CN117801132B - Polygonatum cyrtonema leaf polysaccharide and preparation method and application thereof - Google Patents
Polygonatum cyrtonema leaf polysaccharide and preparation method and application thereof Download PDFInfo
- Publication number
- CN117801132B CN117801132B CN202410002991.0A CN202410002991A CN117801132B CN 117801132 B CN117801132 B CN 117801132B CN 202410002991 A CN202410002991 A CN 202410002991A CN 117801132 B CN117801132 B CN 117801132B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- polygonatum cyrtonema
- leaves
- leaf
- polygonatum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 97
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 97
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 97
- 241001468611 Polygonatum cyrtonema Species 0.000 title claims abstract description 95
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 102100025101 GATA-type zinc finger protein 1 Human genes 0.000 claims abstract description 28
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 claims abstract description 28
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims abstract description 28
- 230000000694 effects Effects 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 24
- 230000028327 secretion Effects 0.000 claims abstract description 14
- 230000001737 promoting effect Effects 0.000 claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 61
- 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 description 36
- 239000008103 glucose Substances 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 239000006228 supernatant Substances 0.000 claims description 17
- 239000003480 eluent Substances 0.000 claims description 16
- 108090000526 Papain Proteins 0.000 claims description 14
- 239000004365 Protease Substances 0.000 claims description 14
- 229940055729 papain Drugs 0.000 claims description 14
- 235000019834 papain Nutrition 0.000 claims description 14
- 239000002244 precipitate Substances 0.000 claims description 14
- 102000004139 alpha-Amylases Human genes 0.000 claims description 12
- 108090000637 alpha-Amylases Proteins 0.000 claims description 12
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 12
- 239000012498 ultrapure water Substances 0.000 claims description 12
- 229940024171 alpha-amylase Drugs 0.000 claims description 11
- 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 description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 238000005349 anion exchange Methods 0.000 claims description 9
- 238000003809 water extraction Methods 0.000 claims description 9
- 229920005654 Sephadex Polymers 0.000 claims description 8
- 239000012507 Sephadex™ Substances 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 238000005238 degreasing Methods 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- 230000003544 deproteinization Effects 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 239000006286 aqueous extract Substances 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 claims description 5
- 239000003472 antidiabetic agent Substances 0.000 claims description 3
- 238000012869 ethanol precipitation Methods 0.000 claims 1
- 229940126904 hypoglycaemic agent Drugs 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 50
- 239000008280 blood Substances 0.000 description 15
- 210000004369 blood Anatomy 0.000 description 15
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 241000700159 Rattus Species 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 8
- 230000001965 increasing effect Effects 0.000 description 7
- 150000002772 monosaccharides Chemical class 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 230000037396 body weight Effects 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 229940093181 glucose injection Drugs 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- UFLHIIWVXFIJGU-ARJAWSKDSA-N (Z)-hex-3-en-1-ol Chemical compound CC\C=C/CCO UFLHIIWVXFIJGU-ARJAWSKDSA-N 0.000 description 4
- 241000037831 Polygonatum sibiricum Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 210000003240 portal vein Anatomy 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 230000002440 hepatic effect Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- -1 sugar alcohol acetate derivative Chemical class 0.000 description 3
- 238000009777 vacuum freeze-drying Methods 0.000 description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- 238000004566 IR spectroscopy Methods 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 238000003287 bathing Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000000569 multi-angle light scattering Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 150000003214 pyranose derivatives Chemical class 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 108010067722 Dipeptidyl Peptidase 4 Proteins 0.000 description 1
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical group C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 241000756042 Polygonatum Species 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 125000003668 acetyloxy group Chemical group [H]C([H])([H])C(=O)O[*] 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 238000004192 high performance gel permeation chromatography Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002356 laser light scattering Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000005173 quadrupole mass spectroscopy Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 150000005856 steroid saponins Chemical class 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Abstract
The invention relates to the technical field of extraction of polygonatum cyrtonema leaf polysaccharide, in particular to polygonatum cyrtonema leaf polysaccharide, a preparation method and application thereof. The preparation method provided by the invention has the advantages of simple and stable process, and the prepared polysaccharide of the polygonatum cyrtonema leaves has high purity, is a uniform polysaccharide, has the function of promoting GLP-1 secretion activity, and improves the utilization rate of the polygonatum cyrtonema leaves.
Description
Technical Field
The invention relates to the technical field of extraction of polygonatum cyrtonema leaf polysaccharide, in particular to polygonatum cyrtonema leaf polysaccharide, a preparation method and application thereof.
Background
Polygonatum cyrtonema Fabricius as a medicine and food homologous traditional Chinese medicine has the effects of tonifying spleen and replenishing qi, nourishing kidney and moistening lung and the like. Polygonatum cyrtonema Fabricius contains various bioactive components such as polysaccharide, steroid saponin, flavone, etc. The polysaccharide is used as one of main medicinal components in Polygonatum cyrtonema Fabricius, and has various pharmacological effects of reducing blood lipid and blood sugar, enhancing immunity, resisting oxidation, resisting tumor, etc. In recent years, wild resources of Polygonatum sibiricum Red are excessively excavated, resources of each main production area are generally reduced, and the demand of Polygonatum sibiricum red is increased.
At present, most researches are developed around the root of Polygonatum sibiricum Red, so that few reports on separation, purification and activity of Polygonatum sibiricum Red leaves are related, and the separation method is complex and has more impurities.
Disclosure of Invention
In order to solve the problems, the invention provides a polygonatum cyrtonema leaf polysaccharide, a preparation method and application thereof. The method provided by the invention has simple and stable process, and the prepared polygonatum cyrtonema leaf polysaccharide has high purity, is a uniform polysaccharide and has the function of promoting GLP-1 secretion activity.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a preparation method of polygonatum cyrtonema leaf polysaccharide, which comprises the following steps:
sequentially degreasing and extracting Polygonatum cyrtonema leaves with ethanol to obtain an aqueous extract;
Mixing the water extract with alpha-amylase, performing first enzymolysis, and performing alcohol precipitation to obtain an alcohol precipitate;
Redissolving the alcohol precipitate to obtain a redissolution; mixing the compound solution with papain, and performing second enzymolysis to obtain supernatant;
Deproteinizing the supernatant by using a sevag method, and dialyzing to retain components with molecular weight greater than 3500Da to obtain Polygonatum cyrtonema leaf crude polysaccharide;
performing primary separation on the polygonatum cyrtonema leaf crude polysaccharide through an anion exchange column, eluting with ultrapure water to obtain a first eluent;
Purifying the first eluent by using a sephadex column, eluting by using ultrapure water to obtain eluent containing polygonatum cyrtonema leaf polysaccharide.
Preferably, the volume concentration of the ethanol solution used in the ethanol degreasing is 60-90%, and the mass-volume ratio of the polygonatum cyrtonema leaves to the ethanol solution is 1g: 5-15 mL.
Preferably, the temperature of the water extraction is 60-100 ℃, the times are 1-4, and the time of each water extraction is 1-3 h.
Preferably, the mass-volume ratio of the polygonatum cyrtonema leaves to the alpha-amylase is 0.5-5 kg:1mL, wherein the enzyme activity of the alpha-amylase is 46U/mL; the temperature of the first enzymolysis is 30-70 ℃ and the time is 0.5-10 h.
Preferably, the volume concentration of the ethanol solution used in the alcohol precipitation is 50-90%.
Preferably, the volume-to-mass ratio of the compound solution to the papain is 1mL: 1-10 mg, wherein the enzyme activity of the papain is more than or equal to 800U/mg; the temperature of the second enzymolysis is 30-70 ℃ and the time is 0.5-10 h.
Preferably, the number of deproteinization is 5 to 10.
The invention provides the polysaccharide of the polygonatum cyrtonema leaves prepared by the preparation method in the technical scheme, and the molecular weight of the polysaccharide of the polygonatum cyrtonema leaves is 3.5 multiplied by 10 3～5×104 Da, and the polysaccharide comprises mannose and glucose; the molar ratio of mannose to glucose is 97.55:2.02.
The invention provides application of the polygonatum cyrtonema leaf polysaccharide in preparation of a medicament for promoting GLP-1 secretion.
The invention provides application of the polygonatum cyrtonema leaf polysaccharide in preparation of hypoglycemic drugs.
The beneficial effects are that:
Degreasing polygonatum cyrtonema leaves by using an ethanol solution, then extracting water to obtain an aqueous extract containing polygonatum cyrtonema leaves polysaccharide, removing starch in the aqueous extract by using alpha-amylase, and then performing alcohol precipitation to obtain an alcohol precipitate containing protein and polygonatum cyrtonema leaves crude polysaccharide; the papain is combined with the sevag method to remove the protein in the alcohol sediment, so that the frequency of removing the protein by the sevag method can be reduced, and the yield of polysaccharide is improved; finally, eluting and separating by an anion exchange column and purifying by a sephadex column to obtain the polysaccharide of the polygonatum cyrtonema She Junyi with higher purity, wherein the polysaccharide structure is analyzed for the first time, and three glycosidic bonds of t-Man (p), 4-Man (p) and 4-Glc (p) are in a molar ratio of 7.663:89.958: 2.379. The preparation method provided by the invention has the advantages of simple and stable process, and the prepared polysaccharide of the polygonatum cyrtonema leaves has high purity, is a uniform polysaccharide, has the function of promoting GLP-1 secretion activity, and improves the utilization rate of the polygonatum cyrtonema leaves.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a DEAE elution profile of Polygonatum cyrtonema leaf polysaccharide;
FIG. 2 is a G75 elution profile of Polygonatum cyrtonema leaf polysaccharide;
FIG. 3 is an ultraviolet spectrogram of rhizoma Polygonati She Junyi polysaccharide;
FIG. 4 is an infrared spectrum of polysaccharide She Junyi of Polygonatum cyrtonema;
FIG. 5 is an absolute molecular weight distribution diagram of polysaccharide of Polygonatum cyrtonema She Junyi;
FIG. 6 is a graph showing the spatial conformation analysis of polysaccharide of Polygonatum cyrtonema She Junyi;
FIG. 7 is a microstructure analysis chart of polysaccharide She Junyi from Polygonatum cyrtonema;
FIG. 8 is a monosaccharide composition map and standard map of Polygonatum cyrtonema rhizome She Junyi polysaccharide;
FIG. 9 is a general ion flow diagram of a methylated sugar alcohol acetate derivative of Polygonatum cyrtonema She Junyi polysaccharide;
FIGS. 10-12 are secondary proton chromatograms of Polygonatum cyrtonema She Junyi polysaccharide;
FIGS. 13 to 15 are results of the effect of single administration of Polygonatum cyrtonema She Junyi polysaccharide solution on glucose tolerance in rats and GLP-1 content in hepatic portal vein;
FIG. 16 is a graph showing the effect of polysaccharide solution of Polygonatum cyrtonema-rhizoma She Junyi on NCI-H716 cell viability after administration;
FIG. 17 is a graph showing the results of an in vitro GLP-1 secretion promoting activity of polysaccharide She Junyi of Polygonatum cyrtonema;
wherein p <0.05, p <0.01, p <0.001, p <0.0001, compared to the blank.
Detailed Description
The invention provides a preparation method of polygonatum cyrtonema leaf polysaccharide, which comprises the following steps:
sequentially degreasing and extracting Polygonatum cyrtonema leaves with ethanol to obtain an aqueous extract;
Mixing the water extract with alpha-amylase, performing first enzymolysis, and performing alcohol precipitation to obtain an alcohol precipitate;
Redissolving the alcohol precipitate to obtain a redissolution; mixing the compound solution with papain, and performing second enzymolysis to obtain supernatant;
Deproteinizing the supernatant by using a sevag method, and dialyzing to retain components with molecular weight greater than 3500Da to obtain Polygonatum cyrtonema leaf crude polysaccharide;
performing primary separation on the polygonatum cyrtonema leaf crude polysaccharide through an anion exchange column, eluting with ultrapure water to obtain a first eluent;
Purifying the first eluent by using a sephadex column, eluting by using ultrapure water to obtain eluent containing polygonatum cyrtonema leaf polysaccharide.
The invention preferably pulverizes the Polygonatum cyrtonema leaf to obtain Polygonatum cyrtonema leaf powder.
After the polygonatum cyrtonema leaf powder is obtained, the polygonatum cyrtonema leaf powder is degreased by ethanol solution, and the degreased polygonatum cyrtonema leaf powder is obtained. In the present invention, the volume concentration of the ethanol solution is preferably 60% to 90%, more preferably 70% to 80% or 85%, still more preferably 85%; the mass volume ratio of the polygonatum cyrtonema leaves to the ethanol solution is preferably 1g:5 to 15mL, more preferably 1g:7 to 13mL, more preferably 1g:10mL; the degreasing time is preferably 24 hours.
After the defatted Polygonatum cyrtonema leaf powder is obtained, the defatted Polygonatum cyrtonema leaf powder is subjected to water extraction to obtain water extract. In the present invention, the temperature of the water extraction is preferably 60 to 100 ℃, more preferably 70 to 80 ℃, and even more preferably 80 ℃; the number of water extractions is preferably 1 to 4, more preferably 2; the time of each water extraction is preferably 1 to 3 hours, more preferably 2 hours; the ratio of the feed liquid in each water extraction is preferably 1g:15 to 30mL, more preferably 1g:20 to 30mL, more preferably 1g:30mL.
After the water extract is obtained, the water extract is preferably concentrated to obtain a concentrated solution. In the present invention, the concentration of the concentrated solution is preferably 0.2 to 2g/mL.
After the concentrated solution is obtained, the concentrated solution is mixed with alpha-amylase, and the first enzymolysis and the alcohol precipitation are carried out to obtain an alcohol precipitate. In the invention, the mass-volume ratio of the polygonatum cyrtonema leaves to the alpha-amylase is preferably 0.5-5 kg:1mL, preferably 46U/mL of the alpha-amylase enzyme activity; the temperature of the first enzymolysis is preferably 30-70 ℃, more preferably 50-65 ℃, and even more preferably 65 ℃; the time of the first enzymolysis is preferably 0.5 to 10 hours, more preferably 1 to 5 hours, and even more preferably 1 hour; the volume concentration of the ethanol solution used in the alcohol precipitation is preferably 50% to 90%, more preferably 60% to 85%, and even more preferably 80%.
After obtaining an alcohol precipitate, the invention redissolves the alcohol precipitate to obtain a redissolution. In the present invention, the complex solution consists of an alcohol precipitate and water; the mass ratio of the alcohol sediment to the water is preferably 1g:40 to 80mL, more preferably 50 to 70mL, still more preferably 1g:50mL.
After the compound solution is obtained, the compound solution and papain are mixed, and the second enzymolysis is carried out to obtain the supernatant. In the invention, the volume-to-mass ratio of the compound solution to the papain is preferably 1mL:1 to 10mg, more preferably 1mL:1 to 5mg, more preferably 1mL:1mg; the enzyme activity of the papain is preferably more than or equal to 800U/mg; the temperature of the second enzymolysis is preferably 30-70 ℃, more preferably 40-60 ℃, and even more preferably 60 ℃; the time for the second enzymolysis is preferably 0.5 to 10 hours, more preferably 1 to 5 hours, and even more preferably 3 hours.
After the supernatant is obtained, the sevag method is utilized to deproteinize the supernatant, and then the supernatant is dialyzed to retain components with molecular weight larger than 3500Da, so that the polygonatum cyrtonema leaf crude polysaccharide is obtained. In the present invention, the number of times of deproteinization is preferably 5 to 10 times, more preferably 7 to 10 times; the deproteinizing agent includes chloroform and n-butanol; the volume ratio of the supernatant to chloroform to n-butanol is preferably 30:5:1.
The invention utilizes papain combined with sevag method to remove protein in the polygonatum cyrtonema leaf alcohol sediment, which not only can reduce the times of removing protein by sevag method, but also can improve the yield of polysaccharide.
After the crude polysaccharide of the polygonatum cyrtonema leaves is obtained, the crude polysaccharide of the polygonatum cyrtonema leaves is subjected to preliminary separation by an anion exchange column, and is eluted by ultrapure water to obtain a first eluent. In the present invention, the anion exchange column preferably comprises a DEAE-52 anion exchange column.
After the first eluent is obtained, the first eluent is purified by a sephadex column and eluted by ultrapure water to obtain eluent containing polygonatum cyrtonema leaf polysaccharide. In the present invention, the Sephadex column preferably comprises a Sephadex G-75 chromatographic column.
The invention adopts a separation and purification method combining anion exchange chromatography with gel chromatography to finally obtain the polygonatum cyrtonema She Junyi polysaccharide with the function of improving GLP-1 secretion activity. In vitro GLP-1-promoting activity indicates that PCPl prepared has GLP-1-promoting secretion activity. The preparation method provided by the invention has stable process and can obtain the uniform polysaccharide for promoting GLP-1 activity.
The invention also provides the polysaccharide of the polygonatum cyrtonema leaves prepared by the preparation method of the technical proposal, and the molecular weight of the polysaccharide of the polygonatum cyrtonema leaves is 3.5 multiplied by 10 3～5×104 Da, comprising mannose and glucose; the molar ratio of mannose to glucose is 97.55:2.02.
Based on the advantages, the invention also provides application of the polygonatum cyrtonema leaf polysaccharide in preparation of a medicament for promoting GLP-1 secretion.
Based on the advantages, the invention also provides the application of the polygonatum cyrtonema leaf polysaccharide in the preparation of the hypoglycemic drug.
For further explanation of the present invention, the present invention provides a polysaccharide from Polygonatum cyrtonema leaves, and its preparation method and application, which are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A polysaccharide of Polygonatum cyrtonema leaves is prepared by the following steps:
1. Taking 150g of freeze-dried polygonatum cyrtonema leaves, crushing and mixing with an ethanol solution with the volume concentration of 85%; the mass volume ratio of the polygonatum cyrtonema leaves to the ethanol solution is 1g:10mL; extracting at room temperature for 24h for 1 time, and volatilizing at room temperature until no alcohol smell exists to obtain rhizoma Polygonati She Tuozhi powder;
2. 126.13g of rhizoma polygonati She Tuozhi powder is added with 30 times of water, heated and extracted twice, each time for 2 hours, and the extracting solutions are combined; recovering at 60deg.C under reduced pressure to 600mL, adding alpha-amylase (available from Shanghai Ala Latin Biotechnology Co., ltd., product No. A109181-100 g) 200 μl, water-bathing at 65deg.C, stirring for 1 hr, inactivating at 100deg.C for 10min, removing starch, adding ethanol to final concentration of 80%, precipitating with ethanol for 24 hr, centrifuging to obtain Polygonatum cyrtonema leaf alcohol precipitate;
3. Dissolving Polygonatum cyrtonema leaf alcohol precipitate with distilled water, adding papain (product number is BS190-25g from Beijing blue Jie Ke technology Co., ltd.) at a ratio of 1mg/mL, water-bathing at 60deg.C, stirring for 3h, and inactivating at 100deg.C with hot water for 15min; centrifuging at 10000 Xg for 15min to obtain supernatant;
4. Mixing and shaking the supernatant, chloroform and n-butanol for 30min, and centrifuging at a centrifugation speed of 10000 Xg for 15min, wherein the volume ratio of the supernatant, chloroform and n-butanol is 30:5:1, a step of; removing protein 10 times after centrifugation, concentrating under reduced pressure at 60 ℃ to remove residual organic solvent, dialyzing with dialysis bag with molecular weight cut-off of 3500Da for 48h, dialyzing with deionized water for 24h, vacuum freeze-drying concentrated solution, and concentrating to obtain polysaccharide 2.44g;
5. Dissolving 200mg of Polygonatum cyrtonema leaf crude polysaccharide with a small amount of distilled water, filtering, loading the filtrate on a balanced DEAE-52 anion exchange column, eluting with ultrapure water at an eluting speed of 2mL/min, collecting the eluent according to a 10 mL/tube, concentrating under reduced pressure, dialyzing with dialysis bag running water with a molecular weight cutoff of 3500Da for 48 hours, dialyzing with deionized water for 24 hours, and vacuum freeze-drying the concentrated solution to obtain a Polygonatum cyrtonema leaf water-washed component;
6. Dissolving 50mg of Polygonatum cyrtonema leaf water-washing component with a small amount of distilled water, filtering, loading the filtrate on a well-balanced Sephadex G-75 chromatographic column, eluting with ultrapure water at the eluting speed of 0.2mL/min, collecting the eluent according to 2 mL/tube, detecting the absorbance value of each tube by a phenol-sulfuric acid method, and drawing a G75 eluting curve. Combining the two tubes according to the highest absorption value and its vicinity of the elution curve (figure 2), collecting eluate containing polysaccharide, concentrating under reduced pressure, and vacuum freeze drying to obtain rhizoma Polygonati She Junyi polysaccharide (PCPl) with elution yield of 94.55 + -0.79%
Comparative example 1
A polysaccharide from leaves of polygonatum cyrtonema of example 1, which is similar to example 1, except that step 5 is:
Dissolving 200mg of Polygonatum cyrtonema leaf crude polysaccharide with a small amount of distilled water, filtering, loading the filtrate on a balanced DEAE-52 anion exchange column, eluting sequentially with ultrapure water and NaCl at an eluting speed of 2mL/min, collecting the eluent according to a 10 mL/tube, detecting the absorbance value of each tube by a phenol-sulfuric acid method, and drawing a DEAE eluting curve.
As can be seen from FIG. 1, the yield of polysaccharide eluted with ultra pure water was 93.28.+ -. 0.58%
Comparative example 2
A Polygonatum cyrtonema leaf polysaccharide similar to example 1 is distinguished in that step 3 is not performed, the Polygonatum cyrtonema leaf alcohol precipitate is dissolved with distilled water and then deproteinized in step 4 directly, and the number of deproteinization times is 17.
Test example 1
The polysaccharide yields of example 1 and comparative example 2 were measured, crude polysaccharide yield = crude polysaccharide mass/drug mass x 100%, and 3 parallel replicates were performed, the results of which are shown in table 1.
Table 1 comparison of two methods before and after deproteinization
| Group of | Comparative example 2 | Example 1 |
| Number of deproteinization times | 17 Times | 10 Times |
| Polysaccharide yield | 1.59±14.57％ | 2.44±5.10％ |
As shown in Table 1, compared with the method for removing protein by using sevag alone, the method provided by the invention has the advantages that the times of deproteinizing after papain is added are obviously reduced, and the yield of polysaccharide is increased by 0.85%.
Test example 2
The PCPl prepared in example 1 was subjected to structure and activity assays, as follows:
1.2PCPl determination of physicochemical Properties
1.2.1 Total carbohydrate, protein content determination
The content of total carbohydrate and protein is measured by phenol sulfuric acid method and Coomassie brilliant blue method respectively.
1.2.2 UV Spectrometry and IR Spectrometry
PCPl was formulated as a 0.1mg/mL solution and scanned over a wavelength range of 200 to 800 nm.
PCPl was dried thoroughly, 2mg was weighed and scanned with an infrared spectrometer over the 4000-400 cm -1 region.
1.2.3 Absolute molecular weight determination
Molecular weight distribution of PCPl was determined by gel permeation chromatography (HPGPC), using a gel chromatography-differential-multi-angle laser light scattering system, a liquid phase system of U3000 (Thermo, USA), an differential detector of Optilab T-rEX (Wyatttechnology, CA, USA), and a laser light scattering detector of DAWN HELEOS II (Wyatt technology, CA, USA).
1.2.4 Congo Red experiments
PCPl (2.5 mg/mL) was formulated and thoroughly mixed with Congo red solution (80. Mu. Mol/L) in a 1:1 (v/v) ratio, and the mixture was gradually adjusted to different NaOH concentrations (0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5 mol/L) by adding NaOH solution (4M). The maximum absorption wavelength of congo red polysaccharide solutions of different concentrations was recorded by uv scanning (200-600 nm).
1.2.5 Scanning electron microscope experiments
The dried PCPl powder (3 mg) was spread on a conductive gel, and then a conductive film was sprayed on the surface of the polysaccharide sample by a vacuum spraying apparatus, and the microscopic morphology of the five polysaccharides was observed by a scanning electron microscope.
1.3PCPl structural resolution
1.3.1 Monosaccharide composition
A clean chromatographic vial was taken, an appropriate amount of PCPl samples were weighed, 1mL of 2M TFA acid solution was added, and heated at 121℃for 2 hours. And (5) introducing nitrogen and drying. Adding 99.99% methanol for cleaning, drying, and repeating methanol cleaning for 2-3 times. Adding sterile water for dissolving, and transferring into chromatographic bottle for testing. The chromatographic system employs a Thermo ICS5000+ ion chromatographic system (ICS 5000+, thermo FISHER SCIENTIFIC, USA) and uses an electrochemical detector to analyze and detect monosaccharide components.
1.3.2 Analysis of glycosidic bond types
1Mg of a sample to be measured is accurately weighed, and 500 mu L of DMSO is added for dissolution. 1mg NaOH was added and incubated for 30min. 50. Mu.L of methyl iodide solution was added thereto and reacted for 1 hour. 1mL of water and 2mL of methylene chloride were added, vortexed, mixed well, centrifuged, and the aqueous phase was discarded. The water wash was repeated 3 times. The lower dichloromethane phase was taken up and evaporated to dryness. 100 mu L0.5MTFA was added and reacted at 70℃for 3 hours. Evaporating to dryness at 30 ℃. 50. Mu.L of 2M ammonia water and 50. Mu.L of 1MNaBD4 were added, and the mixture was stirred and reacted at room temperature for 2.5 hours. The reaction was terminated by adding 20. Mu.L of acetic acid, and then purged with nitrogen, 250. Mu.L of methanol was purged twice, and purged with nitrogen. 250 μl of acetic anhydride was added, and the mixture was vortexed and reacted at 100deg.C for 2.5h. 1mL of water was added and the mixture was allowed to stand for 10min. 500 μl of dichloromethane was added, vortexed, mixed well, centrifuged and the aqueous phase discarded. The water wash was repeated 3 times. And taking the dichloromethane phase of the lower layer, and detecting on a machine. The chromatographic system adopts an Agilent weather chromatographic system (Agilent 7890A; agilent technologies, USA, HP-5MS capillary column (30 m×0.25mm×0.25 μm, agilent J & W SCIENTIFIC, folsom, CA, USA), the carrier gas is high purity helium (purity not less than 99.999%), the flow rate is 1.0mL/min, the temperature of the sample inlet is 260 ℃ and the sample amount is 1 mu L, the split sample ratio is 10:1, the solvent delay is 2.2min, the temperature programming is 50 ℃ and keeps 1.0min,50 ℃/min and keeps the temperature to 130 ℃,3 ℃/min and keeps the temperature to 230 ℃, the mass spectrometry system adopts a quadrupole mass spectrometry detection system (Agilent 5977B; agilent technologies, USA) of American Aiglent company, and is provided with an electron bombardment ion source (EI) and a MassHunter workstation, the electron bombardment ion source (EI), the sample inlet temperature is 230 ℃, the electron energy is 70 ℃ and the scanning mass scanning mode is in a full scanning mode (600 m-600).
2. Effect of Single lavage PCPl solution on GLP-1 content in rats
2.1 Effect of Single oral PCPl solution on glucose tolerance in rats
Rats were fasted overnight (without water break), and the low dose (0.5 g/kg body weight), PCPl high dose (2 g/kg body weight) and blank control groups were each of the same volume of physiological saline (0.9% NaCl) in the stomach-filled test group PCPl, and after 15min of administration, glucose solution (2 g/kgbodyweight) was orally administered and blood was collected before administration (-15 min), before administration of glucose and after administration of glucose for 15min, 30min, 60min, 90min, 120min, and the change in blood glucose level was measured by a glucometer, and the total area under the curve (AUC) indicated the change in blood glucose level after glucose injection.
2.2 Changes in GLP-1 content in hepatic portal vein after single administration PCPl of solution
Rats were fasted overnight (6 rats per group), a low dose (0.5 g/kg body weight), a PCPl high dose (2 g/kg body weight) and a blank control group (0.9% NaCl) of PCPl of the intragastric experimental group, glucose solution (2 g/kg body weight) was intraperitoneally injected under pentobarbital sodium anesthesia, blood was injected for 0min before glucose injection and 15min, 30min, 60min, 90min, 120min after glucose injection, and a basal level group (i.e., the content of GLP-1 in hepatic portal vein of rats without any treatment) was set, and the GLP-1 content (-15 min) was measured. Blood was collected in a syringe containing EDTA (final concentration: 1 mg/mL), aprotinin (final concentration: 500 kIU/mL) and DPP-IV inhibitor (final concentration: 100. Mu.M), centrifuged at 160 g at 4℃for 15min, stored at-80℃and the GLP-1 content in the plasma was measured by the kit.
3. In vitro experiments
3.1 Cytotoxicity assays
NCI-H716 cells in the logarithmic growth phase were adjusted to a density of 1X 10 6/mL with complete medium. After adding 90. Mu.L of cell suspension to each well of a 96-well plate and culturing in an incubator for 48 hours, 10. Mu.L of polysaccharides of different concentrations (final concentrations 25, 50, 100, 200, 400. Mu.g/mL) were added to each well, and the blank group was complete medium. Mixing, continuously culturing in an incubator for 24 hours, adding 10 mu L of cck-8 solution into each hole, reacting in the incubator for 1-2 hours after mixing, measuring OD value at 450nm wavelength, and analyzing whether polysaccharide has cytotoxicity to NCI-H716 cells so as to facilitate subsequent experiments.
3.2PCPl Effect on NCI-H716 cells promoting GLP-1 secretion
NCI-H716 cell density was adjusted to 1X 10 6/mL. 0.5mL of the cell suspension was added to each well of the 48-well plate, and incubated in an incubator for 48 hours. After 48h, polysaccharide solutions with a final concentration of 200 mug/mL are respectively added, the incubation is carried out for 2h, the negative control is a complete culture solution, after the incubation is finished, centrifugation is carried out for 15min at 1000rpm and 4 ℃, PMSF (with a final concentration of 50 mug/mL) is added into the supernatant, and the supernatant is preserved at 80 ℃. GLP-1 content in the supernatant was measured by ELISA kit method.
4. Statistical method
GraphPad 8.0 software was used for mapping and one-way analysis of variance. Results are expressed as mean±sd; p values less than 0.05 are considered statistically significant.
5. Experimental results
5.2PCPl determination of physicochemical Properties
5.2.1 Total carbohydrate, protein content analysis
The physicochemical properties of PCPl are shown in Table 2: PCPl has carbohydrate content of above 90%, and low protein and uronic acid content.
Physical and chemical Property measurement of Table 2PCPl
5.2.2 Ultraviolet Spectrometry and Infrared Spectrometry analysis
As shown in fig. 3: at 260nm and 280nm, PCPl had no distinct absorption peaks, indicating that no nucleic acids and proteins were contained. Fig. 4 shows that: the peak value of 3399cm -1 is the stretching vibration of-OH, the peak value of 2909cm -1 is the stretching vibration of C-H, the peak value of 1720cm -1 is the stretching vibration of C=O on carboxylic acid (or acetoxy), the peak value of 1402cm -1 is the bending vibration of pyranose, the peak value of 1262cm -1 is the C-O vibration of O-acetyl, the peak value near 1066cm -1 indicates the existence of furan ring structure in polysaccharide, and the peak value of 500-900 cm -1 indicates the existence of pyranose ring skeleton in polysaccharide.
5.2.3 Absolute molecular weight analysis
LS represents the multi-angle laser light scattering signal, RI represents the differential signal, FIG. 5 shows the retention Time (Time, min) detected as the abscissa and molar mass Molar Mass (g/moL) as the ordinate, yielding a weight average Molecular Weight (MW) of PSPSl of 4508Da, number average molecular weight (Mn) of 4267Da, polydispersity Mw/Mn of 1.056.
5.2.4 Spatial conformational analysis
As shown in FIG. 6, PCPl solutions in 0-0.15moL NaOH solution showed a significant red shift in the maximum absorption wavelength, indicating the presence of a triple helix structure.
5.2.5 Microstructure analysis
The topographical features of PCPl were observed by electron microscope scanning, as shown in fig. 7. The microscopic morphology map PCPl under the magnification of about 3000 in fig. 7 shows that the surface of PCPl is mostly in an irregular long strip shape, a plurality of particles are accumulated in the middle, the long strip is partially adhered with the particles and a plurality of filiform substances are mixed nearby, and because repulsive force exists between polysaccharides, intermolecular attractive force is weak, and the sheet-shaped structure is broken. The microscopic morphology map PCPl of fig. 7, b being about 13000, shows that a large number of spherical structures are attached to the surface of PCPl molecules, and the surface of the particles is smooth and flat or is concave, which means that the polysaccharide reduces the protein content during the purification process and eliminates the interference of the impurities, so that PCPl molecules interact strongly with each other and are tightly combined.
5.2.6 Monosaccharide composition
The diagram of monosaccharide composition is shown in FIG. 8. As shown in table 3, PCPl consists essentially of mannose and glucose, in a percentage of 97.59%, 2.02%, in a molar ratio of 97.55:2.02.
Monosaccharide composition analysis of Table 3PCPl
| Monosaccharide composition | Percentage of | Molar ratio of |
| Glucose | 2.02％ | 2.02 |
| Mannose | 97.59％ | 97.55 |
5.2.7 Glycosidic bond type analysis
The total ion flow diagram of PCPl methylated sugar alcohol acetate derivative is shown in figure 9, and the secondary proton chromatograms are shown in figures 10-12. As can be seen from Table 4, PCPl consists essentially of three glycosidic linkages, t-Man (p), 4-Man (p), 4-Glc (p), relative molar ratio 7.663:86.958:2.379.
Table 4PCPl glycosidic bond type data analysis
5.3 Effect of Single lavage PCPl solution on GLP-1 content in rats
5.3.1 Effect of Single oral PCPl solution on glucose tolerance in rats
As shown in fig. 13, there was no significant difference between the blood glucose level before the administration of the control group and the administration group after the oral administration of PCPl solutions, but the blood glucose level was significantly increased after 15 minutes of the oral administration of glucose until the blood glucose level was gradually decreased after 30 minutes, and the administration group was able to significantly decrease the blood glucose level compared to the control group, and the administration group had a significant difference (p < 0.05) at 120 minutes and the high-dose group had a significant difference (p < 0.05) at 90 and 120 minutes.
AUC represents the area under the curve of blood glucose levels for the different groups over 0-120min, fig. 14 shows that the area under the curve of blood glucose levels is lower for the low dose group than for the control group (p < 0.01) and the area under the high dose curve is lower than for the low dose and control groups (p < 0.01). The results show that PCPl solution can reduce the blood glucose concentration of rats after glucose is orally taken, and improve glucose tolerance.
5.3.2 Changes in GLP-1 content in portal vein after Single administration PCPl of solution
As shown in fig. 15, after administration, the GLP-1 content of the low dose group was significantly higher than that of the control group (p < 0.05) and the GLP-1 content of the high dose group was higher than that of the low dose group and the control group (p < 0.05) at various time points after glucose injection before glucose injection. After intraperitoneal injection of glucose, the GLP-1 content of the administered group was continuously increased until the GLP-1 content began to decrease after 30min, and the result shows that PCPl might be to decrease the blood glucose level by increasing the GLP-1 content of rat plasma.
5.4 In vitro experiments
5.4.1PCPl cytotoxicity assays on NCI-H716
As shown in FIG. 16, PCPl was not cytotoxic to NCI-H716 cells in the concentration range of 25-400. Mu.g/mL, and the promotion of NCI-H716 cell proliferation increased with increasing polysaccharide concentration (p < 0.05).
5.4.2 Effect of PCPl on NCI-H716 cells promoting GLP-1 secretion
As shown in FIG. 17, both low-concentration PCPl and high-concentration PCPl solutions promote GLP-1 secretion from NCI-H716 cells (p < 0.0001), and the promotion is concentration-gradient dependent.
In conclusion, the polysaccharide of the polygonatum cyrtonema She Junyi with the GLP-1 secretion improving activity is finally obtained by adopting a separation and purification method combining anion exchange chromatography with gel chromatography. In vitro GLP-1-promoting activity indicates that PCPl prepared has GLP-1-promoting secretion activity. The preparation method provided by the invention has stable process and can obtain the uniform polysaccharide for promoting GLP-1 activity.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (4)
1. The preparation method of the polygonatum cyrtonema leaf polysaccharide is characterized by comprising the following steps of:
Sequentially degreasing and extracting Polygonatum cyrtonema leaves with ethanol to obtain an aqueous extract; the volume concentration of the ethanol solution used in the ethanol degreasing is 60% -90%, and the mass volume ratio of the polygonatum cyrtonema leaves to the ethanol solution is 1g: 5-15 mL; the temperature of the water extraction is 60-100 ℃, the times are 1-4, and the time of each water extraction is 1-3 hours;
Mixing the water extract with alpha-amylase, performing first enzymolysis, and performing alcohol precipitation to obtain an alcohol precipitate; the mass volume ratio of the polygonatum cyrtonema leaves to the alpha-amylase is 0.5-5 kg:1mL, wherein the enzyme activity of the alpha-amylase is 46U/mL; the temperature of the first enzymolysis is 30-70 ℃ and the time is 0.5-10 h; the volume concentration of the ethanol solution used in the ethanol precipitation is 50% -90%;
Redissolving the alcohol precipitate to obtain a redissolution; mixing the compound solution with papain, and performing second enzymolysis to obtain supernatant; the volume mass ratio of the compound solution to the papain is 1mL: 1-10 mg, wherein the enzyme activity of the papain is more than or equal to 800U/mg; the temperature of the second enzymolysis is 30-70 ℃ and the time is 0.5-10 h;
Deproteinizing the supernatant by using a sevag method, and dialyzing to retain components with molecular weight greater than 3500Da to obtain Polygonatum cyrtonema leaf crude polysaccharide; the deproteinization times are 5-10 times;
performing primary separation on the polygonatum cyrtonema leaf crude polysaccharide through an anion exchange column, eluting with ultrapure water to obtain a first eluent;
Purifying the first eluent by using a sephadex column, eluting by using ultrapure water to obtain eluent containing polygonatum cyrtonema leaf polysaccharide; the molecular weight of the polygonatum cyrtonema leaf polysaccharide is 3.5X10 3~5×104 Da, and the polygonatum cyrtonema leaf polysaccharide mainly comprises mannose and glucose; the molar ratio of mannose to glucose is 97.55:2.02; the Polygonatum cyrtonema leaf polysaccharide mainly comprises three glycosidic bonds, namely t-Man (p), 4-Man (p) and 4-Glc (p).
2. The polysaccharide of Polygonatum cyrtonema leaves prepared by the preparation method of claim 1, wherein the molecular weight of the polysaccharide of Polygonatum cyrtonema leaves is 3.5X10 3~5×104 Da, and the polysaccharide of Polygonatum cyrtonema leaves mainly consists of mannose and glucose; the molar ratio of mannose to glucose is 97.55:2.02; the Polygonatum cyrtonema leaf polysaccharide mainly comprises three glycosidic bonds, namely t-Man (p), 4-Man (p) and 4-Glc (p).
3. Use of the polysaccharide of polygonatum cyrtonema leaves of claim 2 for preparing a medicament for promoting GLP-1 secretion.
4. The use of the polysaccharide of Polygonatum cyrtonema leaves of claim 2 in the preparation of hypoglycemic agents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410002991.0A CN117801132B (en) | 2024-01-02 | Polygonatum cyrtonema leaf polysaccharide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410002991.0A CN117801132B (en) | 2024-01-02 | Polygonatum cyrtonema leaf polysaccharide and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117801132A CN117801132A (en) | 2024-04-02 |
| CN117801132B true CN117801132B (en) | 2024-07-16 |
Family
ID=
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102351961A (en) * | 2011-11-15 | 2012-02-15 | 重庆市秀山红星中药材开发有限公司 | Method for extracting rhizoma polygonati polysaccharide from stems and leaves of rhizoma polygonati |
| CN109393549A (en) * | 2018-10-10 | 2019-03-01 | 云南大学 | A kind of combined extraction method and its application of rhizoma polygonati volatile oil and Siberian solomonseal rhizome polysaccharide |
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102351961A (en) * | 2011-11-15 | 2012-02-15 | 重庆市秀山红星中药材开发有限公司 | Method for extracting rhizoma polygonati polysaccharide from stems and leaves of rhizoma polygonati |
| CN109393549A (en) * | 2018-10-10 | 2019-03-01 | 云南大学 | A kind of combined extraction method and its application of rhizoma polygonati volatile oil and Siberian solomonseal rhizome polysaccharide |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10155822B2 (en) | Process for preparation of a grifola frondosa polysaccharide F2 and its hypoglycemic activity | |
| CN112062872A (en) | Polygonatum sibiricum homogeneous polysaccharide and preparation method and application thereof | |
| WO2021143595A1 (en) | Low-molecular-weight tremella aurantialba glucuronic acid-xylomannan, and preparation method therefor and use thereof | |
| CN110128562B (en) | An antitumor fructus Psoraleae polysaccharide, its extraction and separation method, and its application in preparing antitumor drugs | |
| CN111892663B (en) | Hericium erinaceus polysaccharide and preparation method and application thereof | |
| CN112961258B (en) | Red ginseng homogeneous polysaccharide and extraction method and application thereof | |
| CN112062866B (en) | Hericium erinaceus selenium-rich polysaccharide and preparation method and application thereof | |
| CN117801132B (en) | Polygonatum cyrtonema leaf polysaccharide and preparation method and application thereof | |
| CN112321742A (en) | Separation and purification of coriolus versicolor exopolysaccharide and structural characterization thereof | |
| CN115746156B (en) | Lycium barbarum polysaccharide with immunoregulatory function and preparation method thereof | |
| WO2023066163A1 (en) | Exopolysaccharide separated from lactobacillus delbrueckii and streptococcus thermophilus fermented yoghourt and application thereof | |
| CN111320709A (en) | Method for extracting glehnia acidic polysaccharide, extract thereof and application of glehnia acidic polysaccharide in immunoregulation | |
| CN113480674B (en) | Black tea polysaccharide-I active ingredient, preparation method thereof and application thereof in resisting cancer | |
| CN117801132A (en) | Polygonatum cyrtonema leaf polysaccharide and preparation method and application thereof | |
| WO2014173058A1 (en) | Trametes robiniophila polysaccharide protein, preparation method therefor, and application thereof | |
| CN114057907A (en) | Method for extracting, separating and purifying red ginseng polysaccharide | |
| WO2014173059A1 (en) | Trametes robiniophila polysaccharide protein, preparation method therefor, and application thereof | |
| CN111777693A (en) | Ligusticum wallichii homogeneous polysaccharide and extraction method and application thereof | |
| CN115028752B (en) | Uniform water-soluble polysaccharide and preparation method and application thereof | |
| WO2014173056A1 (en) | Trametes robiniophila polysaccharide protein, preparation method therefor, and application thereof | |
| WO2014173057A1 (en) | Trametes robiniophila polysaccharide protein, preparation method therefor, and application thereof | |
| CN111892662B (en) | Sedum sarmentosum homogeneous polysaccharide and preparation method and application thereof | |
| CN114409824B (en) | Mucor exopolysaccharide and preparation method and application thereof | |
| CN115124628B (en) | Bletilla striata polysaccharide, preparation method and application thereof, and immunologic adjuvant and nano vaccine containing bletilla striata polysaccharide | |
| CN111647095B (en) | Polysaccharide of fraxinus chinensis, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |