CN105732250B - A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction - Google Patents
A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction Download PDFInfo
- Publication number
- CN105732250B CN105732250B CN201610082919.9A CN201610082919A CN105732250B CN 105732250 B CN105732250 B CN 105732250B CN 201610082919 A CN201610082919 A CN 201610082919A CN 105732250 B CN105732250 B CN 105732250B
- Authority
- CN
- China
- Prior art keywords
- acid
- purity
- polyphenol
- grifola frondosus
- preparation
- 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
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 54
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 241000222684 Grifola Species 0.000 title claims description 35
- -1 polyphenol compound Chemical class 0.000 claims abstract description 17
- 240000001080 Grifola frondosa Species 0.000 claims abstract description 13
- 235000007710 Grifola frondosa Nutrition 0.000 claims abstract description 13
- 239000011347 resin Substances 0.000 claims abstract description 12
- 229920005989 resin Polymers 0.000 claims abstract description 12
- 238000005516 engineering process Methods 0.000 claims abstract description 9
- 150000004676 glycans Chemical class 0.000 claims abstract description 6
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 6
- 239000005017 polysaccharide Substances 0.000 claims abstract description 6
- 238000002953 preparative HPLC Methods 0.000 claims abstract description 6
- 239000012528 membrane Substances 0.000 claims abstract description 3
- 238000001179 sorption measurement Methods 0.000 claims abstract description 3
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 13
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 12
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims description 12
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 12
- 235000005875 quercetin Nutrition 0.000 claims description 12
- 229960001285 quercetin Drugs 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- COVFEVWNJUOYRL-UHFFFAOYSA-N digallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)=C1 COVFEVWNJUOYRL-UHFFFAOYSA-N 0.000 claims description 10
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 claims description 10
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 238000004440 column chromatography Methods 0.000 claims description 8
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims description 8
- 229930182478 glucoside Natural products 0.000 claims description 8
- 150000008131 glucosides Chemical class 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 claims description 7
- 239000000287 crude extract Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 239000002250 absorbent Substances 0.000 claims description 6
- 230000002745 absorbent Effects 0.000 claims description 6
- 125000002252 acyl group Chemical group 0.000 claims description 6
- 239000000945 filler Substances 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 claims description 5
- 229920002707 Digallic acid Polymers 0.000 claims description 5
- 240000003152 Rhus chinensis Species 0.000 claims description 5
- 235000014220 Rhus chinensis Nutrition 0.000 claims description 5
- 235000004883 caffeic acid Nutrition 0.000 claims description 5
- 229940074360 caffeic acid Drugs 0.000 claims description 5
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- DMFPEGLBGOENBN-UHFFFAOYSA-N m-Digallic acid Natural products Cc1c(O)cc(cc1OC(=O)c2cc(O)c(O)c(O)c2)C(=O)O DMFPEGLBGOENBN-UHFFFAOYSA-N 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- SWGKAHCIOQPKFW-JTNORFRNSA-N Caftaric acid Chemical compound OC(=O)[C@H](O)[C@H](C(O)=O)OC(=O)\C=C\C1=CC=C(O)C(O)=C1 SWGKAHCIOQPKFW-JTNORFRNSA-N 0.000 claims description 4
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 claims description 4
- AFSDNFLWKVMVRB-UHFFFAOYSA-N Ellagic acid Chemical compound OC1=C(O)C(OC2=O)=C3C4=C2C=C(O)C(O)=C4OC(=O)C3=C1 AFSDNFLWKVMVRB-UHFFFAOYSA-N 0.000 claims description 4
- ATJXMQHAMYVHRX-CPCISQLKSA-N Ellagic acid Natural products OC1=C(O)[C@H]2OC(=O)c3cc(O)c(O)c4OC(=O)C(=C1)[C@H]2c34 ATJXMQHAMYVHRX-CPCISQLKSA-N 0.000 claims description 4
- 229920002079 Ellagic acid Polymers 0.000 claims description 4
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 4
- 239000003463 adsorbent Substances 0.000 claims description 4
- SWGKAHCIOQPKFW-GHMZBOCLSA-N caffeoyltartaric acid Natural products OC(=O)[C@H](O)[C@H](C(O)=O)OC(=O)C=CC1=CC=C(O)C(O)=C1 SWGKAHCIOQPKFW-GHMZBOCLSA-N 0.000 claims description 4
- 229960002852 ellagic acid Drugs 0.000 claims description 4
- 235000004132 ellagic acid Nutrition 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 235000004515 gallic acid Nutrition 0.000 claims description 4
- 229940074391 gallic acid Drugs 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- FAARLWTXUUQFSN-UHFFFAOYSA-N methylellagic acid Natural products O1C(=O)C2=CC(O)=C(O)C3=C2C2=C1C(OC)=C(O)C=C2C(=O)O3 FAARLWTXUUQFSN-UHFFFAOYSA-N 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 239000012071 phase Substances 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 235000002906 tartaric acid Nutrition 0.000 claims description 4
- 239000011975 tartaric acid Substances 0.000 claims description 4
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims description 3
- 241001313855 Bletilla Species 0.000 claims description 3
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 3
- 238000006356 dehydrogenation reaction Methods 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 229930003944 flavone Natural products 0.000 claims description 3
- 150000002213 flavones Chemical class 0.000 claims description 3
- 235000011949 flavones Nutrition 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 239000001630 malic acid Substances 0.000 claims description 3
- 235000011090 malic acid Nutrition 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 2
- 230000008025 crystallization Effects 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000001694 spray drying Methods 0.000 claims description 2
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 claims 2
- SJSOFNCYXJUNBT-UHFFFAOYSA-N 3,4,5-trimethoxybenzoic acid Chemical class COC1=CC(C(O)=O)=CC(OC)=C1OC SJSOFNCYXJUNBT-UHFFFAOYSA-N 0.000 claims 1
- 239000012156 elution solvent Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 25
- 239000000178 monomer Substances 0.000 abstract description 10
- 102100024295 Maltase-glucoamylase Human genes 0.000 abstract description 5
- 108010028144 alpha-Glucosidases Proteins 0.000 abstract description 5
- 239000008280 blood Substances 0.000 abstract description 5
- 210000004369 blood Anatomy 0.000 abstract description 5
- 238000000746 purification Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 235000013376 functional food Nutrition 0.000 abstract description 2
- 230000035790 physiological processes and functions Effects 0.000 abstract description 2
- 238000000108 ultra-filtration Methods 0.000 abstract description 2
- 230000032683 aging Effects 0.000 abstract 1
- 230000001093 anti-cancer Effects 0.000 abstract 1
- 230000002526 effect on cardiovascular system Effects 0.000 abstract 1
- 230000007760 free radical scavenging Effects 0.000 abstract 1
- 210000005260 human cell Anatomy 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 abstract 1
- 238000002137 ultrasound extraction Methods 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 239000000523 sample Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 7
- 230000003064 anti-oxidating effect Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 5
- 230000031700 light absorption Effects 0.000 description 5
- PUNOCEUUYUXUGR-UHFFFAOYSA-N 5-nonadecylresorcinol Natural products CCCCCCCCCCCCCCCCCCCC1=CC(O)=CC(O)=C1 PUNOCEUUYUXUGR-UHFFFAOYSA-N 0.000 description 4
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 230000002292 Radical scavenging effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- FBSFWRHWHYMIOG-UHFFFAOYSA-N methyl 3,4,5-trihydroxybenzoate Chemical compound COC(=O)C1=CC(O)=C(O)C(O)=C1 FBSFWRHWHYMIOG-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 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 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000004262 preparative liquid chromatography Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 description 2
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 description 2
- LWROVTMVBSWNIL-ZZXKWVIFSA-N 2,3-dihydroxy-2-[(E)-3-(4-hydroxyphenyl)prop-2-enoyl]butanedioic acid Chemical compound OC(=O)C(O)C(O)(C(O)=O)C(=O)\C=C\C1=CC=C(O)C=C1 LWROVTMVBSWNIL-ZZXKWVIFSA-N 0.000 description 2
- UZDZNIDGWLPLJL-ZZXKWVIFSA-N 2-hydroxy-2-[(E)-3-(4-hydroxyphenyl)prop-2-enoyl]butanedioic acid Chemical compound OC(=O)CC(O)(C(O)=O)C(=O)\C=C\C1=CC=C(O)C=C1 UZDZNIDGWLPLJL-ZZXKWVIFSA-N 0.000 description 2
- SPKZMOSDXTYXLK-UHFFFAOYSA-N Dehydrodiferulic acid Natural products C1=C(O)C(OC)=CC(C=C(C(O)=O)C(=CC=2C=C(OC)C(O)=CC=2)C(O)=O)=C1 SPKZMOSDXTYXLK-UHFFFAOYSA-N 0.000 description 2
- DNJFYRXUTBVKIJ-UHFFFAOYSA-N Dehydrodiferulic dilactone Chemical compound C1=C(O)C(OC)=CC(C2C3C(=O)OC(C3C(=O)O2)C=2C=C(OC)C(O)=CC=2)=C1 DNJFYRXUTBVKIJ-UHFFFAOYSA-N 0.000 description 2
- OVSQVDMCBVZWGM-SJWGPRHPSA-N Hyperin Natural products O[C@H]1[C@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-SJWGPRHPSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229960002632 acarbose Drugs 0.000 description 2
- XUFXOAAUWZOOIT-UHFFFAOYSA-N acarviostatin I01 Natural products OC1C(O)C(NC2C(C(O)C(O)C(CO)=C2)O)C(C)OC1OC(C(C1O)O)C(CO)OC1OC1C(CO)OC(O)C(O)C1O XUFXOAAUWZOOIT-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 description 2
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 2
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 description 2
- 235000001785 ferulic acid Nutrition 0.000 description 2
- 229940114124 ferulic acid Drugs 0.000 description 2
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 description 2
- 235000012041 food component Nutrition 0.000 description 2
- 239000005417 food ingredient Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- IBKQQKPQRYUGBJ-UHFFFAOYSA-N methyl gallate Natural products CC(=O)C1=CC(O)=C(O)C(O)=C1 IBKQQKPQRYUGBJ-UHFFFAOYSA-N 0.000 description 2
- INYJZRKTYXTZHP-UHFFFAOYSA-N p-Coumaroyl tartaric acid Natural products OC(=O)C(O)C(C(O)=O)OC(=O)C=CC1=CC=C(O)C=C1 INYJZRKTYXTZHP-UHFFFAOYSA-N 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- OVSQVDMCBVZWGM-QSOFNFLRSA-N quercetin 3-O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-QSOFNFLRSA-N 0.000 description 2
- BPYKBJYZLASLKL-LKDYQKJYSA-N quercetin-3-O-beta-D-galactopyranoside Natural products CO[C@H]1O[C@@H](OC2=C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)[C@H](O)[C@@H](O)[C@H]1O BPYKBJYZLASLKL-LKDYQKJYSA-N 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 238000010183 spectrum analysis Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- BMRSEYFENKXDIS-OTCYKTEZSA-N trans-5-O-(4-coumaroyl)-D-quinic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C=C1 BMRSEYFENKXDIS-OTCYKTEZSA-N 0.000 description 2
- SWGKAHCIOQPKFW-UHFFFAOYSA-N trans-caffeoyl-tartaric acid Natural products OC(=O)C(O)C(C(O)=O)OC(=O)C=CC1=CC=C(O)C(O)=C1 SWGKAHCIOQPKFW-UHFFFAOYSA-N 0.000 description 2
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 description 2
- QVPHNABUSKBIMG-UHFFFAOYSA-N trans-p-coumaroylmalic acid Natural products OC(=O)CC(C(O)=O)OC(=O)C=CC1=CC=C(O)C=C1 QVPHNABUSKBIMG-UHFFFAOYSA-N 0.000 description 2
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000222341 Polyporaceae Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000000549 coloured material Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 125000004402 polyphenol group Chemical group 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B63/00—Purification; Separation; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/685—Processes comprising at least two steps in series
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
- C07C37/82—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/06—Peri-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/08—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to carbocyclic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
本发明涉及一种高纯度灰树花多酚组分的制备方法，属于功能食品的功能因子和药物领域。通过超声波辅助浸提技术制备灰树花多酚粗组分，利然后用超滤膜截留技术去除其中的多糖和蛋白等生物大分子，最后采用大孔吸附树脂NKA‑Ⅱ柱层析‑制备型高效液相色谱的串联分离纯化技术，制备多种高纯度的灰树花多酚类化合物单体。本发明制备方法快速高效，可同时制备17种灰树花多酚类化合物单体，产品的纯度高。所制备的灰树花多酚类化合物单体均具有较强的DPPH自由基和羟自由基清除率及较强的还原力，大多数多酚类化合物单体能够抑制α‑葡萄糖苷酶活性，具有有效地保护人体细胞组织、心脑血管循环系统及抗癌、延缓衰老和辅助降血糖等生理作用。The invention relates to a preparation method of high-purity grifola frondosa polyphenol components, belonging to the field of functional factors and medicines of functional foods. The crude polyphenol fraction of Grifola frondosa was prepared by ultrasonic-assisted extraction technology, and then the biomacromolecules such as polysaccharides and proteins were removed by ultrafiltration membrane interception technology, and finally macroporous adsorption resin NKA‑Ⅱ column chromatography‑preparative High-performance liquid chromatography tandem separation and purification technology to prepare a variety of high-purity polyphenol compound monomers from Grifola frondosa. The preparation method of the present invention is fast and efficient, can simultaneously prepare 17 kinds of Grifola frondosa polyphenol compound monomers, and the product has high purity. The prepared polyphenolic compound monomers of Grifola frondosa have strong DPPH free radical and hydroxyl free radical scavenging rate and strong reducing power, most of the polyphenolic compound monomers can inhibit the activity of α-glucosidase, It has physiological functions such as effectively protecting human cell tissue, cardiovascular and cerebrovascular circulatory system, anti-cancer, delaying aging and assisting in lowering blood sugar.
Description
Technical field
The present invention relates to a kind of preparation method of high-purity grifola frondosus weight polyphenol fraction, belong to functional food function factor and
Drug field.
Background technology
Grifola frondosus（Grifola frondosa）It is true to belong to Basidiomycotina Hymenomycetes Aphyllophorales Polyporaceae tree Pseudomonas
Bacterium, subtropical zone to temperate zone is mainly grown in, the ground such as Fujian, Hebei, Jilin, Guangxi and Sichuan are distributed mainly in China.As
A kind of precious food, medicine dual-purpose gill fungus bacterium, the various nutrients of grifola frondosus and physiological function are known as into first of the various edible mushrooms that live apart
" edible mushroom prince " and the good reputation of " North China ginseng ".Correlative study shows that grifola frondosus has strengthen immunity, suppresses tumour, be anti-
Viral, anti-oxidant anti-aging, stabilizing blood pressure, reducing blood sugar and blood lipid, improve fat metabolism and alleviate hepatitis symptom, enhancing memory etc.
A variety of medicinal health care functions.Numerous studies work shows that polyphenols has anti-oxidant, antibacterial, anti-mutagenesis, antiviral, anti-
The multiple functions such as tumour, reducing blood sugar and blood fat activity.Research report both at home and abroad for ash tree flower polyphenol is less.Chen XiangDong et al.
Using the macroporous absorbent resin of low pole, separation prepares Polyphenols component from maitake mushroom mycelia crude extract, and the component of acquisition is
Brown powder shape product, empirical tests have anti-oxidant, antibacterial and alpha-amylase suppresses isoreactivity, but its obtained ash tree flower polyphenol
It is mixture, is not further purified and identifies that grifola frondosus weight polyphenol fraction needs into one to the component of grifola frondosus polyphenols
Walk separating-purifying（Chen XiangDong, Liu Xiaowen, the extraction of Wu Wutong ash tree flower polyphenols and activity research food and biotechnics
Report, 2005,24 (4): 26-30.）.
The present invention is proposed using Grifola Frondosa sporophore as raw material, and ash tree flower polyphenol group is prepared by Ultrasonic Wave-Assisted Extraction technology
Point, then profit removes the large biological molecules such as polysaccharide and albumen therein with milipore filter retention technology, finally using macroporous absorption tree
The separation purification technique of II column chromatographies of fat NKA--preparative high performance liquid chromatography, the grifola frondosus for preparing a variety of high-purities are more
Phenolic compound monomer.Compared to polyphenols is extracted from maitake mushroom mycelia in the past, the invention uses real with ash tree beggar
Body is raw material, and raw material sources are easier to obtain and polyphenol content comes high compared to mycelium, and the extraction yield of polyphenol is also higher；With
Existing research report is compared, and is eliminated using the grifola frondosus weight polyphenol fraction obtained by ultrafiltration membrance filter in Effects of Extracts of Grifola frondosa on Active
The macromolecular substances such as polysaccharide, albumen, it is made through column chromatography-preparative liquid chromatography series connection thick weight polyphenol fraction of purification techniques
The weight polyphenol fraction purity obtained is higher（Purity is all higher than 95%）, also conspicuousness improves polyphenol antioxidation activity in vitro after purification, its
Middle reducing power than etc. mass concentration（1 mg/mL）Ascorbic acid it is strong.
The content of the invention
It is an object of the invention to provide it is a kind of rapidly and efficiently, the grifola frondosus polyphenol compound that purity is high, preparation amount is larger
The preparation technology of monomer.
A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction, its preparation process are as follows：
（1）Pretreatment：Grifola Frondosa sporophore dried product is ground into powder, it is standby to cross 40~80 mesh sieves；
（2）Extraction：A certain amount of grifola frondosus powder is taken, is extracted 2-4 times using ultrasonic assistant extractive technique, merges extraction
Liquid, residue is removed through plate-frame filtering；
（3）Membrane filtration：Filtered by film filter, retain egg bletilla polysaccharide, the concentrating filter liquor of acquisition, drying
Ash tree flower polyphenol crude extract is obtained afterwards；
（4）Isolate and purify：Ash tree flower polyphenol crude extract is redissolved in deionized water, it is efficient using column chromatography-preparative
Liquid chromatography tandem separating and purifying technology is isolated and purified.
The condition of ultrasonic wave added of the present invention extraction is：Using water as Extraction solvent, feed liquid weight ratio is 1：10-1：
50,30-70 DEG C of ultrasonic temperature, ultrasonic time 30-90 min, ultrasonic power 100-400w.
Filter membrane of the present invention is the milipore filter that molecular cut off is 5 kD, and drying means is true used by filtrate
Vacuum freecing-dry or centrifugal spray drying.
The filler of column chromatography of the present invention is the type macroporous absorbent resins of NKA- II, the BV/h of loading speed 3, during absorption
Between 2 h, eluting solvent is 20%-60% ethanol solutions, the BV/h of elution speed 3；Through adsorbing, eluting collection eluent, it is concentrated under reduced pressure
To crude product is done to obtain, through 0.45 μm of membrane filtration after being redissolved with 50% methanol.
Liquid phase chromatogram condition of the present invention is：The anti-phase C18 chromatographic columns of preparative, chromatographic column specification：The mm of internal diameter 20,
5 μm, Detection wavelength 280nm, the mL of sample size 1, flow velocity 5-10 mL/min of 250 mm, C18 silica filler particle diameter of length, column temperature
30 DEG C, using A:Methanol-B:0.1% formic acid carries out linear gradient elution, and elution program is
（1）0~30 min：A: 5%+B: 95%→A: 60%+B: 40%；
（2）30~35 min：A: 60%+B: 40%→A: 5%+B: 95%；
（3）35~45 min：A: 5%+B: 95%.
High-purity grifola frondosus weight polyphenol fraction of the present invention is purified by preparative high performance liquid chromatography, according to ultraviolet
Detector spectrogram collects grifola frondosus weight polyphenol fraction and carries out crystallization operation acquisition respectively, and 17 kinds of high-purity weight polyphenol fractions reflect through mass spectrum
It is set to：Ellagic acid（ellagic acid）, Quercetin（quercetin）, 4-HBA（4-hydroxybenzoic
acid）, gallic acid -3-O- (6`-O- nutgall acyls) glucoside（gallic acid-4-O-(6`-O-galloyl)
glucoside）, caftaric acid（caffeoyl tartaric acid）, caffeic acid（caffeic acid）, epicatechin
Gallate（(-)-epicatechin 3-O-gallate）, 5,6,7,3', 4'- pentamethoxyl flavones（sinenstein）、
To coumaroyl guinic acid（p-coumaroyl-quinic acid）, m-digallic acid（m-digallic acid）, dehydrogenation two
Forulic acid（dehydrodiferulic acid）, Quercetin -3-O- β-D- galactopyranosides（quercetin-3-O-β-D-
galactopyranoside）, 3,4,5- trimethoxybenzoic acids（methyl gallate）, 5- nonadecyl resorcinols（5-
nonadecylresorcin）, forulic acid（ferulic acid）, to coumaric acyl malic acid（p-coumaroyl malic
acid）And to coumaric acyl tartaric acid（p-coumaroyl tartaric acid）, purity is all higher than 95%.
Using DPPH radical scavenging activities, Hydroxyl radical-scavenging ability and reducing power as foundation, it was demonstrated that the ash tree of purifying
Flower polyphenol component has stronger antioxidation activity in vitro, can prepare the food with antioxidation activity, food ingredient or guarantor
Strong product.
The grifola frondosus weight polyphenol fraction for proving purifying through experiment in vitro has stronger suppression to the activity of α-glucuroide
Effect, wherein Quercetin, gallic acid -3-O- (6`-O- nutgall acyls) glucosides and to Quercetin -3-O- β-D- pyrans half
The IC of lactoside50Respectively 3.94 μ g/mL, 3.24 μ g/mL and 7.23 μ g/mL, it is remote to the inhibition of alpha-glucosidase
It is much better than acarbose（IC50=1.04 mg/mL）.
The advantage of the invention is that：The present invention is mutually tied using macroporous adsorbent resin column chromatography with preparative high performance liquid chromatography
The technology of conjunction, a kind of preparation method of high-purity grifola frondosus polyphenol compound monomer rapidly and efficiently is built, there is separating effect
Good, the time is short, prepares the advantages such as purity height.
Embodiment
Embodiment one
The preparation of grifola frondosus fine powder：Taking Grifola Frondosa sporophore, it is standby that pulverizer crushed 80 mesh sieves in 50 DEG C of drying.
More phenol extractions：A certain amount of grifola frondosus fine powder is taken, using water as Extraction solvent, solid-liquid ratio 1：30, ultrasonic temperature
Under conditions of 70 DEG C, ultrasonic time 60min, ultrasonic power 300w, extracted 2 times using ultrasonic assistant extractive technique, merging carries
Liquid is taken, filtered, concentration, dries preparation grifola frondosus Polyphenols crude extract.
Membrane filtration：Grifola frondosus Polyphenols crude extract will be prepared to redissolve, filled by the milipore filter that molecular cut off is 5 kD
Put and filtered, retain egg bletilla polysaccharide.Drying means is vacuum freeze drying used by filtrate, and routinely drying condition enters
Row drying.
Macroporous adsorbent resin column chromatography：Take the macroporous absorbent resin wet method dress posts of NKA- II pre-processed, after balance on
Sample, macroreticular resin loading mass concentration 5 mg/mL, the BV/h of loading speed 3, the h of adsorption time 2, with distilled water with 2 mL/min
Flow velocity is rinsed to neutrality, then with 40% ethanol solution（pH 6.0）Eluted with the BV/h of elution speed 3, collect eluent, subtract
Pressure concentration, lyophilized, the preparation thick component of grifola frondosus polyphenol compound, through 0.45 μm of membrane filtration after being redissolved with 50% methanol.
It is prepared by high-purity polyphenol compound monomer：The type macroporous adsorbent resin column chromatography isolates of NKA- II further across
Preparative high performance liquid chromatography is purified, and grifola frondosus weight polyphenol fraction, concentrated drying, system are collected respectively according to UV-detector spectrogram
Standby high-purity grifola frondosus polyphenol compound monomer.Preparative liquid chromatography condition is defined as：The anti-phase C18 chromatographic columns of preparative,
Chromatographic column specification：The mm of internal diameter 20,5 μm, Detection wavelength 280nm, the mL of sample size 1 of 250 mm, C18 silica filler particle diameter of length,
Flow velocity 5-10 mL/min, 30 DEG C of column temperature, using A:Methanol-B:0.1% formic acid carries out linear gradient elution, and elution program is：
（1）0~30 min：A: 5%+B: 95%→A: 60%+B: 40%；
（2）30~35 min：A: 60%+B: 40%→A: 5%+B: 95%；
（3）35~45 min：A: 5%+B: 95%.
The HPLC-MS analysis of high-purity grifola frondosus polyphenol compound monomer：Liquid phase chromatogram condition：
The anti-phase C18 column chromatographies post specification of preparative：5 μm mm of internal diameter 4.6, length 250 of mm, C18 silica filler particle diameter, detection
Wavelength 280nm, sample size 20 μ L, the mL/min of flow velocity 0.8,30 DEG C of column temperature, using A:Methanol-B:0.1% formic acid enters line
Property gradient elution, elution program are：
（1）0~30 min：A: 5%+B: 95%→A: 60%+B: 40%；
（2）30~35 min：A: 60%+B: 40%→A: 5%+B: 95%；
（3）35~45 min：A: 5%+B: 95%.
Mass Spectrometry Conditions are：Spraying（IS）Voltage is 3.0 kV, hole voltage 20V, 130 DEG C of the temperature of ESI ion guns, is dissociated
350 DEG C of temperature, mass spectral analysis is carried out in negative ion mode with reference to spectrum, mass spectral analysis scope 50 arrives 600m/z.
By corresponding mass spectrogram, the retention time and Information in Mass Spectra of Main Basiss chromatographic peak including molecular weight information etc.,
Pass through European Phenol-Explorer polyphenol database website http://phenol-explorer.eu/ and documentation ＆ info are carried out
The identification of phenolic compound, the results showed that the weight polyphenol fraction that macroporous absorbent resin is adsorbed in -40% ethanol eluate includes：Tan is spent
Acid（ellagic acid）, Quercetin（quercetin）, 4-HBA（4-hydroxybenzoic acid）, do not eat
Sub- acid -3-O- (6`-O- nutgall acyls) glucoside（gallic acid-4-O-(6`-O-galloyl)glucoside）, coffee
Coffee acyl tartaric acid（caffeoyl tartaric acid）, caffeic acid（caffeic acid）, L-Epicatechin gallate
（(-)-epicatechin 3-O-gallate）, 5,6,7,3', 4'- pentamethoxyl flavones（sinenstein）, to coumaric acyl Kui
Buddhist nun's acid（p-coumaroyl-quinic acid）, m-digallic acid（m-digallic acid）, the forulic acid of dehydrogenation two
（dehydrodiferulic acid）, Quercetin -3-O- β-D- galactopyranosides（quercetin-3-O-β-D-
galactopyranoside）, 3,4,5- trimethoxybenzoic acids（methyl gallate）, 5- nonadecyl resorcinols（5-
nonadecylresorcin）, forulic acid（ferulic acid）, to coumaric acyl malic acid（p-coumaroyl malic
acid）And to coumaric acyl tartaric acid（p-coumaroyl tartaric acid）.
The macroporous absorbent resin of table 1 adsorbs the Mass Spectrometric Identification result of weight polyphenol fraction in -40% ethanol eluate
The antioxidation in vitro experiment of the ash tree flower polyphenol of test example 1
Using DPPH radical scavenging activities, Hydroxyl radical-scavenging ability and reducing power as foundation, it was demonstrated that the ash tree of purifying
Flower polyphenol component has stronger antioxidation activity in vitro, can prepare the food with antioxidation activity, food ingredient or guarantor
Strong product.
（1）Remove the measure of DPPH free radical abilities（DPPH methods）
2.0mL various concentrations testing sample solutions are taken, add the mL of 0.2 mmol/L DPPH ethanol solutions 2.0, mixing
The min of avoid light place 30 after uniformly, determines its light absorption value at the nm of wavelength 517.Sample is replaced as sky using the % ethanol of 2.0 mL 95
White control；Using 2.0 mL samples and the % alcohol mixeding liquids of 2.0 mL 95 as sample controls, to eliminate the shadow of sample itself color
Ring.The calculation formula of DPPH clearance rates is：
The IC50 of 17 kinds of grifola frondosus polyphenolic substances is respectively less than 0.20 mg/mL after measured.This result can be seen that grifola frondosus
Polyphenol has certain scavenging action to DPPH free radicals.
（2）The measure of hydroxyl radical free radical ability
The mL of various concentrations testing sample solution 1 is taken, adds 9 mmol/L FeSO41 mL, 9 mmol/L salicylic acids-second
The mL of alcoholic solution 1, finally add 8.8 mmol/L H2O2 1 mL starts reaction, 30 min is reacted under 37 DEG C of water-baths, in 510 nm
The light absorption value of lower each strength solution of measure.Due to H2O2With Fe2+Mixing can produce OH, and salicylic acid can catch OH and produce coloured
Material, because sample itself has light absorption value, then with 9 mmol/L FeSO41 mL, 9 mmol/L salicylic acids-ethanol solution 1
ML each adds the control absorption value of the mL of sample solution 1 and the mL of distilled water 1 of various concentrations as sample（That is distilled water generation
For 8.8 mmol/L H2O2）.OH clearance rates are calculated as follows：
The IC50 of 17 kinds of grifola frondosus polyphenolic substances is less than 0.12 mg/mL after measured, and polyphenol is to H2O2/Fe2+ System passes through
OH caused by Fenton reactions has scavenging action.
（3）The measure of reducing power
0.2 mol/L pH 6.6 phosphate buffer 2.5mL and the testing sample of various concentrations are separately added into test tube
Solution 1mL, adds the potassium ferricyanide solutions of 2.5 mL 1%, well mixed after reacting 20 min in 50 DEG C of water-baths.It is fast after taking-up
Quickly cooling is but and the addition % trichloroacetic acid terminating reactions of 2. 5mL 10,3500 r/min centrifuge 10 min.The mL of supernatant 2.5 is taken,
2.5 mL distilled water and the % liquor ferri trichloridis of 0.5 mL 0.1 are added, 10 min are stood after mixing, are determined at 700 nm each
The light absorption value of solution.Light absorption value is bigger, and explanation reducing power is stronger.The calculation formula of reducing power is：Reducing power=A samples-A is empty
In vain.
After measured the reducing power of 17 kinds of grifola frondosus polyphenolic substances than etc. mass concentration（1 mg/mL）Ascorbic acid
By force.
The external alpha-glucosaccharase enzyme level experiment of the ash tree flower polyphenol of test example 2
Alpha-glucosidase activity determines：In 10 mM that 1 mL is prepared with 50 mM phosphate buffers (pH 7.4)
In pNPG substrate systems, add 5 μ L alpha-glucosaccharase enzyme solutions, it is rapid to mix, by Shimadzu Corporation UV-2600 is ultraviolet can
See that extinction value changes per minute carry out Indicator Reaction speed ν values at spectrophotometer detection 400nm, so as to characterize the big of enzyme activity
It is small.Measurement temperature is 37 DEG C.
By the high-purity grifola frondosus weight polyphenol fraction of preparation, the concentration soluble in water that is configured to is 50 mg/mL solution respectively, with 50
MM pH 6.5 PBS buffer solutions are diluted to concentration as 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 respectively
mg/mL、0.1 mg/mL、0.2 mg/mL、0.4 mg/mL、0.6 mg/mL、0.8 mg/mL、1.0 mg/mL、2.0 mg/mL、
4.0 mg/mL, 6.0 mg/mL, 8.0 mg/mL and 10.0 mg/mL.Take the μ L of solution 10 of each concentration and isometric α-grape
Glucosides enzyme liquid mixes, and enzyme activity system is made, enzyme activity is determined after 30 DEG C of insulation 2h.Enzyme activity determination method is same as above.α during measure-
Final concentration of 0.2 U/mL of glucuroide, substrate pNPG concentration are 10 mM.Each enzyme activity system is calculated according to measurement result
Enzyme activity.Enzyme activity × 100% of enzyme activity/alpha-glucosidase of enzyme activity=each enzyme activity system.
The grifola frondosus weight polyphenol fraction for proving purifying through experiment in vitro has stronger suppression to the activity of α-glucuroide
Effect, wherein Quercetin, gallic acid -3-O- (6`-O- nutgall acyls) glucosides and to Quercetin -3-O- β-D- pyrans half
The IC of lactoside50Respectively 3.94 μ g/mL, 3.24 μ g/mL and 7.23 μ g/mL, it is remote to the inhibition of alpha-glucosidase
It is much better than acarbose（IC50=1.04 mg/mL）.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
Claims (2)
- A kind of 1. preparation method of high-purity grifola frondosus weight polyphenol fraction, it is characterised in that：Comprise the following steps：（1）Pretreatment：Grifola Frondosa sporophore dried product is ground into powder, it is standby to cross 40~80 mesh sieves；（2）Extraction：Take grifola frondosus powder to add in distilled water, extracted 2-4 times using ultrasonic assistant extractive technique, merge extraction Liquid, residue is removed through plate-frame filtering；（3）Membrane filtration：Filtered by film filter, retain egg bletilla polysaccharide, the concentrating filter liquor of acquisition, obtained after drying Obtain ash tree flower polyphenol crude extract；（4）Isolate and purify：Ash tree flower polyphenol crude extract is redissolved in deionized water, using column chromatography-preparative efficient liquid phase Chromatographic tandem separating and purifying technology is isolated and purified；The condition of described ultrasonic wave added extraction is：Using water as Extraction solvent, feed liquid weight ratio is 1：10-1：50, ultrasonic temperature 30-70 DEG C, ultrasonic time 30-90 min, ultrasonic power 100-400w；Described filter membrane is the milipore filter that molecular cut off is 5 kD, and drying means is vacuum freeze drying used by filtrate Or centrifugal spray drying；The filler of described column chromatography is：The type macroporous absorbent resins of NKA- II, the BV/h of loading speed 3, the h of adsorption time 2, elution Solvent is 20%-60% ethanol solutions, the BV/h of elution speed 3；Through adsorbing, eluting collection eluent, it is concentrated under reduced pressure into slightly dry Product, through 0.45 μm of membrane filtration after being redissolved with 50% methanol；The anti-phase C18 chromatographic columns of preparative, chromatographic column specification：5 μm mm of internal diameter 20, length 250 of mm, C18 silica filler particle diameter, Detection wavelength 280nm, the mL of sample size 1, flow velocity 5-10 mL/min, 30 DEG C of column temperature, using A:Methanol-B:0.1% formic acid is carried out Linear gradient elution, elution program are：（1）0~30 min：A: 5%+B: 95%→A: 60%+B: 40%；（2）30~35 min：A: 60%+B: 40%→A: 5%+B: 95%；（3）35~45 min：A: 5%+B: 95%.
- A kind of 2. preparation method of high-purity grifola frondosus weight polyphenol fraction according to claim 1, it is characterised in that：NKA- II type macroporous adsorbent resin column chromatography isolate purifies further across preparative high performance liquid chromatography, is composed according to UV-detector Figure collects grifola frondosus weight polyphenol fraction and carries out crystallization operation respectively, obtains 17 kinds of high-purity weight polyphenol fractions：Ellagic acid, Quercetin, 4- Hydroxybenzoic acid, gallic acid -3-O- (6`-O- nutgall acyls) glucoside, caftaric acid, caffeic acid, epicatechin Gallate, 5,6,7,3', 4'- pentamethoxyls flavones, to coumaroyl guinic acid, m-digallic acid, the forulic acid of dehydrogenation two, It is Quercetin -3-O- β-D- galactopyranosides, 3,4,5- trimethoxybenzoic acids, 5- nonadecyls resorcinol, forulic acid, right Coumaric acyl malic acid and 95% is all higher than to coumaric acyl tartaric acid, purity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610082919.9A CN105732250B (en) | 2016-02-06 | 2016-02-06 | A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610082919.9A CN105732250B (en) | 2016-02-06 | 2016-02-06 | A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105732250A CN105732250A (en) | 2016-07-06 |
| CN105732250B true CN105732250B (en) | 2018-03-09 |
Family
ID=56241936
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610082919.9A Active CN105732250B (en) | 2016-02-06 | 2016-02-06 | A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105732250B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106946833A (en) * | 2017-05-02 | 2017-07-14 | 湖南华诚生物资源股份有限公司 | A kind of method that high-purity sinensetin is extracted from Mao Xu Cao |
| CN110437289B (en) * | 2019-09-02 | 2020-07-07 | 广东省农业科学院茶叶研究所 | Preparation method of tetragalloyl glucose |
| CN112225827A (en) * | 2020-10-15 | 2021-01-15 | 广东粤微生物科技有限公司 | Extraction method of active polysaccharide of grifola frondosa, extracted active polysaccharide and application |
| CN113854558B (en) * | 2021-09-13 | 2024-01-09 | 青岛农业大学 | Polyphenol compound with antioxidant and amylase inhibiting functions and preparation method and application thereof |
| CN115836730A (en) * | 2022-12-20 | 2023-03-24 | 天津科技大学 | A method for enriching polyphenols with the function of regulating intestinal flora from waste liquid extracted from Grifola frondosa polysaccharides |
| CN116139705B (en) * | 2023-02-27 | 2024-12-10 | 吉林大学 | A method for preparing a MOFs mixed matrix membrane and its application in polyphenol purification |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101407833A (en) * | 2008-11-10 | 2009-04-15 | 浙江工业大学 | Preparation of edible fungus beta-dextran |
| CN101736053A (en) * | 2010-02-01 | 2010-06-16 | 南京泽朗医药科技有限公司 | Technique for extracting Grifola frondosa water-soluble polysaccharide |
-
2016
- 2016-02-06 CN CN201610082919.9A patent/CN105732250B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101407833A (en) * | 2008-11-10 | 2009-04-15 | 浙江工业大学 | Preparation of edible fungus beta-dextran |
| CN101736053A (en) * | 2010-02-01 | 2010-06-16 | 南京泽朗医药科技有限公司 | Technique for extracting Grifola frondosa water-soluble polysaccharide |
Non-Patent Citations (2)
| Title |
|---|
| 灰树花多酚物质抑菌作用的研究;柴丽等;《中国酿造》;20121231;第31卷(第3期);91-93 * |
| 灰树花多酚的提取和活性研究;陈向东等;《食品与生物技术学报》;20050730;第24卷(第4期);26-30 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105732250A (en) | 2016-07-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105732250B (en) | A kind of preparation method of high-purity grifola frondosus weight polyphenol fraction | |
| CN102229632B (en) | Preparation method of cyanidin-3-O-glucoside | |
| CN101863871B (en) | Total glycosides of Rhodiola rosea, medical application and preparation method thereof | |
| CN101220062A (en) | Method for preparing stevioside and rebaudiodside A simultaneously | |
| CN105566414B (en) | The method that four kinds of flavone glycosides are isolated and purified from waxberry flesh | |
| CN109293712A (en) | The industrialized utilization method and its steviol glycoside and chlorogenic acid of a kind of STEVIA REBAUDIANA | |
| CN113181254A (en) | Application of apricot flower bee pollen in extraction of phenol amine compound and method for extracting phenol amine compound from apricot flower bee pollen | |
| CN102836202A (en) | A method for comprehensive development and utilization of aboveground parts of licorice | |
| CN102078345A (en) | Preparation method of actinidia arguta flavonoids | |
| CN101229335A (en) | Method for Enzymatically Preparing Smilax Smilax Total Saponins Extract | |
| CN102617669B (en) | Method for separating and purifying mangiferin from mango pericarp | |
| CN102229638B (en) | Method for extracting oleanolic acid from chaenomeles fruit and preparing oleanolic acid standard | |
| CN102372750A (en) | Method for simultaneously preparing albiflorin and paeoniflorin | |
| CN112159451A (en) | Gynostemma pentaphylla saponin extract and preparation method thereof | |
| CN103664855A (en) | Preparation method of high-purity oligomer lotus seedpod procyanidine | |
| CN104761596A (en) | Method for preparing anthocyanin standard substance | |
| CN107805270A (en) | A kind of ginsenoside Rh2Extracting method | |
| CN105434539A (en) | Composition of lotus flavones | |
| CN102617674B (en) | Preparation method of scopolin monomer in anisodus tanguticus root | |
| CN101823964A (en) | Technology for preparing chlorogenic acid in viburnum sargentii koehne leaves | |
| CN114426478B (en) | A high-content gallic acid and its preparation method | |
| CN106187975B (en) | A kind of preparation for improving rice bran aldehydes matter bioactivity and purification process | |
| CN112321664B (en) | Method for extracting, separating and purifying Inonotinol | |
| CN101456803A (en) | Method for purifying hypericin | |
| CN105061455B (en) | It is a kind of can massively separating high purity gambogicacid and Neo-garcinolic acid simultaneously method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201010 Address after: 354400 No.5, No.1 Road, Yangping Industrial Park, Shancheng Town, Taining County, Sanming City, Fujian Province (business place: kiln side pit, Shengyi village, Shancheng Town, Taining County, Sanming City) Patentee after: FUJIAN JUNZHITANG BIOTECHNOLOGY Co.,Ltd. Address before: Cangshan District of Fuzhou City, Fujian Province, on the 350002 Road No. 15 Patentee before: FUJIAN AGRICULTURE AND FORESTRY University |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A preparation method of high purity Grifola frondosa polyphenol component Effective date of registration: 20220210 Granted publication date: 20180309 Pledgee: Agricultural Bank of China Limited Taining County sub branch Pledgor: FUJIAN JUNZHITANG BIOTECHNOLOGY CO.,LTD. Registration number: Y2022350000018 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Granted publication date: 20180309 Pledgee: Agricultural Bank of China Limited Taining County sub branch Pledgor: FUJIAN JUNZHITANG BIOTECHNOLOGY CO.,LTD. Registration number: Y2022350000018 |