regulation, anti-oxidant, anti-tumor aspect.Substantial amounts of research
Show that polysaccharide has antioxidation activity, such as mushroom polysaccharide, GL-B etc..Therefore, polysaccharide has huge in terms of food, medicine
Development volue be widely used in the fields such as biomedicine, food nutrition health.
Grey Craterellus cornucopioides (L.ex Fr.) Pers are a kind of macro fungis of thalline grey black, be mushroom mesh, chicken fat cordycepps, one of Cantharellus into
Member.Grey Craterellus cornucopioides (L.ex Fr.) Pers are originated in the remote, thickly forested mountains in Sichuan and Yunnan, edible, are a kind of very popular wild edible fungus.Mesh
Preceding this edible mushroom is liked by Europe deeply, is referred to as " delicious food " by them.
Grey Craterellus cornucopioides (L.ex Fr.) Pers are nutritious, according to the literature, and total fat accounts for 4.88% in every hectogram dry product, and crude protein is accounted for
69.45%, ash content accounts for 12.22%, and carbohydrate accounts for 13.44%, mainly contains mannose, trehalose etc., with very high
Nutritive value.The antibacterial activity for being mostly focused on grey loudspeaker coarse extraction, immunocompetence, the active anticancer to cancer cell are studied at present
In terms of nutrition, extraction separation and the research of bioactivity to grey loudspeaker granulose there are no document report.
The content of the invention
The present invention is intended to provide grey loudspeaker granulose CCPP-1 of a kind of natural and its production and use, mesh
Be that a kind of active polysaccharide of high antioxidant is isolated and purified from grey Craterellus cornucopioides (L.ex Fr.) Pers, be named as CCPP-1, can be as a kind of natural
Antioxidant, be widely used in food, medicine and the field such as cosmetics.
The present invention is the active polysaccharide ingredient that inoxidizability is isolated and purified from grey loudspeaker granulose, is as a result found, grey loudspeaker
Granulose CCPP-1 is almost suitable with VC to the Scavenging activity of DPPH and hydroxyl in 400ug/ml, with very high researching value
And development prospect.
Determined by HPLC, CCPP-1 of the present invention molecular weight is 1.85 × 106Da, its monose composition is constituted in molar ratio
For glucose：Rhamnose：Mannose：Xylose：Glucuronic acid=0.47：0.05：1：0.86：0.006.
The grey loudspeaker granulose CCPP-1 of natural of the present invention preparation method, including extract and isolate and purify each list
First process：
The extraction process comprises the following steps：
1st, the grey Craterellus cornucopioides (L.ex Fr.) Pers fructification dries pulverizings of 100g are weighed and 80 mesh sieves are crossed and obtain powder；
2nd, the powder for obtaining step 1 is added in distilled water, and 4h, solid-liquid ratio 1g are extracted at 75-85 DEG C：30mL, extracts three
It is secondary, merge extract solution and be concentrated into 100mL, concentrate is centrifuged 10 minutes through 4000r/min, takes supernatant；
3rd, the supernatant for obtaining step 2 is added in 95% ethanol, is centrifuged under 4 DEG C of standings 8-12h, 4000r/min
20min, collects sediment；
4th, the sediment that step 3 is obtained is dissolved with distilled water, Rotary Evaporators revolving removes the ethanol of residual, obtains thick
Polysaccharide solution；Sevage reagents are added into Thick many candies solution, volume ratio is 3:1, magnetic agitation 1 hour, 4000r/min centrifugations
10 minutes, retain supernatant；Sevage reagents repeat removing protein 5 times, merge supernatant, and concentration freezes 2h, freezing after -80 DEG C
After be put into freeze dryer under -56 DEG C, 0.10mbar freeze 2 days, obtain grey Craterellus cornucopioides (L.ex Fr.) Pers Thick many candies；
The process that isolates and purifies comprises the following steps：
The 1st, grey Craterellus cornucopioides (L.ex Fr.) Pers Thick many candies are made into 10% solution with distilled water, with DEAE-Sephorase F.F gel columns point
It is 10ml from, applied sample amount, 0-1.5mol/L NaCl solution gradient elutions, flow velocity is 1ml/min, is monitored in real time with AKTA instruments,
After 3500Da bag filter dialysis, concentration, -80 DEG C of freezing 2h are subsequently placed into freeze dryer under -56 DEG C, 0.10mbar
It is lyophilized 2 days, obtain component 1；
The 2nd, obtained component 1 is made into 0.1% solution with distilled water, 0.22 μm of filter membrane is crossed, uses Superdex-75 posts
Chromatography, distilled water elution, flow velocity 1ml/min is monitored in real time with AKTA instruments, and collection obtains homogeneous grey loudspeaker granulose
CCPP-1。
The CCPP-1 obtained to isolating and purifying carries out antioxidation activity in vitro experiment, as a result shows：What the present invention was extracted
CCPP-1 there is very high radicals scavenging to act on DPPH, hydroxyl radical free radical.
Analyze and determine through HPLC, CCPP-1 molecular weight is 1.85 × 106Da, monose composition is configured to grape in molar ratio
Sugar：Rhamnose：Mannose：Xylose：Glucuronic acid=0.47：0.05：1：0.86：0.006.
Ash loudspeaker granulose CCPP-1 of the invention purposes, is as antioxidant in food, medicine or cosmetic field
Use, there is significant scavenging action to DPPH, hydroxyl radical free radical.
Beneficial effects of the present invention are embodied in：
Ash Craterellus cornucopioides (L.ex Fr.) Pers CCPP-1 extracting and developing simple purifications of the invention, are a kind of natural high in antioxidants, extract and obtain
CCPP-1 have obvious scavenging action to DPPH, hydroxyl radical free radical, when concentration be 400ug/ml when, clearance rate all reach with
VC quite, can be as efficiency natural antioxidant, applied to fields such as food, medicine and cosmetics.
Brief description of the drawings
Fig. 1 is CCPP-1 molecular weight determination HPLC chromatograms.As can be seen that ash loudspeaker granulose CCPP-1 is from Figure 1B
Sterling, CCPP-1 retention time is 4.518min；From Figure 1A standard curves calculate CCPP-1 molecular weight for 1.85 ×
106Da。
Fig. 2 is influence of the number of processes to albumen clearance and polysaccharide loss rate.From figure 2 it can be seen that with processing
The increase of number of times, protein removal rate and polysaccharide loss rate all increase therewith.Preceding 5 protein removal rates and polysaccharide loss rate are all
Increased gather way quickly, after 5 times is slowed by.It is 5 times to consider selection number of processes, and now albumen clearance is
72%, polysaccharide loss rate is 36%.
Fig. 3 is the influence that sample liquid compares albumen clearance and polysaccharide loss rate.From figure 3, it can be seen that with sample liquid ratio
Increase, protein removal rate and polysaccharide loss rate are all gradually reduced.Albumen clearance is minimum at 5: 1, in 3: 1 to 4: 1 mistake
Cheng Zhong, it is most that clearance is reduced, and at 3: 1 to 5: 1, the reduction of polysaccharide loss rate is slower, is 30.2% at 3: 1.It is comprehensive
The sample liquid of the upper selection 3: 1 than being adapted to the most.
Fig. 4 is HPLC chromatogram (1 solvent peak of CCPP-1 hydrolysates；2GlcA；3Rha；4Man；5Xyl).Can from Fig. 4
Go out CCPP-1 monose composition and monose ratio with qualitative and quantitative analysis.It can be seen that from left to right, 1 represents solvent peak, CCPP-
1 has five kinds of monose, and its mol ratio is respectively glucose：Rhamnose：Mannose：Xylose：Glucuronic acid=0.47：0.05：
1：0.86：0.006.
Fig. 5 is Scavenging activities of the grey loudspeaker granulose CCPP-1 to DPPH.As can be seen from Figure 5 grey loudspeaker granulose
CCPP-1 from 25 μ g/ml to 400 μ g/ml in the range of, the Scavenging activity of DPPH free radicals strengthens with the increase of concentration, present
Certain concentration dependent.In 400 μ g/ml, clearance rate is up to 81.2%, with VC clearance rate 86.32% closely, by
This can be seen that CCPP-1 is notable to DPPH Scavenging activity, suitable with VC.
Fig. 6 is grey loudspeaker granulose CCPP-1 to hydroxyl radical free radical Scavenging activity.From fig. 6 it can be seen that in 25 μ g/ml
Ascendant trend is presented to OH Scavenging activity to 500 μ g/ml, there is certain concentration dependent.In 500 μ g/ml
CCPP-1 and VC clearance rate is respectively 73.74% and 84.4%, and showing CCPP-1 has very high removing energy to OH
Power.
Fig. 7 is grey loudspeaker granulose CCPP-1 infrared spectrograms.Grey Craterellus cornucopioides (L.ex Fr.) Pers are can be seen that through infrared spectrum (FT-IR) figure
Polysaccharide CCPP-1 has five typical absworption peaks 3396,2923,1643,1043cm-1Neighbouring position.Wherein, in 3396cm-1
The strong and wide absworption peak of phone, is another weak absorbing peak 2923cm caused by O-H stretching vibrations-1Place, is C-H stretching vibrations
It is caused.It is due to caused by O-H change angular oscillation, to show there is pyranoid ring at 1043, illustrate CCPP-1 in 1643 absworption peak
It is pyranose.In 854cm-1And 891cm-1The absworption peak at place shows that the existing α configurations of CCPP-1 also have beta comfiguration.
Embodiment
Embodiment 1：Sevage method removing proteins most preferably remove technique
(1) Sevage reagents extraction times
1st, grey Craterellus cornucopioides (L.ex Fr.) Pers fructification 100g dries pulverizings are weighed and 80 mesh sieves are crossed and obtain powder；
2nd, the powder for obtaining step 1 is added in distilled water, and 4h, solid-liquid ratio 1g are extracted at 75-85 DEG C：30mL, extracts three
It is secondary, merge extract solution and be concentrated to 100ml, concentrate is centrifuged 10 minutes through 4000r/min, takes supernatant；
3rd, 95% ethanol of three times volume is added after the supernatant for obtaining step 2,4 DEG C stand 8-12h, 4000r/
20min is centrifuged under min, sediment is collected；
4th, the sediment that step 3 is obtained is dissolved with distilled water, Rotary Evaporators revolving removes the ethanol of residual, obtains thick
Polysaccharide solution；Sevage reagents are added into Thick many candies solution, volume ratio is 3:1 magnetic agitation 1 hour, is extracted with Sevage reagents
Take 7 times, record protein removal rate and polysaccharide loss rate are shown in Fig. 2.
As shown in Figure 2, with the increase of number of processes, protein removal rate and polysaccharide loss rate all increase therewith.First 5 times
Protein removal rate and polysaccharide loss rate all increased gather way quickly, after 5 times are slowed by.Consider selection processing time
Number is 5 times, and now albumen clearance is 72%, and polysaccharide loss rate is 36%.
(2) sample liquid compares the influence of albumen clearance and polysaccharide loss rate
The volume ratio of sample and Sevage reagents is sample liquid ratio.It is respectively adopted 1: 1,2: 1,3: Isosorbide-5-Nitrae: 1,5: 1 sample liquid
Than with Sevage agent treatments polysaccharide solution 5 times, albumen clearance and polysaccharide loss rate that record is 5 times are shown in Fig. 3.
From the figure 3, it may be seen that with sample liquid than increase, protein removal rate and polysaccharide loss rate are all gradually reduced.Albumen is gone
Except rate at 5: 1 it is minimum, during 3: 1 to 4: 1, clearance reduction it is most, at 3: 1 to 5: 1, polysaccharide loss rate subtracts
It is few relatively slower, it is 30.2% at 3: 1.3: 1 sample liquid ratio is selected to be adapted to the most in summary.
Embodiment 2：Isolate and purify
1st, grey Craterellus cornucopioides (L.ex Fr.) Pers Thick many candies prepared by embodiment 1 are made into 10% (w/w) solution, carry out anion-exchange column layer
Analysis, gel column is DEAE-Sephorase F.F, and applied sample amount is 10ml, 0-1.5mol/L NaCl solution gradient elutions, and flow velocity is
1ml/min, is monitored in real time with AKTA instruments, after 3500Da bag filter dialysis, concentration, 2h is freezed at -80 DEG C, after freezing
It is put into freeze dryer in being freezed 2 days under -56 DEG C, 0.10mbar, obtains grey loudspeaker component 1.
The 2nd, above-mentioned obtained component 1 is made into 0.1% solution with distilled water, 0.22um filter membrane is crossed, uses Superdex
75 column chromatography for separation, distilled water elution, flow velocity 1ml/min is monitored in real time with AKTA instruments, and collection obtains grey loudspeaker polysaccharide
CCPP-1。
Embodiment 3：Antioxidation activity in vitro is determined
1st, the measure of DPPH free radical scavenging activities
Precision weighs DPPH 10mg, is dissolved and is settled in 200ml brown volumetric flasks with methanol, and obtaining concentration is
0.3mmol/L DPPH solution, keeps in dark place, standby.
Take respectively various concentrations CCPP-1 solution (25,50,100,200,300,400ug/ml) 2ml, put 15ml centrifugation
Guan Zhong, adds 2ml DPPH, room temperature lucifuge reaction 30min, light absorption value is determined at 517nm wavelength.Calculated by following equation
DPPH free radical scavenging activities.
DPPH free radical scavenging activities (%)=A0-(AS-AC)/A0× 100%
In formula, the light absorption value of A0-2ml DPPH solution+2ml distilled water
The light absorption value of As -2ml sample solution+2ml DPPH solution
The light absorption value of Ac -2ml sample solution+2ml methanol solutions
It will test in triplicate, and try to achieve the average value of clearance rate.As a result Fig. 5 is seen.As can be seen from Figure 5 grey Craterellus cornucopioides (L.ex Fr.) Pers are more
Sugared CCPP-1 from 25 μ g/ml to 400 μ g/ml in the range of, the Scavenging activity of DPPH free radicals strengthens with the increase of concentration, is in
Now certain concentration dependent.In 400 μ g/ml, clearance rate is up to 81.2%, with VC clearance rate 86.32% closely,
It can be seen that CCPP-1 is notable to DPPH Scavenging activity, it is suitable with VC.
2nd, the measure of Scavenging action to hydroxyl free radical
Fenton reactions are the OH extracorporeal modes reactions existed in organism.The method reacted with reference to Fenton sets up reaction
System model, utilizes H2O2With Fe2+Mixing produces OH (reaction equation H2O2+Fe2+→Fe3++OH+OH-)。Fe2+With 1,10- phenanthrolines
Stable orange red complex compound can be formed in the range of pH 2-9, there is absorption maximum at 510nm.When adding, grey Craterellus cornucopioides (L.ex Fr.) Pers are more
During sugared CCPP-1, CCPP-1 can remove the OH dissociated in solution, thus can calculate the grey loudspeaker of sample indirectly by measuring A values
Clearance rates of the granulose CCPP-1 to OH.
Clearance rate (%)=(A2-A1)/(A0-A1) × 100%
In formula：A0-be not added with H2O2, and A values when adding sample.
A1-addition H2O2, but A values when being not added with sample.
A2-addition H2O2A values during with sample.
Hydroxy radical is produced using Fenton reactions, 0.2mL1,10- phenanthrolines are sequentially added in 10mL tool colorimetric cylinders
(5mmol/L), 1mL Tris-HCl cushioning liquid (50mmol/L, PH 6.0), 0.2mL ferrous sulfate (7.5mmol/L),
0.2mL H2O2(1%) and different volumes grey loudspeaker granulose CCPP-1, absolute ethyl alcohol is settled to 10mL, then super in 37 DEG C
60min is reacted in level water bath with thermostatic control, A values are measured at 510nm, parallel 3 times, Fig. 6 are as a result seen.From fig. 6 it can be seen that 25
Ascendant trend is presented to OH Scavenging activity in μ g/ml to 500 μ g/ml, there is certain concentration dependent.In 500 μ g/ml
When CCPP-1 and VC clearance rate be respectively 73.74% and 84.4%, show CCPP-1 has very high removing to OH
Ability.
Claims (8)
1. a kind of grey loudspeaker granulose CCPP-1 of natural, it is characterised in that：The grey loudspeaker granulose CCPP-1's
Molecular weight is 1.85 × 106Da, its monose composition is configured to glucose in molar ratio：Rhamnose：Mannose：Xylose：Glucose
Aldehydic acid=0.47：0.05：1：0.86：0.006.
2. a kind of grey loudspeaker granulose CCPP-1 of natural described in claim 1 preparation method, including extract and
Isolate and purify each unit process, it is characterised in that the extraction process comprises the following steps：
(1) the grey Craterellus cornucopioides (L.ex Fr.) Pers fructification dries pulverizings of 100g are weighed and 80 mesh sieves are crossed and obtain powder；
(2) powder for obtaining step (1) is added in distilled water, and 4h is extracted at 75-85 DEG C, is extracted three times, merges extract solution simultaneously
Concentration, supernatant is left and taken after centrifugation；
(3) supernatant for obtaining step (2) is added in 95% ethanol, 4 DEG C of standing 8-12h, is centrifuged and is collected sediment；
(4) sediment for obtaining step (3) is dissolved with distilled water, and Rotary Evaporators revolving removes the ethanol of residual, obtains thick
Polysaccharide solution；Sevage reagents are added into Thick many candies solution, volume ratio is 3:1, magnetic agitation 1 hour retains after centrifugation
Clear liquid；Sevage reagents repeat removing protein 5 times, merge supernatant, and concentration freezes 2h after -80 DEG C, freeze dryer is put into after freezing
It is lyophilized, obtain grey Craterellus cornucopioides (L.ex Fr.) Pers Thick many candies.
3. preparation method according to claim 2, it is characterised in that：
In step (2), the powder that step (1) is obtained controls solid-liquid ratio when adding in distilled water be than 1g：30mL.
4. preparation method according to claim 2, it is characterised in that：
In step (4), freeze dryer freezes parameter and is：- 56 DEG C of temperature, air pressure 0.10mbar, 2 days time.
5. a kind of grey loudspeaker granulose CCPP-1 of natural described in claim 1 preparation method, including extract and
Isolate and purify each unit process, it is characterised in that the process that isolates and purifies comprises the following steps：
(1) grey Craterellus cornucopioides (L.ex Fr.) Pers Thick many candies are made into 10% solution with distilled water, with DEAE-Sephorase F.F gel post separations,
Applied sample amount is 10ml, 0-1.5mol/L NaCl solution gradient elutions, and flow velocity is 1ml/min, is monitored in real time with AKTA instruments, thoroughly
Concentrated after analysis, -80 DEG C of freezing 2h are subsequently placed into freeze dryer and freezed, obtain component 1；
(2) obtained component 1 is made into 0.1% solution with distilled water, 0.22 μm of filter membrane is crossed, with Superdex-75 posts layer
Analysis separation, distilled water elution, flow velocity 1ml/min is monitored in real time with AKTA instruments, and collection obtains homogeneous grey loudspeaker granulose
CCPP-1。
6. preparation method according to claim 5, it is characterised in that：
In step (1), 3500Da bag filter is used during dialysis.
7. preparation method according to claim 5, it is characterised in that：
In step (1), freeze dryer freezes parameter and is：- 56 DEG C of temperature, air pressure 0.10mbar, 2 days time.
8. the purposes of the grey loudspeaker granulose CCPP-1 of natural described in claim 1 a kind of, it is characterised in that：It is described
Grey loudspeaker granulose CCPP-1 is used in food, medicine or cosmetic field as antioxidant, is had to DPPH, hydroxyl radical free radical
There is significant scavenging action.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107474149A (en) * | 2017-08-03 | 2017-12-15 | 西华师范大学 | A kind of new natural products cockscomb granulose LSM X and its application |
| CN108309825A (en) * | 2018-04-27 | 2018-07-24 | 安徽大学 | Antioxidant capable of inhibiting melanin generation and preparation method and application thereof |
-
2017
- 2017-03-27 CN CN201710186705.0A patent/CN106946998A/en active Pending
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| Title |
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| WEI-WEI YANG ET AL.: ""Structural characterization and antioxidant activities of a novel polysaccharide fraction from the fruiting bodies of Craterellus cornucopioides"", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| 关海宁等: "《龙江地产农产品加工技术（第1版）》", 31 January 2013, 黑龙江大学出版社 * |
| 范晓丹等: ""喇叭菌多糖的结构和抗肿瘤活性"", 《现代食品科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107474149A (en) * | 2017-08-03 | 2017-12-15 | 西华师范大学 | A kind of new natural products cockscomb granulose LSM X and its application |
| CN107474149B (en) * | 2017-08-03 | 2019-09-06 | 西华师范大学 | A kind of new natural products cockscomb granulose LSM-X and its application |
| CN108309825A (en) * | 2018-04-27 | 2018-07-24 | 安徽大学 | Antioxidant capable of inhibiting melanin generation and preparation method and application thereof |
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