CN113336808A - Glucoside compound extracted and separated from lily, and method and application thereof - Google Patents
Glucoside compound extracted and separated from lily, and method and application thereof Download PDFInfo
- Publication number
- CN113336808A CN113336808A CN202110830176.XA CN202110830176A CN113336808A CN 113336808 A CN113336808 A CN 113336808A CN 202110830176 A CN202110830176 A CN 202110830176A CN 113336808 A CN113336808 A CN 113336808A
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- China
- Prior art keywords
- lily
- extracting
- separating
- concentrated
- glucoside
- Prior art date
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- Granted
Links
- 241000234435 Lilium Species 0.000 title claims abstract description 46
- -1 Glucoside compound Chemical class 0.000 title claims abstract description 43
- 229930182478 glucoside Natural products 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 57
- 229930182470 glycoside Natural products 0.000 claims abstract description 28
- 238000004440 column chromatography Methods 0.000 claims abstract description 18
- 230000002829 reductive effect Effects 0.000 claims abstract description 18
- 238000010992 reflux Methods 0.000 claims abstract description 18
- 238000000605 extraction Methods 0.000 claims abstract description 17
- 239000011347 resin Substances 0.000 claims abstract description 16
- 229920005989 resin Polymers 0.000 claims abstract description 16
- 238000000926 separation method Methods 0.000 claims abstract description 15
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 12
- 201000005202 lung cancer Diseases 0.000 claims abstract description 12
- 208000020816 lung neoplasm Diseases 0.000 claims abstract description 12
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 150000002338 glycosides Chemical class 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 48
- 239000006185 dispersion Substances 0.000 claims description 26
- 239000000047 product Substances 0.000 claims description 26
- 239000000843 powder Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 15
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 15
- 150000008131 glucosides Chemical class 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 238000011068 loading method Methods 0.000 claims description 9
- 238000012856 packing Methods 0.000 claims description 9
- 230000002441 reversible effect Effects 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- 238000000227 grinding Methods 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- 241000234280 Liliaceae Species 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 5
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- WABPQHHGFIMREM-UHFFFAOYSA-N lead(0) Chemical compound [Pb] WABPQHHGFIMREM-UHFFFAOYSA-N 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 12
- 239000000126 substance Substances 0.000 abstract description 11
- 230000000144 pharmacologic effect Effects 0.000 abstract description 9
- 239000003560 cancer drug Substances 0.000 abstract description 3
- 239000002246 antineoplastic agent Substances 0.000 abstract description 2
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 2
- 238000013375 chromatographic separation Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 52
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 36
- 150000001875 compounds Chemical class 0.000 description 15
- 150000004676 glycans Chemical class 0.000 description 13
- 229920001282 polysaccharide Polymers 0.000 description 13
- 239000005017 polysaccharide Substances 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000003463 adsorbent Substances 0.000 description 6
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- 230000000875 corresponding effect Effects 0.000 description 6
- 238000004811 liquid chromatography Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 238000010298 pulverizing process Methods 0.000 description 6
- 238000004007 reversed phase HPLC Methods 0.000 description 6
- 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 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 206010011224 Cough Diseases 0.000 description 3
- 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 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 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 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000059156 Toddalia Species 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- HIMXGTXNXJYFGB-UHFFFAOYSA-N alloxan Chemical compound O=C1NC(=O)C(=O)C(=O)N1 HIMXGTXNXJYFGB-UHFFFAOYSA-N 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
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- 238000006243 chemical reaction Methods 0.000 description 2
- 208000013116 chronic cough Diseases 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- UKMSUNONTOPOIO-UHFFFAOYSA-N docosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCC(O)=O UKMSUNONTOPOIO-UHFFFAOYSA-N 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
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- 239000000203 mixture Substances 0.000 description 2
- 230000005311 nuclear magnetism Effects 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000004017 serum-free culture medium Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 238000003809 water extraction Methods 0.000 description 2
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 1
- QXRAHTFDPBQKIM-HWKANZROSA-N 1-feruloyl-sn-glycerol Chemical compound COC1=CC(\C=C\C(=O)OCC(O)CO)=CC=C1O QXRAHTFDPBQKIM-HWKANZROSA-N 0.000 description 1
- 229960002666 1-octacosanol Drugs 0.000 description 1
- PBUWCVBYMDVPCL-UHFFFAOYSA-N 3-anilinophthalic acid Chemical compound OC(=O)C1=CC=CC(NC=2C=CC=CC=2)=C1C(O)=O PBUWCVBYMDVPCL-UHFFFAOYSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 208000000616 Hemoptysis Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 241001648859 Lilium candidum Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241001474728 Satyrodes eurydice Species 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 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
- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 description 1
- HZYXFRGVBOPPNZ-UHFFFAOYSA-N UNPD88870 Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)=CCC(CC)C(C)C)C1(C)CC2 HZYXFRGVBOPPNZ-UHFFFAOYSA-N 0.000 description 1
- 208000031971 Yin Deficiency Diseases 0.000 description 1
- 241000234314 Zingiber Species 0.000 description 1
- 235000006886 Zingiber officinale Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 1
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940076810 beta sitosterol Drugs 0.000 description 1
- 125000003404 beta-D-xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004141 dimensional analysis Methods 0.000 description 1
- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 description 1
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 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
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000008397 ginger Nutrition 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- HOVAGTYPODGVJG-VEIUFWFVSA-N methyl alpha-D-mannoside Chemical compound CO[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O HOVAGTYPODGVJG-VEIUFWFVSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- CNNRPFQICPFDPO-UHFFFAOYSA-N octacosan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCCCCCO CNNRPFQICPFDPO-UHFFFAOYSA-N 0.000 description 1
- 230000001662 opsonic effect Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
- 229940032091 stigmasterol Drugs 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
- 235000016831 stigmasterol Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000002936 tranquilizing effect Effects 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
- 239000000341 volatile oil Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 150000008505 β-D-glucopyranosides Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C07H1/08—Separation; Purification from natural products
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Steroid Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a glucoside compound extracted and separated from lily, a method and application thereof, wherein the structural formula of the glucoside compound is shown as (I). The glucoside compound is obtained by taking dried lily as a raw material and carrying out extraction and separation through the steps of ethanol reflux extraction, reduced pressure concentration, AB-8 resin column chromatography, C18 reversed phase chromatographic separation and the like, is a novel glucoside compound with novel structure and pharmacological activity, can be used for preparing anti-lung cancer drugs, and provides reliable basis for preparing new anti-cancer drugs, glycoside substance pharmaceutical research and the like.
Description
Technical Field
The invention belongs to the technical field of phytochemistry, and particularly relates to a glycoside compound with pharmacological activity extracted and separated from lily, and a preparation method and application thereof.
Background
The Liliaceae Lilium plant has about 115 varieties all over the world, is mainly distributed in northern temperate regions and subtropical mountain regions, and is made into 39 varieties and 26 varieties, wherein 25 varieties and 19 varieties are special products in China. The bulb of the lily plant contains starch, and can be eaten and used as a medicine in some species; the fresh flower contains volatile oil, and can be used as perfume. The lily medicinal material collected from 2020 edition of China pharmacopoeia is derived from herba Clausenae LansiiLilium lancifoliumThunb, lily bulbL．brownii F. E. Brown var. viridulumBaker and Lilium tenuifoliumL．PumilumThe dry bulb of DC has effects of nourishing yin, moistening lung, clearing heart fire and tranquilizing mind, and can be used for treating chronic cough due to yin deficiency, chronic cough with hemoptysis, vexation, pavor, insomnia, dreaminess and absentmindedness. The edible lily is commonly used for dietary therapy, and has health promotion effects of relieving cough and asthma, lowering blood sugar, resisting tumor, improving sleep, enhancing immunity, preventing senile dementia, etc., and has high nutritive value.
A large number of researches show that the liliaceae plants mainly contain polysaccharides, flavonoids, polyphenols, saponins, amino acids, alkaloids, steroids and other components, and the existing literature for researching the chemical components of the liliaceae plants mainly comprises the following components:
(I) ZhouZhou, etc. "study of chemical composition of Toddalia chinensis" (the university of Beijing Chinese medicine, 1 month 2010, 33, No. 1 Chinese medicinal chemistry) reported that 15 compounds were separated from the bulb of Toddalia chinensis, and were respectively identified as (beta) -D-glucosyl- (1 → 4) -beta-D-glucopyranoside, ((beta) -D-fructofuranosyl-alpha-D-glucopyranoside, ((methyl) -alpha-D-glucopyranoside), (methyl-alpha-D-mannopyranoside), (adenosine, rhodanoside, seventy 1-O-p-coumaroyl glyceride, (diosgenin) 3-O- { O-alpha-L-rhamnosyl- (1 → 2) -O- [ beta-D-xylosyl (1 → 3) ] -beta-D-glucopyranoside -glucoside }, ninthly (25R) -3 beta, 17 alpha-dihydroxy-5 alpha-spirostan-6-one-3-O-alpha-L-rhamnosyl- (1 → 2) -beta-D-glucoside, ninthly (25R) -3 beta-hydroxy-5 alpha-spirostan-6-one-3-O-alpha-L-rhamnosyl- (1 → 2) -beta-D-glucoside, (11) (25R) -spirostan-5-en-3 beta-O-alpha-L-rhamnopyranose- (1 → 2) - [ beta-D-glucopyranose- (1 → 6) ] beta-D-glucopyranoside, (12) montanyl alcohol, (13) n-docosanoic acid, (14) stigmasterol, and (15) beta-sitosterol.
(II) Zhao national Hua, etc. "chemical Structure and antitumor Activity of Lily polysaccharide" (food and biotechnology, 1 month in 2002, vol. 21, 1 st stage), and reports the research on the separation and purification, chemical structure and antitumor activity of LBPS-I as the main component of Lily polysaccharide. 1) Homogeneous LBPS-I polysaccharide is obtained by water extraction, ethanol precipitation and column chromatography, and is pure non-starch glucose, which is glucose with alpha-D- (1,4) -Glcp and alpha-D- (1,3) -Glcp alternately forming a main chain in a ratio of 2:1 and alpha-D- (1,6) -Glcp side chains. 2) The research of the mouse transplanted solid tumor shows that LBPS-I has stronger inhibiting effect on transplanted melanin B16 and Lewis lung cancer.
And thirdly, the ginger shavings and the like are separated from the lily decoction pieces for the first time by adopting a hot water extraction ethanol precipitation method to obtain the water-soluble polysaccharide BHP. Molecular weight 75000, acid hydrolysis. Thin layer development was performed for analysis of polysaccharide components. And (3) developing color by using aniline phthalic acid. Spots such as D-galactose, L-arabinose, D-mannose, D-glucose, L-rhamnose and the like are presented. The polysaccharide acts on the immune system of the body. Has obvious opsonic effect on the immune function of mice.
And (IV) separating two polysaccharides LP1 and LP2 from fresh Lilium Candidum's bulb leaves produced in Sichuan. Respectively accounting for 0.55 percent and 0.25 percent of the fresh weight. In the component analysis of polysaccharide, LP1 consists of glucose and mannose in the ratio of 1 to 2.46 and has molecular weight of 79400, and LP2 consists of glucose, mannose, arabinose and galacturonic acid. The ratio of the two polysaccharides is 1:0.73:2.61:1.8:0.84, the molecular weight is 181500, the two polysaccharides have obvious blood sugar reducing function on mice with hyperglycemia caused by alloxan, and the two polysaccharides are positively correlated with the concentration.
From the content reported in the above documents, the existing research on chemical components of liliaceae plants mainly focuses on polysaccharide and steroid saponin compounds, and relatively few reports are made on other structural types of compounds. The inventor is in the process of preparing the pelletL. lancifoliumWhen active ingredients are researched, a novel medicinal compound is discovered and separated, the compound is a chlorphenyl glycoside compound, pharmacological activity research shows that the compound has certain anti-lung cancer activity, and scientific basis is provided for researching and developing novel anti-lung cancer medicaments.
Disclosure of Invention
The invention aims to provide a glucoside compound extracted and separated from lily. The novel compound with novel structure and pharmacological activity is obtained by extracting and separating from the lily medicinal material, further deepens the systematic research of the chemical components of the lily and provides a material basis for the research of the pharmacological action of the lily.
The second purpose of the invention is to provide a method for extracting and separating glucoside compounds from lily. The method takes dried lily as a raw material, and obtains the glucoside compound with a novel structure and pharmacological activity by the steps of ethanol reflux extraction, reduced pressure concentration, AB-8 resin column chromatography, C18 reversed phase chromatographic separation and the like.
The invention also aims to provide the application of the glucoside compound extracted and separated from the lily in preparing the anti-lung cancer medicament, and provide a reliable basis for preparing a new anti-lung cancer medicament.
The purpose of the invention is realized by the following technical scheme: a glycoside compound extracted and separated from lily, wherein the glycoside compound has a structural formula shown as (I):
the glycoside compound with the structural formula shown in the formula (I) is obtained by extracting and separating dried lily, wherein the name of the glycoside compound is as follows: 2, 4-dichloro-3, 5-dimethoxy-benzyl-1-O-beta-D-glucopyranosyl- ((1 → 6) -beta-D-glucopyranoside, belonging to the chlorophenyl glycoside compounds with the name of lily neo-glycoside
。
The Bulbus Lilii is Liliaceae plant herba CentellaeLilium lancifoliumDried bulbs of thunb.
The molecular weight of the lily neoside I is 561, and the molecular formula is C21H30O13Cl2。
A method for extracting and separating glycoside compounds from lily comprises the following steps:
A. reflux extraction
Taking dried lily as a raw material, crushing, and performing reflux extraction by using 80-90% ethanol to obtain an extracting solution;
B. concentrating under reduced pressure
B, decompressing the extracting solution obtained in the step A to-0.08-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain a water dispersion;
C. resin column chromatography
B, carrying out wet loading on the aqueous dispersion obtained in the step B, carrying out column chromatography separation by using AB-8 macroporous adsorption resin, collecting a chromatographic solution containing glucoside components, and then carrying out reduced pressure concentration until no alcohol exists to obtain a concentrated solution;
c18 reverse phase chromatography column separation
And C, filtering the concentrated solution obtained in the step C, and separating the filtrate by using C18 reverse phase chromatographic packing under high pressure: a: acetonitrile B: 0.1% V/V formic acid water, a: B = 22: 78V/V as mobile phase; the detection wavelength is 215 nm;
E. concentrating
And D, concentrating the product collected liquid obtained in the step D at 50 ℃ under reduced pressure of-0.08 to-0.09 MPa until no acetonitrile exists, freeze-drying the concentrated liquid, grinding the dried solid into powder, and then carrying out vacuum drying at 45 ℃ to obtain a white powder, wherein the white powder is lily neo-glycoside I which is a glycoside compound and has a structural formula shown in the formula (I).
In the step A, the particle size of the crushed raw materials is 60-80 meshes.
In the step A, during reflux extraction, the weight of the ethanol is 8-10 times that of the raw materials.
In the step A, the reflux extraction is carried out for 3-5 times, and each time lasts for 1 hour.
In the step B, water is added into the concentrated extracting solution according to the volume ratio of 1: 6-1: 10 for dispersing treatment.
In the step C, the mobile phase used for the AB-8 macroporous adsorption resin column chromatography separation is methanol-water =50: 50V/V.
The white powder obtained by extraction and separation of the invention has positive Molish reaction, and is further proved to be a glucoside compound.
On the basis, the further analysis results are as follows:
ESI-MS (electrospray ionization mass spectrometry) of glycoside compounds with the structural formula shown in (I) shows that: m/z 560.09 [ M-H]- ；562.33 [M+H]+，584.36 [M+Na]+The molecular weight of the compound is 561, the molecular formula is C21H30O13Cl2。IRνmax（KBr，cm-1）：3400.6，1649.6，1026.0，999.7， 8272，766.2 cm-1；UV λmax201 (3.72) nm。
2 methoxy groups are seen on the hydrogen spectrumδ H3.82, 3.88 (each 3H, s). Shows two glycosyl-terminated hydrogen signals in the low-field part: (δ H4.29, d =7.6 Hz, 1H; 4.26, d =7.6 Hz, 1H). Full attribution of hydrocarbons by HSQC, discovery of delta by HMBCH 4.26 (H-1') and δC66.8 (C-6'), descriptionThe sugar unit being attached to C-6, deltaH 4.29 (H-1') and deltaC68.7 (C-6 ') which indicates that the sugar unit is attached to C-6'. Combining literature nuclear magnetic data, the compound is determined to be:
2,4-dichloro-3,5-dimethoxyl-benzyl-1-O-β-D-glucopyranosyl-((1→6)-β-D-glucopyranoside, chinese name: 2, 4-dichloro-3, 5-dimethoxy-benzyl-1-O-beta-D-glucopyranosyl- ((1 → 6) -beta-D-glucopyranoside, a related report of the compound is not found through the search of a scifinder, and the compound is determined to be a new glycoside structure and is named as lily new glycoside I.
1H-NMR and13the C-NMR data are shown in Table 1 below.
By passing1H-NMR、13C-NMR and DEPT135 ℃ and nuclear magnetism two-dimensional analysis technical means such as HSQC, HMBC, H-HCOSY, NOESY and the like determine that the compound is as follows: the structural formula of the lily neoside I (neoglucoside compound) is shown as the formula (I).
Meanwhile, pharmacological experiments prove that the glucoside compound extracted and separated by the method disclosed by the invention and having the structural formula shown in the formula (I) has certain anti-lung cancer activity and can be applied to preparation of anti-lung cancer drugs.
The invention has the beneficial technical effects that:
1. the novel glycoside compound provided by the invention is obtained by extracting and separating from dried lily, has a structural formula shown in (I), is determined in structure, and provides scientific basis for systematic research on chemical components and pharmacological action of lily.
2. The invention uses liliaceae plant lilium tigrinumL. lancifoliumThe dried bulb is taken as a raw material, and glucoside compounds are obtained through the technical steps of ethanol reflux extraction, AB-8 column chromatography separation and purification, C18 reversed phase chromatographic packing high-pressure preparation and separation and the likeThe purity of I is more than 99 percent, the time consumption of the whole production process is short, and the method is suitable for industrial production.
3. The invention reports the structure of the lily neoside I for the first time, determines the relative configuration according to relevant data such as nuclear magnetism two-dimension and the like, and is a novel glucoside compound; pharmacological research proves that the compound has certain anti-lung cancer activity, can be used as a latent structure for development and utilization, and provides reliable basis for the aspects of large-scale tissue culture for producing glucoside substances, activity research of the glucoside substances, research and development of new anti-cancer drugs and the like.
Detailed Description
The present invention will be described in further detail with reference to examples. It should be noted, however, that the following examples are not to be construed as limiting the scope of the present invention, and that many insubstantial modifications and variations of the invention can be made by those skilled in the art without departing from the spirit and scope of the invention as set forth herein.
Example 1:
a glycoside compound extracted from Bulbus Lilii is white powder, has positive Molish reaction, molecular weight of 561, and molecular formula C21H30O13Cl2Having the formula (I):
the glucoside compound is sudandanL. lancifoliumThe dried bulb is obtained by extraction and separation, and the specific steps are as follows:
A. reflux extraction
Taking dried lily as a raw material, crushing, and performing reflux extraction by using 80-90% ethanol to obtain an extracting solution;
B. concentrating under reduced pressure
B, decompressing the extracting solution obtained in the step A to-0.08-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain a water dispersion;
C. resin column chromatography
B, carrying out wet loading on the aqueous dispersion obtained in the step B, carrying out column chromatography separation by using AB-8 macroporous adsorption resin, collecting a chromatographic solution containing glucoside components, and then carrying out reduced pressure concentration until no alcohol exists to obtain a concentrated solution;
c18 reverse phase chromatography column separation
And C, filtering the concentrated solution obtained in the step C, and separating the filtrate by using C18 reverse phase chromatographic packing under high pressure: a: acetonitrile B: 0.1% V/V formic acid water, a: B = 22: 78V/V as mobile phase; the detection wavelength is 215 nm;
E. concentrating
And D, decompressing the collected liquid of the product obtained in the step D to-0.08 to-0.09 MPa at 50 ℃ until no acetonitrile exists, freeze-drying the concentrated liquid, grinding the dried solid into powder, and then carrying out vacuum drying at 45 ℃ to obtain white powder, namely the glucoside compound with the structural formula shown in the formula (I).
Example 2:
collecting dried Bulbus Lilii (herba Zosterae Marinae)L. lancifoliumDried bulb) of 10kg, pulverizing to 60 mesh, adding 8 times of 80wt% ethanol, reflux extracting for 3 times, each for 1 hr, and mixing extractive solutions; decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:6 for dispersion treatment to obtain 20L of aqueous dispersion; loading the aqueous dispersion by wet method, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V is mobile phase), collecting chromatography liquid containing glucoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 5L; filtering the concentrated solution, separating the filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B: 0.1% V/V formic acid water, A: B = 22: 78V/V is mobile phase, detection wavelength is 215 nm), collecting corresponding chromatographic peaks, concentrating the product collected solution at 50 ℃ under reduced pressure to-0.08 to-0.09 MPa until no acetonitrile exists, freeze-drying the concentrated solution, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain 6g of white powder product which is the glycoside compound with the structural formula shown in (I).
The whole production process takes about 5 days;
the purity of the product was determined to be 99.36% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V formic acid water, A: B = 46: 54V/V as the mobile phase; detection wavelength 215 nm).
Example 3:
collecting dried Bulbus Lilii (herba Zosterae Marinae)L. lancifoliumDried bulb) of 20kg, pulverizing to 70 mesh, adding 8.5 times of 85wt% ethanol, reflux extracting for 4 times, each for 1 hr, and mixing extractive solutions; decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:7 for dispersion treatment to obtain 36L of aqueous dispersion; loading the aqueous dispersion by wet method, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V is mobile phase), collecting chromatography liquid containing glucoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 12L; filtering the concentrated solution, separating the filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B: 0.1% V/V formic acid water, A: B = 22: 78V/V is mobile phase, detection wavelength is 215 nm), collecting corresponding chromatographic peaks, concentrating the product collected solution at 50 ℃ under reduced pressure to-0.08 to-0.09 MPa until no acetonitrile exists, freeze-drying the concentrated solution, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain 13g of white powder product which is the glycoside compound with the structural formula shown in (I).
The whole production process takes about 6 days;
the purity of the product was determined to be 99.08% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V formic acid water, A: B = 46: 54V/V as the mobile phase; detection wavelength 215 nm).
Example 4:
collecting dried Bulbus Lilii (herba Zosterae Marinae)L. lancifoliumDried bulb) of 30kg, pulverizing to 80 mesh, adding 9 times of ethanol with concentration of 90wt%, reflux extracting for 5 times, each for 1 hr, and mixing extractive solutions; decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:8 for dispersion treatment to obtain 58L of aqueous dispersion; loading the aqueous dispersion by wet method, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V is mobile phase), collecting chromatography liquid containing glucoside components, and concentrating under reduced pressure until no alcohol exists to obtain 23L of concentrated solution; filtering the concentrated solution, and separating the filtrate by C18 reverse phase chromatography under high pressure(A: acetonitrile B: 0.1% V/V formic acid water, A: B = 22: 78V/V is mobile phase; detection wavelength is 215 nm), corresponding chromatographic peaks are collected, a product collection liquid is concentrated to be acetonitrile-free after being decompressed to-0.08 to-0.09 MPa at 50 ℃, a concentrated solution is freeze-dried, a dried solid is ground into powder, and the powder is vacuum-dried at 45 ℃, so that 20g of a white powder product is obtained, and the glycoside compound is the glycoside compound with the structural formula shown in (I).
The whole production process takes about 7 days;
the purity of the product was determined to be 99.16% by rechecking the product by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V formic acid water, A: B = 46: 54V/V as the mobile phase; detection wavelength 215 nm).
Example 5:
A. collecting dried Bulbus Lilii (herba Zosterae Marinae)L. lancifoliumDried bulb) of 50kg, pulverizing to 60 mesh, adding 8 times of 80wt% ethanol, reflux extracting for 3 times, each for 1 hr, and mixing extractive solutions;
B. decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:6 for dispersion treatment to obtain 90L of aqueous dispersion;
C. loading the aqueous dispersion by wet method, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V is mobile phase), collecting chromatography liquid containing glucoside components, and concentrating under reduced pressure until no alcohol exists to obtain 30L concentrated solution;
D. filtering the concentrated solution, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: acetonitrile B: 0.1% V/V formic acid water, A: B = 22: 78V/V is mobile phase; detection wavelength is 215 nm), and collecting corresponding chromatographic peak;
E. and (3) decompressing the collected liquid of the product to-0.08 to-0.09 MPa at 50 ℃ and concentrating to be dry, grinding the solid into powder, and then drying by air blowing at 45 ℃ to obtain 32g of white powder product, namely the glucoside compound with the structural formula shown in the formula (I).
The whole production process takes about 7 days;
the purity of the product was found to be 99.21% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V formic acid water, A: B = 46: 54V/V as the mobile phase; detection wavelength 215 nm).
Example 6:
A. collecting dried Bulbus Lilii (herba Zosterae Marinae)L. lancifoliumDried bulb) of 50kg, pulverizing to 70 mesh, adding 10 times of ethanol with concentration of 85wt%, reflux extracting for 5 times, each for 1 hr, and mixing extractive solutions;
B. decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, adding water into the concentrated extracting solution according to the volume ratio of 1:10, and dispersing to obtain 120L of aqueous dispersion;
C. loading the aqueous dispersion by wet method, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V is mobile phase), collecting chromatography liquid containing glucoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 50L;
D. filtering the concentrated solution, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: acetonitrile B: 0.1% V/V formic acid water, A: B = 22: 78V/V is mobile phase; detection wavelength is 215 nm), and collecting corresponding chromatographic peak;
E. and (3) concentrating the collected liquid of the collected liquid product at 50 ℃ under reduced pressure to-0.08 to-0.09 MPa until no acetonitrile exists, freeze-drying the concentrated liquid, grinding dried solid into powder, and then carrying out vacuum drying at 45 ℃ to obtain 38g of a white powder product, namely the glucoside compound with the structural formula shown in the formula (I).
The whole production process takes about 9 days;
the purity of the product was determined to be 99.34% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V formic acid water, A: B = 46: 54V/V as the mobile phase; detection wavelength 215 nm).
Example 7:
A. collecting dried Bulbus Lilii (herba Zosterae Marinae)L. lancifoliumDried bulb of (1) by a weight ratio, pulverizing to 80 mesh, adding 9 times of ethanol with a concentration of 90wt%, reflux-extracting for 4 times, each for 1 hour, and mixing the extractive solutions;
B. decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:9 for dispersion treatment to obtain 100L of aqueous dispersion;
C. wet loading the aqueous dispersion, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V as mobile phase), collecting chromatography liquid containing total flavone component, and concentrating under reduced pressure until no alcohol exists to obtain 40L concentrated solution;
D. filtering the concentrated solution, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: acetonitrile B: 0.1% V/V formic acid water, A: B = 22: 78V/V is mobile phase; detection wavelength is 215 nm), and collecting corresponding chromatographic peak;
E. and (3) decompressing the collected liquid of the collected liquid product to-0.08 to-0.09 MPa at 50 ℃ and concentrating until no acetonitrile exists, freeze-drying the concentrated liquid, grinding the dried solid into powder, and then carrying out vacuum drying at 45 ℃ to obtain 35g of white powder product, namely the glucoside compound with the structural formula shown in the formula (I).
The whole production process takes about 8 days;
the purity of the product was determined to be 99.19% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V formic acid water, A: B = 46: 54V/V as the mobile phase; detection wavelength 215 nm).
Example 8:
the following experiment was carried out by arbitrarily selecting the white powdery compound having the structural formula shown in (I) obtained by extraction and separation in the above example 4 (i.e., so-called neoliloside I in the following experiment):
anti-lung cancer activity test of the compound:
(1) experimental materials and instruments
Cell: a. the549Cells (Wuhan Punuousel life science and technology limited)
Drugs and reagents: lily neoside I (purity > 99.0%, self-made); the DMSO solution is dissolved, the concentration of the prepared mother liquor is 200 mu mol/L, 1 mu L of the mother liquor is diluted to 1ml by serum-free culture medium each time, and the volume ratio of the mother liquor to the serum-free culture medium is 1: 4 for dilution. RPMI-1640 medium (Gibco, batch No. 8120139, USA), FBS (Procell, USA, batch No. SA 201126), PBS buffer powder (Wuxi Aorui Dongyuan Biotech, Inc.), pancreatin (bioflorox, batch No. EZ6688C 183), penicillin-streptomycin solution (bioharp, batch No. 1334GR 005), MTT (bioflorox, batch No. EZ6688D 183), DMSO (Doudotolong chemical, batch No. 67-68-5)
The instrument comprises the following steps: thermo Varlosks microplate reader (Thermo Fisher Scientific, USA), Allegra X-30R centrifuge (Beckman Coulter, USA), ultra clean bench (Changhong Mei Ling Ltd.), inverted microscope (Zeiss, Germany), CO2Constant temperature incubator (siemens electronic limited, suzhou).
(2) Experimental methods
Using 10% 1640 complete medium at 37 ℃ with 5% CO2The culture was carried out under concentration conditions. Digesting the cells in logarithmic growth phase to prepare cell suspension, and then preparing the cell suspension by 1 × 104One/ml was inoculated in 96-well plates in CO2Culturing in a constant temperature incubator for 24 h. Discarding the culture medium, washing with PBS for 1 time, adding different concentrations of neoliensin I (each group has 3 multiple wells), and adding 3-well blank culture medium as normal control group; it is put back into CO2Culturing in a constant temperature incubator for 24 h. Then 20 mu L of MTT solution is put into the culture medium for culture for 4h, the culture solution is discarded, 150 mu L of DMSO solution is added into each hole, the mixture is put into a microplate reader for oscillation for 5min, and then the absorbance (OD) is detected at the wavelength of 490 nm.
Data were processed using SPSS 26.0 statistical software and results were expressed as mean. + -. standard deviation (). One way ANOVA (One way-ANOVA) was used for comparisons between groups.
(3) Results of the experiment
As can be seen in the following table 1, the lily neoside I has inhibitory activity on the proliferation of A549 cells cultured in vitro, the maximum inhibition rate in the research dosage is 65.10%,IC 50is 0.029 (0.012-0.102) μmol/L.
Therefore, the glucoside compound with the structural formula shown in (I) extracted and separated by the invention can be applied to the preparation of anti-lung cancer drugs.
Claims (8)
1. A glycoside compound extracted and separated from lily, wherein the glycoside compound has a structural formula shown as (I):
the glycoside compound represented by formula (I) is prepared from Liliaceae plant Plumbum PreparatiumLilium lancifoliumDried bulbs of thunb, under the literal name: 2, 4-dichloro-3, 5-dimethoxy-benzyl-1-O-beta-D-glucopyranosyl- ((1 → 6) -beta-D-glucopyranoside, molecular weight 561, molecular formula C21H30O13Cl2The name is as follows: and (3) lily neoside I.
2. The method for preparing the glycoside compound extracted and separated from lily as claimed in claim 1, which mainly comprises the following steps:
A. reflux extraction
Taking dried lily as a raw material, crushing, and performing reflux extraction by using 80-90% ethanol to obtain an extracting solution;
B. concentrating under reduced pressure
B, decompressing the extracting solution obtained in the step A to-0.08-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain a water dispersion;
C. resin column chromatography
B, carrying out wet loading on the aqueous dispersion obtained in the step B, carrying out column chromatography separation by using AB-8 macroporous adsorption resin, collecting a chromatographic solution containing glucoside components, and then carrying out reduced pressure concentration until no alcohol exists to obtain a concentrated solution;
c18 reverse phase chromatography column separation
And C, filtering the concentrated solution obtained in the step C, and separating the filtrate by using C18 reverse phase chromatographic packing under high pressure: a: acetonitrile B: 0.1% V/V formic acid water, a: B = 22: 78V/V as mobile phase; the detection wavelength is 215 nm;
E. concentrating
And D, decompressing the collected liquid of the product obtained in the step D to-0.08 to-0.09 MPa at 50 ℃ until no acetonitrile exists, freeze-drying the concentrated liquid, grinding the dried solid into powder, and then carrying out vacuum drying at 45 ℃ to obtain white powder, namely the glucoside compound with the structural formula shown in the formula (I).
3. The method for extracting and separating glycosides from lily as claimed in claim 2, wherein: in the step A, the particle size of the crushed raw materials is 60-80 meshes.
4. The method for extracting and separating glycosides from lily as claimed in claim 2, wherein: in the step A, during reflux extraction, the weight of the ethanol is 8-10 times that of the raw materials.
5. The method for extracting and separating glycosides from lily as claimed in claim 2, wherein: in the step A, the reflux extraction is carried out for 2-3 times, and each time lasts for 1 hour.
6. The method for extracting and separating glycosides from lily as claimed in claim 2, wherein: in the step B, water is added into the concentrated extracting solution according to the volume ratio of 1: 6-1: 10 for dispersing treatment.
7. The method for extracting and separating glycosides from lily as claimed in claim 2, wherein: in the step C, the mobile phase used for the AB-8 macroporous adsorption resin column chromatography separation is methanol-water =50: 50V/V.
8. Use of the glycoside compound extracted from Bulbus Lilii of claim 1 in preparing anti-lung cancer medicine.
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| CN111187323A (en) * | 2019-12-20 | 2020-05-22 | 成都普思生物科技股份有限公司 | Hosta plantaginea flower extract and extraction method and application thereof |
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| JP2003113088A (en) * | 2001-10-09 | 2003-04-18 | Pola Chem Ind Inc | Carcinogenesis promoter-suppressant and composition containing the same |
| CN101029073A (en) * | 2007-03-29 | 2007-09-05 | 复旦大学 | Steroid saponin compound and its use in preparation antineoplastic |
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| JPS5498339A (en) * | 1978-01-18 | 1979-08-03 | Rikagaku Kenkyusho | Carcinostatic agent |
| JP2003113088A (en) * | 2001-10-09 | 2003-04-18 | Pola Chem Ind Inc | Carcinogenesis promoter-suppressant and composition containing the same |
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| CN111187323A (en) * | 2019-12-20 | 2020-05-22 | 成都普思生物科技股份有限公司 | Hosta plantaginea flower extract and extraction method and application thereof |
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