CN110305094B - 马齿苋中两种黄酮类化合物及其提取分离方法与用途 - Google Patents
马齿苋中两种黄酮类化合物及其提取分离方法与用途 Download PDFInfo
- Publication number
- CN110305094B CN110305094B CN201910640045.8A CN201910640045A CN110305094B CN 110305094 B CN110305094 B CN 110305094B CN 201910640045 A CN201910640045 A CN 201910640045A CN 110305094 B CN110305094 B CN 110305094B
- Authority
- CN
- China
- Prior art keywords
- methanol
- elution
- ethyl acetate
- extraction
- compounds
- 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
- -1 flavonoid compounds Chemical class 0.000 title claims abstract description 62
- 235000001855 Portulaca oleracea Nutrition 0.000 title claims abstract description 25
- 241000219304 Portulacaceae Species 0.000 title claims abstract description 25
- 238000000605 extraction Methods 0.000 title claims abstract description 23
- 238000000926 separation method Methods 0.000 title claims abstract description 23
- 229930003935 flavonoid Natural products 0.000 title claims abstract description 17
- 235000017173 flavonoids Nutrition 0.000 title claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000004440 column chromatography Methods 0.000 claims abstract description 9
- 239000004952 Polyamide Substances 0.000 claims abstract description 6
- 229920002647 polyamide Polymers 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 45
- 238000010828 elution Methods 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 27
- 239000000463 material Substances 0.000 claims description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 11
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- 238000004809 thin layer chromatography Methods 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 9
- 239000013078 crystal Substances 0.000 claims description 8
- 239000002024 ethyl acetate extract Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000001704 evaporation Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 5
- 229920005654 Sephadex Polymers 0.000 claims description 4
- 239000012507 Sephadex™ Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 2
- UREBWPXBXRYXRJ-UHFFFAOYSA-N ethyl acetate;methanol Chemical compound OC.CCOC(C)=O UREBWPXBXRYXRJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000010829 isocratic elution Methods 0.000 claims 3
- 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 abstract description 34
- 229930003944 flavone Natural products 0.000 abstract description 34
- 235000011949 flavones Nutrition 0.000 abstract description 34
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 abstract description 34
- 150000001875 compounds Chemical class 0.000 abstract description 26
- 238000001228 spectrum Methods 0.000 abstract description 12
- 229940079593 drug Drugs 0.000 abstract description 9
- KLPMIPWOCDHPDO-UHFFFAOYSA-N 3-[(2-hydroxyphenyl)methyl]-6,8-dimethoxychromen-4-one Chemical compound OC1=C(CC2=COC3=C(C=C(C=C3C2=O)OC)OC)C=CC=C1 KLPMIPWOCDHPDO-UHFFFAOYSA-N 0.000 abstract description 7
- 230000000259 anti-tumor effect Effects 0.000 abstract description 7
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 abstract description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 abstract description 6
- 206010061218 Inflammation Diseases 0.000 abstract description 6
- 230000003064 anti-oxidating effect Effects 0.000 abstract description 6
- 229930014805 neoflavone Natural products 0.000 abstract description 6
- 230000000144 pharmacologic effect Effects 0.000 abstract description 5
- 238000000746 purification Methods 0.000 abstract description 5
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 abstract description 4
- 238000010898 silica gel chromatography Methods 0.000 abstract description 4
- DCPSPIQNWXPYQK-UHFFFAOYSA-N 3-[(2-hydroxyphenyl)methyl]-6,8-dimethoxy-2,3-dihydrochromen-4-one Chemical compound OC1=C(CC2COC3=C(C=C(C=C3C2=O)OC)OC)C=CC=C1 DCPSPIQNWXPYQK-UHFFFAOYSA-N 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000001953 recrystallisation Methods 0.000 abstract description 3
- 150000003839 salts Chemical class 0.000 abstract description 3
- 229940126680 traditional chinese medicines Drugs 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 238000001425 electrospray ionisation time-of-flight mass spectrometry Methods 0.000 abstract 1
- 150000002611 lead compounds Chemical class 0.000 abstract 1
- 230000003334 potential effect Effects 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 31
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 210000002540 macrophage Anatomy 0.000 description 9
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 8
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 7
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 6
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000002757 inflammatory effect Effects 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 6
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 125000006290 2-hydroxybenzyl group Chemical group [H]OC1=C(C([H])=C([H])C([H])=C1[H])C([H])([H])* 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 229930013930 alkaloid Natural products 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 229930182555 Penicillin Natural products 0.000 description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 229940049954 penicillin Drugs 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 230000003698 anagen phase Effects 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000013375 chromatographic separation Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000006481 glucose medium Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 238000001896 rotating frame Overhauser effect spectroscopy Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000001026 1H--1H correlation spectroscopy Methods 0.000 description 2
- 125000003816 2-hydroxybenzoyl group Chemical group OC1=C(C(=O)*)C=CC=C1 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- 101000605172 Aspergillus niger (strain CBS 513.88 / FGSC A1513) Probable endopolygalacturonase E Proteins 0.000 description 2
- 101000605171 Aspergillus niger Endopolygalacturonase E Proteins 0.000 description 2
- 101000941281 Bos taurus Gastric triacylglycerol lipase Proteins 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 206010008342 Cervix carcinoma Diseases 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 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
- POJWUDADGALRAB-UHFFFAOYSA-N allantoin Chemical compound NC(=O)NC1NC(=O)NC1=O POJWUDADGALRAB-UHFFFAOYSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 201000010881 cervical cancer Diseases 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- VILAVOFMIJHSJA-UHFFFAOYSA-N dicarbon monoxide Chemical compound [C]=C=O VILAVOFMIJHSJA-UHFFFAOYSA-N 0.000 description 2
- 238000002072 distortionless enhancement with polarization transfer spectrum Methods 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 208000001848 dysentery Diseases 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 201000005249 lung adenocarcinoma Diseases 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 description 1
- BVCOHOSEBKQIQD-UHFFFAOYSA-N 2-tert-butyl-6-methoxyphenol Chemical compound COC1=CC=CC(C(C)(C)C)=C1O BVCOHOSEBKQIQD-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- POJWUDADGALRAB-PVQJCKRUSA-N Allantoin Natural products NC(=O)N[C@@H]1NC(=O)NC1=O POJWUDADGALRAB-PVQJCKRUSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 201000000297 Erysipelas Diseases 0.000 description 1
- XWDDIZKKSZLMEB-UHFFFAOYSA-N Feruloyl tyramine Natural products COc1cc(C=CC(=O)Oc2ccc(CCN)cc2)ccc1O XWDDIZKKSZLMEB-UHFFFAOYSA-N 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- 208000012671 Gastrointestinal haemorrhages Diseases 0.000 description 1
- 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 1
- 206010054787 Haemorrhoidal haemorrhage Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 208000006877 Insect Bites and Stings Diseases 0.000 description 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 1
- 206010027514 Metrorrhagia Diseases 0.000 description 1
- NPNNKDMSXVRADT-WEVVVXLNSA-N N-feruloyltyramine Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)NCCC=2C=CC(O)=CC=2)=C1 NPNNKDMSXVRADT-WEVVVXLNSA-N 0.000 description 1
- AVBCARAQLFOQID-UHFFFAOYSA-N N-trans-feruloyltyramine Natural products COc1cc(C=CC(=O)CNCc2ccc(O)cc2)ccc1O AVBCARAQLFOQID-UHFFFAOYSA-N 0.000 description 1
- 244000234609 Portulaca oleracea Species 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 241000270295 Serpentes Species 0.000 description 1
- 208000004078 Snake Bites Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 229960000458 allantoin Drugs 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013592 cell lysate 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
- 238000004587 chromatography analysis Methods 0.000 description 1
- NPNNKDMSXVRADT-UHFFFAOYSA-N cis-N-feruloyl tyramine Natural products C1=C(O)C(OC)=CC(C=CC(=O)NCCC=2C=CC(O)=CC=2)=C1 NPNNKDMSXVRADT-UHFFFAOYSA-N 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 150000004775 coumarins Chemical class 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000000431 effect on proliferation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000017095 negative regulation of cell growth Effects 0.000 description 1
- 150000002802 neoflavones Chemical class 0.000 description 1
- 229930014802 neoflavonoid Natural products 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 description 1
- 229960002748 norepinephrine Drugs 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 229930191164 oleracein Natural products 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 150000007964 xanthones Chemical class 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明涉及中药提取、分离领域,尤其涉及从马齿苋中提取、分离和鉴别出的两种新的黄酮类化合物及其提取分离方法。所述的两种新黄酮化合物,分子式依次为C18H16O5、C18H18O5,命名分别为3‑(2‑hydroxybenzyl)‑6,8‑dimethoxy‑4H‑chromen‑4‑one、3‑(2‑hydroxybenzyl)‑6,8‑dimethoxychroman‑4‑one。还提供上述新化合物的提取分离方法,依次采用水煎煮提取、硅胶柱层析、聚酰胺柱层析、ODS中压柱纯化、Sephadex LH‑20及重结晶进行分离纯化与制备。其结构采用HR‑ESI‑TOF‑MS、1H‑NMR、13C‑NMR及二维核磁波谱解析的方法鉴定为两种新黄酮类化合物。新黄酮化合物具有潜在的抗炎、抗肿瘤和抗氧化等活性,本发明新黄酮化合物及其盐或衍生物可以作为其他化合物合成先导物,以及新药开发和药理活性研究的原料,用于制备抗炎、抗肿瘤和抗氧化的药物。
Description
技术领域
本发明涉及中药提取、分离领域,尤其涉及从马齿苋药材中提取、分离和鉴别出的黄酮类化合物及其提取分离方法。
背景技术
马齿苋(Portulaca oleracea L.),又名长命菜、马苋菜,为马齿苋科植物。马齿苋耐旱耐涝,且耐光耐阴,分布广泛、资源丰富,作为药食两用的野生植物备受关注。2015版《中华人民共和国药典》中收载马齿苋的干燥地上部分入药,具有清热解毒、凉血止血、止痢等功效,用于热毒血痢、痈肿疔疮、湿疹、丹毒、蛇虫咬伤、便血、痔血、崩漏下血等。
马齿苋现代药理学研究表明,其具有抗炎止痛、抗菌抗病毒、降血压、降血脂、抗氧化、抗癌、松弛骨骼肌和平滑肌、调节免疫功能等作用。研究表明马齿苋众多化学成分为其多样的药理作用提供了物质基础,马齿苋主要化学成分包括黄酮类、香豆素类、萜类、甾类、有机酸类、挥发油、生物碱类、氨基酸类、各种色素类和矿物质类等。其中生物碱是马齿苋中一类主要的化学成分,目前已报道的生物碱类成分有去甲肾上腺素、多巴胺、少量多巴、腺苷、尿嘧啶、腺嘌呤、N,N-二环己基脲、尿囊素、N-反式-阿魏酰基酪胺;还有环二肽生物碱和酰胺类生物碱:马齿苋酰胺A-I、K、L、N-S。
目前从马齿苋中分离出的化学成分大多数是已知的,且结构新颖性较低,因此,对马齿苋中新化合物的开发和分离是亟待需要的。
发明内容
针对上述问题,本发明提供从马齿苋中提取的两种新黄酮类化合物,经研究发现本发明的两种新化合物具有抗炎、抗肿瘤和抗氧化的作用,同时提供一种针对本发明新化合物的简便、快速、环保、纯度高的提取分离方法。
为实现本发明的上述目的,本发明提供从马齿苋中分离出的两种黄酮类化合物,分子式分别为C18H16O5、C18H18O5,分别命名为3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one,化学结构式分别为:
为实现本发明的上述目的,本发明还提供一种马齿苋中黄酮类化合物的提取分离方法,具体步骤为:
步骤1:取马齿苋干燥药材,采用水提取(水用量为药材的8~16倍)两次,水提液滤过,合并滤液直接加热浓缩,放凉至室温,得药液备用。
步骤2:将步骤1中药液蒸干后上硅胶柱,用乙酸乙酯洗脱,减压回收乙酸乙酯至浸膏,得到乙酸乙酯提取物。
步骤3:将步骤2中乙酸乙酯提取物经聚酰胺柱分离,采用乙醇-水梯度洗脱,50%乙醇部分蒸干后上硅胶柱,依次用乙酸乙酯洗脱,回收乙酸乙酯至浸膏,得到乙酸乙酯提取物。
步骤4:将步骤3中所得物再经预处理的ODS柱(Octadecylsilyl,十八烷基硅烷键合硅胶填料)层析分离,用甲醇-水梯度洗脱,得到若干洗脱部位,经薄层色谱进行检测、显色,将各显色的洗脱部位分别减压浓缩至干,得到浓缩物备用。
步骤5:将步骤4中所得物再经预处理的葡聚糖凝胶柱(Sephadex LH-20)层析分离,用甲醇洗脱,得到若干洗脱部位,经薄层色谱进行检测,显色,合并显色的洗脱部位,将合并后的洗脱部位经减压浓缩至干,备用。
步骤6:将步骤5静置析出晶体,多次用甲醇洗净晶体后挥干甲醇,经薄层色谱进行检测、显色,最终得到本发明所述的两种新黄酮类化合物。
所述ODS与葡聚糖凝胶的预处理过程为甲醇浸泡过24小时,上柱,用甲醇洗至滴入水中无混浊,再以初始流动相平衡。
与现有技术相比本发明的有益效果在于:本发明中所述马齿苋新黄酮类化合物的分离和药理活性研究未被现有论文期刊所报道。本发明提供来源于马齿苋的两种黄酮类化合物及一种针对本发明新化合物的提取分离方法,依次采用水煎煮提取、硅胶柱层析、聚酰胺柱层析、ODS中压柱纯化、Sephadex LH-20及重结晶进行分离纯化与制备,成功提取分离出两种新的黄酮类化合物。该方法操作步骤仅为六步,操作方法简便及快速,提取分离过程主要采用水煎煮提取及乙酸乙酯洗脱,工艺方法环保,且经该方法分离得到的化合物纯度较高均大于90%。此外经研究表明以上化合物具有抗炎、抗肿瘤和抗氧化作用,因此本发明新化合物及其盐和衍生物可以作为其他化合物合成先导物,以及新药开发和药理活性研究的原料,亦可用于制备抗炎、抗肿瘤和抗氧化的药物。
附图说明
图1为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one的高分辨质谱图。
图2为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的高分辨质谱图。
图3为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的1H-NMR光谱图。
图4为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的13C-NMR光谱图。
图5为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的核磁共振碳谱(DEPT)光谱图。
图6为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的核磁共振1H-1HCOSY光谱图。
图7为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的核磁共振HMBC光谱图。
图8为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的核磁共振HSQC光谱图。
图9为本发明新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的核磁共振ROESY光谱图。
具体实施方式
下面结合具体实施对本发明做详细的说明。
本发明提供两种新化合物,分子式分别为C18H16O5、C18H18O5,分别命名为3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one,化学结构式分别为:
所述两种新化合物根据结构分别命名为3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one、3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one,表1、2分别为两种黄酮化合物的核磁数据:1H-NMR与13C-NMR在CCl3D中。
表1本发明新化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one的核磁数据
序号 | δ<sub>C</sub> | 类型 | δ<sub>H</sub>(J in Hz) |
1 | O | ||
2 | 151.11 | CH | 7.87,s |
3 | 125.51 | C | |
4 | 178.73 | C | |
5 | 92.58 | CH | 6.42,d,(2.2) |
6 | 164.76 | C | |
6-OMe | 55.81 | CH<sub>3</sub> | 3.83,s |
7 | 96.60 | CH | 6.35,d,(2.2) |
8 | 161.31 | C | |
8-OMe | 56.61 | CH<sub>3</sub> | 3.9,s |
9 | 160.65 | C | |
10 | 108.89 | C | |
11 | 27.48 | CH<sub>2</sub> | 3.66,s |
1′ | 126.21 | C | |
2′ | 155.43 | C | |
2′-OH | 8.23,brs | ||
3′ | 118.69 | CH | 6.94,q,(8.1,14.1) |
4′ | 128.56 | CH | 7.12,m |
5′ | 120.31 | CH | 6.82,t,(7.1) |
6 | 130.24 | CH | 7.04,dd,(0.9,7.1) |
3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one:白色粉末,难溶于甲醇,易溶于氯仿。点样于硅胶薄层板后,喷香草酚绿试液斑点呈淡黄色。HRESI(+)TOFMS给出m/z:313.1071[M+H]+的准分子离子峰,分子量为312.0998。结合1H-NMR,13C-NMR以及DEPT数据,推测该化合物可能的分子式为C18H16O5,不饱和度为11。13C-NMR谱和DEPT谱显示18个碳信号,分别为2个CH3(δ:55.81,56.61)、1个CH2(δ:27.48)、7个CH(δ:92.58,96.60,118.69,120.31,128.56,130.24,151.11)、8个季碳(一个羰基碳,δ:178.73;四个连O的双键碳,δ:155.43,160.65,161.31,164.76;三个双键碳,δ:108.89,125.51,126.21)。
1H-NMR谱显示1个活泼H信号δ8.23(1H,brs),表明可能存在一个羟基基团;2个甲基信号,为δ3.83(3H,s)和δ3.93(3H,s);1个亚甲基信号,分别为δ3.66(2H,s);7个次甲基信号分别为δ6.35(1H,d,J=2.2),δ6.42(1H,d,J=2.2),δ6.82(1H,t,J=7.1),δ6.94(1H,q,J=8.1,14.1),δ7.04(1H,dd,J=0.9,7.1),δ7.12(1H,m),δ7.87(1H,s)。根据1H-1H COSY谱可知,亚甲基δ3.66与次甲基δ7.87相耦合;两个次甲基δ6.35和δ6.42相耦合;次甲基δ6.82,δ7.04,δ7.12,δ6.94相互耦合,说明有苯环的存在。根据HMBC谱相关峰表明H-5,H-7分别与C-9,C-10相耦合,且H-5,H-7相互耦合,说明C-5与C-7相关联,C-5,C-7分别与C-9,C-10相关联;两个甲氧基中的δH3.83,δH3.93分别与C-6,C-8相耦合,且C-6(δ164.76),C-8(δ161.31)位于低场区,提示与O相连,说明两个甲氧基分别与苯环上的C-6,C-8相连;根据ROESY谱相关峰表明,两个甲氧基中的δH3.83,δH3.93分别与H-5,H-7相耦合,说明C-6与C-5,C-8与C-7相关联;H-5与C-9相耦合,且C-9(δ160.65)位于低场区,提示C-5与C-9相关,且C-9与O相连;同时,H-5,H-7与C-10相关,说明C-5,C-7与C-10相关;H-2与H-11相耦合,且H-2,H-11与C-3相耦合,说明C-2,C-11与C-3相关,同时C-2(δ151.11)位于低场区,提示与O相连,同时,H-2与C-9相耦合,说明C-2,C-9中间与O相连;H-2,H-5,H-11与C-4的羰基C相耦合,说明C-2,C-5,C-11与C-4相关联。H-3',H-4',H-5'和H-6'相互耦合,其中H-3',H-5',H-6'与C-1'相耦合,H-3',H-4',H-5',H-6'与C-2'相耦合,提示有苯环的存在且C-1'与C-2'邻位取代;其中C-2'(δ155.43)处于低场区,提示与O相连,说明C-2'连有一个羟基基团;H-2,H-11与C-1'相耦合,说明C-2,C-11与C-1'相关,H-11与C-2',C-6'相耦合,说明C-11与C-2',C-6'相关,H-6'与C-1'相耦合,说明C-6'与C-1'相关,综上说明C-11与C-1'相连。根据以上信息,可确定此新化合物为上述结构。
表2本发明新化合物3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one的核磁数据
3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one:白色粉末,难溶于甲醇,易溶于氯仿。点样于硅胶薄层板后,喷香草酚绿试液斑点呈淡黄色。HRESI(+)TOFMS给出m/z:315.1224[M+H]+的准分子离子峰,分子量为314.1154。结合1H-NMR,13C-NMR以及DEPT数据,推测该化合物可能的分子式为C18H18O5,不饱和度为10。13C-NMR谱和DEPT谱显示18个碳信号,分别为2个CH3(δ:55.97,56.33)、2个CH2(δ:26.85,70.52)、7个CH(δ:47.80,93.14,93.40,117.76,120.43,128.62,131.04)、7个季碳(一个羰基碳,δ:194.09;四个连O的双键碳,δ:155.12,162.79,165.56,166.70;两个双键碳,δ:105.45,125.17)。
1H-NMR谱显示1个活泼H信号δ9.65(1H,brs),表明可能存在一个羟基基团;2个甲基信号,为δ3.88(6H,s);2个亚甲基信号,分别为δ2.79(1H,q,J=6.6,16.7),δ3.06(1H,m);δ4.17(1H,t,J=11.3),δ4.53(1H,dd,J=4.9,11.2);7个次甲基信号分别为δ3.04(1H,m),δ6.05(2H,q,J=2.3,3.7),δ6.82(1H,t,J=7.1),δ6.94(1H,q,J=8.1,14.1),δ7.11(1H,m),δ7.12(1H,m)。根据1H-1H COSY谱可知,亚甲基中的Hδ4.17,δ4.53,δ2.79,δ3.06分别与次甲基δ3.04相耦合;两个次甲基δ6.05和δ6.05相耦合;次甲基δ6.82,δ6.94,δ7.11,δ7.12相互耦合,说明有苯环的存在。根据HMBC谱相关峰表明H-5,H-7分别与C-9,C-10相耦合,且H-5,H-7相互耦合,说明C-5与C-7相关联,C-5,C-7分别与C-9,C-10相关联;两个甲氧基中的δH3.88(6H,s)与C-6,C-8相耦合,且C-6(δ166.70)和C-8(δ162.79)位于低场区,提示与O相连,说明两个甲氧基分别与苯环上的C-6,C-8相连;根据ROESY谱表明,两个甲氧基中的δH3.88(6H,s)与H-5,H-7相耦合,说明C-6,C-8与C-5,C-7相关联;H-5与C-9相耦合,且C-9(δ160.65)位于低场区,提示C-5与C-9相关,且C-9与O相连;H-2,H-3,H-11相互耦合,且C-2(δ70.52)位于低场区,提示与O相连,同时,H-2与C-9相耦合,说明C-2,C-9中间与O相连;H-2,H-3,H-11与C-4的羰基C相耦合,说明C-2,C-3,C-11与C-4相关联。H-3',H-4',H-5'和H-6'相互耦合,其中H-3',H-5',H-6'与C-1'相耦合,H-3',H-4',H-5',H-6'与C-2'相耦合,提示有苯环的存在且C-1'与C-2'邻位取代;其中C-2'(δ155.12)处于低场区,提示与O相连,说明C-2'连有一个羟基基团;H-2,H-3,H-11与C-1'相耦合,说明C-2,C-3,C-11与C-1'相关,H-11与C-2',C-6'相耦合,说明C-11与C-2',C-6'相关,H-6'与C-1'相耦合,说明与C-1'相关,综上说明C-11与C-1'相连。根据以上信息,可确定此新化合物为上述结构。
本发明还提供上述两种黄酮化合物的提取分离方法,具体步骤为:
步骤1:称取马齿苋干燥药材150kg,采用水回流提取,水用量(v/v)为药材的10倍,回流提取两次,每次2h,加热浓缩,放凉至室温,得药液备用。
步骤2:将步骤1中所得药液蒸干后经硅胶柱层析分离,用乙酸乙酯(115L)等度洗脱,其中硅胶为100-200目,40℃以下减压回收乙酸乙酯至浸膏,得到乙酸乙酯提取物。
步骤3:将步骤2中乙酸乙酯提取物经聚酰胺柱分离,采用乙醇-水(0/100,30/70,50/50,70/30,100/0,v/v)梯度洗脱,50%(体积百分数)乙醇蒸干后经硅胶柱层析分离,其中硅胶为200~300目,依次用乙酸乙酯、乙酸乙酯-甲醇(5/1、2/1、1/2,v/v)梯度洗脱,共得到19个部位(即共得到19个瓶,每瓶300mL),经薄层色谱进行检测,显色,将乙酸乙酯洗脱得到部位合并,并于室温以上,40℃以下减压浓缩至干,备用。
步骤4:将步骤3中所得物再经预处理的ODS中压柱层析分离(Octadecylsilyl,十八烷基硅烷键合硅胶填料),其中填料粒度为20~40μm,用甲醇-水(60/40,70/30,80/20,90/10、100/0,v/v)梯度洗脱(加压,使流速为1mL/min,温度为室温),得到16个部位(即梯度洗脱得16个瓶,每瓶100mL),经薄层色谱进行检测,显色,将显色的3~5部位保留,50℃以下减压浓缩至干,备用。
步骤5:将步骤4中所得物再经预处理的葡聚糖凝胶柱层析分离(Sephadex LH-20),用甲醇洗脱,得到26个洗脱部位(即共得到26个瓶,每瓶50mL),经薄层色谱进行检测,显色,将显色的13、14部位保留,50℃以下减压浓缩至干,备用,得到新化合物。
步骤6:将步骤5中所得物静置,待析出淡黄色晶体,经薄层色谱进行检测、显色,多次用甲醇洗净晶体,洗至甲醇上清液呈无色后蒸干甲醇,最终得到本发明所述的两种新黄酮化合物。
所述ODS与葡聚糖凝胶的预处理过程为甲醇浸泡过24h,上柱,以初始流动相平衡。
本发明新黄酮化合物的抗炎作用实验。
1主要材料
1.1药品和试剂:实验所用两种新黄酮化合物由上述方法制备,纯度为90~99%,精密称取,用DMSO稀释至下述各剂量组所需溶液。DMEM高糖培养基、胎牛血清(美国Hyclone公司);青霉素、链霉素(杭州四季青公司);LPS(美国Sigma公司);IL-6、TNF-α、PGE2的ELISA试剂盒(美国Cayman公司);细胞裂解液、Griess试剂(碧云天生物技术有限公司)。
1.2细胞株:RAW264.7巨噬细胞(美国ATCC细胞库)。
1.3分组:分为对照组、LPS组和实验组,各一组。
2实验方法
2.1细胞培养:DMEM高糖培养基,加入l0%的胎牛血清,l%抗菌素(100U/mL青霉素和100μg/mL链霉素),置于37.5%,CO2培养箱中培养。
2.2MTT比色法测定细胞活力:上述三组分别取对数生长期RAW264.7巨噬细胞接种于96孔培养板中,细胞密度为1×104个/mL,每孔100μL,温度37℃,5%CO2条件下培养过夜后,实验组加入不同浓度的本发明两种新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one(1-100μM)或3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one(1-100μM),孵育1h后向LPS组和实验组分别加入终浓度为1μg/mL的LPS,另设调零组(含DMSO溶媒的培养液),每组设3个复孔,考察加入药物后对细胞的影响。上述各组细胞培养24h后,在各孔细胞中加入5mg/mL MTT20μL,温度37℃,5%CO2条件下继续孵育4h后,终止培养,吸弃孔内液体,每孔加入100μL二甲基亚砜(DMSO),振荡10min,使细胞内结晶充分溶解,酶标仪570nm波长处测定各孔吸光值。
2.3利用格里斯(Griess)法测定NO的含量,考察本发明新黄酮化合物对LPS诱导的小鼠巨噬细胞RAW264.7的NO产生量的抑制作用。小鼠巨噬细胞RAW264.7传代后在含10%胎牛血清的高糖细胞培养基DMEM中培养,实验组加入不同浓度的本发明两种新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one(1-50μM)或3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one(1-50μM),在37℃,5%CO2条件下孵育1h后用LPS(终浓度为1μg/mL)诱导炎症反应,24h后收集上清液,每组处理重复3孔。Griess法测定细胞上清液中NO的含量,根据不同浓度本发明两种新黄酮化合物对LPS诱导的RAW264.7细胞释放NO的影响,用以反映NO水平。
2.4ELISA法测定炎症因子IL-6、TNF-α和炎症介质PGE2:将对数生长期RAW264.7巨噬细胞接种于24孔培养板中,细胞密度为1×105个/mL,每孔1mL,温度37℃,5%CO2条件下培养过夜,实验组加入本发明两种新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one(1-50μM)或3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one(1-50μM),培育1h后,在每孔加入LPS(终浓度为1μg/mL),共孵育24h,每组处理重复3孔。ELISA法测定两种马齿苋来源新黄酮化合物处理后的RAW264.7巨噬细胞分泌的IL-6、TNF-α和PGE2的含量。
3实验结果
实验结果表明本发明两种新黄酮化合物对LPS诱导的巨噬细胞RAW264.7的增殖无影响,安全无毒;并可有效抑制LPS诱导的巨噬细胞RAW264.7所产生过量炎症细胞因子IL-6、TNF-α和炎症介质NO、PGE2,且呈浓度依赖。
细胞相对存活率实验结果如表3所示。
表3本发明对RAW264.7巨噬细胞相对存活率的影响
注:*P<0.05与对照组比较(高浓度组有显著性差异)
利用格里斯(Griess)法测定NO的含量实验结果见表4。
表4本发明对LPS诱导的RAW264.7细胞释放NO的影响(均数±标准差,n=3)
注:*P<0.05与对照组比较,#P<0.05与LPS组比较。
ELISA法测定炎症因子IL-6、TNF-α和炎症介质PGE2结果如表5所示。
表5本发明对LPS诱导的RAW264.7细胞分泌的IL-6、TNF-α和PGE2含量的影响(均数±标准差,n=3)
注:*P<0.05与对照组比较,#P<0.05与LPS组比较。
本发明新黄酮化合物的抗肿瘤作用。
1主要材料
1.1药品和试剂:实验所用两种新黄酮化合物由上述方法制备,纯度为90~99%,精密称取,用DMSO稀释至下述各剂量组所需溶液。DMEM高糖培养基、胎牛血清(美国Hyclone公司);青霉素、链霉素(杭州四季青公司)。
1.2细胞株:人结肠癌细胞Caco-2、人乳腺癌细胞MCF-7、人胃癌细胞BGC-823、人肺腺癌细胞SPC-A1、人肝癌细胞BEL-7402、人***细胞Hela-229、卵巢癌细胞Ho-8910、人类口腔表皮样癌细胞KB(中科院上海细胞库)。
1.3分组:分为对照组、实验组和调零组(含DMSO溶媒的培养液)。
2实验方法
2.1细胞培养:DMEM高糖培养基,加入l0%的胎牛血清,l%抗菌素(100U/mL青霉素和100μg/mL链霉素),置于37℃、5%CO2培养箱中培养。
2.2MTI法检测细胞增殖:取对数生长期细胞接种于96孔培养板中,细胞密度为1×104个/mL,每孔100μL,温度37℃,5%CO2条件下培养过夜后,实验组加入不同浓度的本发明两种新黄酮化合物3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one或3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one,每组设3个复孔,加药后置于37℃,5%CO2培养箱中培养48h。将含药培养液吸去,加入体积比为4:1的无血清培养液和MTT(终质量浓度为5mg/L)共100mL,继续孵育4h,小心吸去上清液后,每孔加入DMSO150μL,放于震荡器上震荡以使结晶完全溶解(5min),酶标仪在570nm波长下检测各孔的吸光度(A)值。然后,计算各浓度化合物对细胞生长的抑制率,再应用SPSS软件处理数据,将抑制率对药物浓度作曲线,计算IC50值。
抑制率公式:细胞生长抑制率=(1-A加药孔/A对照孔)×100%
3实验结果
实验结果表明本发明两种新黄酮化合物对人结肠癌细胞Caco-2、人乳腺癌细胞MCF-7、人胃癌细胞BGC-823、人肺腺癌细胞SPC-A1、人肝癌细胞BEL-7402、人***细胞Hela-229、卵巢癌细胞Ho-8910、人类口腔表皮样癌细胞KB的增殖具有抑制作用,且随药物浓度增大,抑制率也明显升高,即呈浓度依赖。本发明两种新黄酮化合物对上述八种肿瘤细胞IC50值见表6。
表6本发明两种新黄酮化合物对肿瘤细胞的抑制作用
本发明新黄酮化合物的抗氧化作用实验。
1主要材料
1.1药品和试剂:实验所用两种新黄酮化合物由上述方法制备,纯度为90~99%,精密称取,用甲醇稀释至下述各剂量组所需溶液。DPPH(1,1-二苯基-2-苦基肼自由基)(Sigma-Fluka公司);BHA(叔丁基羟茴香醚)(上海祥瑞科技有限公司);甲醇,色谱纯(昌泰兴业有限公司)。
1.2分组:分为对照组、实验组和空白组,各一组。
2实验方法
比色法测定消除DPPH自由基的能力:实验组取1mL DPPH溶液(126.80μM)加入到4mL比色皿中,再加入1mL不同浓度的3-(2-hydroxybenzyl)-6,8-dimethoxy-4H-chromen-4-one(8.32,16.61,33.31,50.02,66.61μM)或3-(2-hydroxybenzyl)-6,8-dimethoxychroman-4-one样品溶液(8.32,16.61,33.31,50.02,66.61μM);对照组取1mL甲醇溶液加入到4mL比色皿中,再加入1mL不同浓度的样品溶液;空白组取1mL DPPH溶液加入到4mL比色皿中,再加入1mL甲醇溶液。三组均充分混匀,室温避光静置10min,517nm下测定吸光值,静置30min后,按同样方法操作。每个样品平均测定三次取平均值,阳性对照为不同浓度的BHA溶液。根据以下公式计算样品对DPPH自由基的清除率,并进一步计算其自由基清除率IC50值。
DPPH清除率(%)=1-(A1-A2)/A0×100%
其中,A0为空白组的吸光度值;A1为样品组的吸光度值;A2为对照组的吸光度值。
3实验结果
实验结果表明本发明两种黄酮新化合物对DPPH自由基均具有清除作用,且随药物浓度增大,清除率也明显升高。本发明两种黄酮新化合物对DPPH自由基IC50值见表7。
表7本发明两种新黄酮化合物对DPPH自由基的清除作用
综上所述,本发明提供两种新黄酮化合物及其提取分离方法,依次采用水煎煮提取、硅胶柱层析、聚酰胺柱层析、硅胶柱层析、ODS中压柱及重结晶进行分离纯化与制备,成功的分离得到两种新化合物。该方法简便、快速、环保,且经该方法分离得到的化合物纯度较高。由于所得化合物化学结构独特,从常用中药马齿苋中提取出来,其具有抗炎、抗肿瘤和抗氧化作用,因此本发明的两种新黄酮化合物及其盐和衍生物可以作为天然产物开发中药新药,具有广阔的前景。
Claims (6)
1.从马齿苋药材中分离出的两种黄酮类化合物提取分离方法,其特征在于,具体步骤为:
步骤1:取马齿苋干燥药材,采用水煎煮提取,放凉至室温,得药液备用;
步骤2:将步骤1中药液蒸干后上硅胶柱,用乙酸乙酯洗脱,减压回收乙酸乙酯至浸膏,得到乙酸乙酯提取物;
步骤3:将步骤2中乙酸乙酯提取物经聚酰胺柱分离,采用乙醇-水梯度洗脱,50%乙醇部分蒸干后上硅胶柱,依次用乙酸乙酯、乙酸乙酯-甲醇梯度洗脱,回收乙酸乙酯至浸膏,得到乙酸乙酯提取物;
步骤4:将步骤3中所得物经预处理的ODS柱层析分离,用甲醇-水梯度洗脱,得到若干洗脱部位,经薄层色谱进行检测,显色,将各显色的洗脱部位分别减压浓缩至干,得到浓缩物备用;
步骤5:将步骤4中所得物再经预处理的葡聚糖凝胶柱层析分离,用甲醇洗脱,得到若干洗脱部位,经薄层色谱进行检测,显色,合并显色的洗脱部位,将合并后的洗脱部位经减压浓缩至干,备用;
步骤6:将步骤5中静置析出晶体,多次用甲醇洗净晶体后挥干甲醇,经薄层色谱进行检测、显色,得到两种黄酮类化合物,其化学结构式分别如下:
2.如权利要求1所述的提取分离方法,其特征在于,所述步骤1中水煎煮提取两次,每次煎煮2小时,水量为药材的8~16倍。
3.如权利要求1所述的提取分离方法,其特征在于,所述步骤2中所用流动相洗脱程序为等度洗脱。
4.如权利要求1所述的提取分离方法,其特征在于,所述步骤3中用水和乙醇的体积比为100/0,70/30,50/50,30/70和0/100梯度洗脱;所述步骤3中所用乙酸乙酯洗脱程序为等度洗脱,乙酸乙酯和甲醇的体积比为5/1,2/1和1/2梯度洗脱;所述步骤4中用甲醇和水的体积比为50/50,60/40,70/30和80/20梯度洗脱。
5.如权利要求1所述的提取分离方法,其特征在于,所述步骤4和步骤5中所用的ODS与葡聚糖凝胶的预处理过程为甲醇浸泡过24小时,上柱,用甲醇洗至滴入水中无混浊,再以初始流动相平衡。
6.如权利要求1所述的提取分离方法,其特征在于,所述步骤5中用甲醇洗脱程序为等度洗脱。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910640045.8A CN110305094B (zh) | 2019-07-16 | 2019-07-16 | 马齿苋中两种黄酮类化合物及其提取分离方法与用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910640045.8A CN110305094B (zh) | 2019-07-16 | 2019-07-16 | 马齿苋中两种黄酮类化合物及其提取分离方法与用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110305094A CN110305094A (zh) | 2019-10-08 |
CN110305094B true CN110305094B (zh) | 2022-06-17 |
Family
ID=68081470
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910640045.8A Active CN110305094B (zh) | 2019-07-16 | 2019-07-16 | 马齿苋中两种黄酮类化合物及其提取分离方法与用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110305094B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115385884B (zh) * | 2022-08-23 | 2023-04-25 | 辽宁中医药大学 | 马齿苋中一种新色酮醇类的提取分离方法及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107698546A (zh) * | 2017-11-28 | 2018-02-16 | 辽宁中医药大学 | 马齿苋中化合物Oleracone D及其提取分离方法 |
CN107746397A (zh) * | 2017-11-28 | 2018-03-02 | 辽宁中医药大学 | 马齿苋中化合物 Oleracone C及其提取分离方法 |
CN108558809A (zh) * | 2018-04-17 | 2018-09-21 | 辽宁中医药大学 | 马齿苋中化合物Oleracone F及其提取分离方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100720151B1 (ko) * | 2005-12-13 | 2007-05-18 | 한국생명공학연구원 | 항바이러스 활성을 갖는 플라보노이드 화합물 |
-
2019
- 2019-07-16 CN CN201910640045.8A patent/CN110305094B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107698546A (zh) * | 2017-11-28 | 2018-02-16 | 辽宁中医药大学 | 马齿苋中化合物Oleracone D及其提取分离方法 |
CN107746397A (zh) * | 2017-11-28 | 2018-03-02 | 辽宁中医药大学 | 马齿苋中化合物 Oleracone C及其提取分离方法 |
CN108558809A (zh) * | 2018-04-17 | 2018-09-21 | 辽宁中医药大学 | 马齿苋中化合物Oleracone F及其提取分离方法 |
Non-Patent Citations (1)
Title |
---|
马齿苋的化学成分与药理作用最新研究进展;解思友等;《现代药物与临床》;20110531;第26卷(第3期);第212-215页 * |
Also Published As
Publication number | Publication date |
---|---|
CN110305094A (zh) | 2019-10-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107459477B (zh) | 一种马齿苋中异吲哚生物碱类化合物及其提取分离方法 | |
CN109897077B (zh) | 马齿苋中化合物Oleraciamide E及其提取分离方法与应用 | |
CN110272342B (zh) | 马齿苋中一种萘酸化合物及其提取分离方法与用途 | |
CN109824568B (zh) | 马齿苋中两种吲哚类新生物碱化合物及其提取分离方法与应用 | |
CN110272369B (zh) | 马齿苋中一种吡咯二羧酸类化合物及其提取分离方法与用途 | |
CN108558809B (zh) | 马齿苋中化合物Oleracone F及其提取分离方法 | |
CN108084060B (zh) | 马齿苋中生物碱oleraurea及其提取分离方法 | |
CN107827726B (zh) | 马齿苋中化合物Oleracone E及其提取分离方法 | |
CN109336747B (zh) | 马齿苋中Oleralignan与其提取分离方法及其应用 | |
CN115716790A (zh) | 马齿苋中一种酰胺酯类生物碱的提取分离方法及其应用 | |
CN113264828B (zh) | 马齿苋中一种苯甲酸类化合物及其提取分离方法 | |
CN112300104B (zh) | 马齿苋中一种木脂素类化合物及其提取分离方法和应用 | |
CN114213473A (zh) | 马齿苋中三种生物碱类化合物及其提取分离方法 | |
CN113321618A (zh) | 马齿苋中三种生物碱类化合物及其提取分离方法 | |
CN110305094B (zh) | 马齿苋中两种黄酮类化合物及其提取分离方法与用途 | |
CN109942481B (zh) | 马齿苋中化合物Oleraisoindole A及其提取分离方法与应用 | |
CN114989084B (zh) | 马齿苋中一种四氢异喹啉类生物碱的提取分离方法及其应用 | |
CN115724812B (zh) | 马齿苋中一种呋喃酯类生物碱的提取分离方法及其应用 | |
CN113968862B (zh) | 马齿苋中两种新生物碱及其提取分离方法 | |
CN114369076B (zh) | 马齿苋中两种茚类化合物及其提取分离方法 | |
CN110194755B (zh) | 马齿苋中化合物Oleracone H及其提取分离方法及其与应用 | |
CN113264829B (zh) | 马齿苋中四种木脂素及其提取分离方法 | |
CN110294733B (zh) | 马齿苋中一种含过氧键化合物Oleracone I及其提取分离方法与应用 | |
CN109824685B (zh) | 马齿苋中化合物oleracone G及其提取分离方法与应用 | |
CN114989064A (zh) | 马齿苋中一种新型吡咯生物碱类化合物及其提取分离方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |