CN113636923A - Method for extracting and separating tsaoko essence from tsaoko amomum fruits - Google Patents
Method for extracting and separating tsaoko essence from tsaoko amomum fruits Download PDFInfo
- Publication number
- CN113636923A CN113636923A CN202111127531.3A CN202111127531A CN113636923A CN 113636923 A CN113636923 A CN 113636923A CN 202111127531 A CN202111127531 A CN 202111127531A CN 113636923 A CN113636923 A CN 113636923A
- Authority
- CN
- China
- Prior art keywords
- tsaoko
- extracting
- ethyl acetate
- separating
- silica gel
- 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.)
- Pending
Links
- 241001127714 Amomum Species 0.000 title claims abstract description 54
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 25
- 150000001875 compounds Chemical class 0.000 claims abstract description 58
- 238000000605 extraction Methods 0.000 claims abstract description 29
- 239000000284 extract Substances 0.000 claims abstract description 26
- 239000000463 material Substances 0.000 claims abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 49
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 31
- 239000000741 silica gel Substances 0.000 claims description 30
- 229910002027 silica gel Inorganic materials 0.000 claims description 30
- 239000002024 ethyl acetate extract Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 238000002156 mixing Methods 0.000 claims description 18
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 14
- 238000010992 reflux Methods 0.000 claims description 14
- 238000012856 packing Methods 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 12
- 238000010898 silica gel chromatography Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 238000007664 blowing Methods 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 7
- 238000011068 loading method Methods 0.000 claims description 7
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000004809 thin layer chromatography Methods 0.000 claims description 7
- 238000007865 diluting Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 239000000178 monomer Substances 0.000 abstract description 36
- 230000003078 antioxidant effect Effects 0.000 abstract description 30
- 239000003963 antioxidant agent Substances 0.000 abstract description 11
- 238000001228 spectrum Methods 0.000 abstract description 11
- 238000011161 development Methods 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 39
- 239000000523 sample Substances 0.000 description 31
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 24
- 235000010354 butylated hydroxytoluene Nutrition 0.000 description 24
- -1 DPPH radicals Chemical class 0.000 description 22
- 239000012224 working solution Substances 0.000 description 20
- 239000012071 phase Substances 0.000 description 17
- 238000012360 testing method Methods 0.000 description 17
- 235000019441 ethanol Nutrition 0.000 description 16
- 239000003814 drug Substances 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- 229940079593 drug Drugs 0.000 description 13
- 230000002000 scavenging effect Effects 0.000 description 12
- 239000012153 distilled water Substances 0.000 description 11
- 238000011160 research Methods 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 238000005481 NMR spectroscopy Methods 0.000 description 10
- 235000006708 antioxidants Nutrition 0.000 description 10
- 230000000875 corresponding effect Effects 0.000 description 9
- 230000007760 free radical scavenging Effects 0.000 description 9
- 238000001819 mass spectrum Methods 0.000 description 9
- ZTOJFFHGPLIVKC-YAFCTCPESA-N (2e)-3-ethyl-2-[(z)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound S\1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C/1=N/N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-YAFCTCPESA-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 8
- 150000003254 radicals Chemical class 0.000 description 8
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 7
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- 241001346334 Amomum tsao-ko Species 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 6
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 6
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 229910001447 ferric ion Inorganic materials 0.000 description 6
- 230000003647 oxidation Effects 0.000 description 6
- 238000007254 oxidation reaction Methods 0.000 description 6
- 235000013824 polyphenols Nutrition 0.000 description 6
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 230000008030 elimination Effects 0.000 description 5
- 238000003379 elimination reaction Methods 0.000 description 5
- 229910001448 ferrous ion Inorganic materials 0.000 description 5
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 5
- 150000008442 polyphenolic compounds Chemical class 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 230000002292 Radical scavenging effect Effects 0.000 description 4
- 239000013543 active substance Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 229930003935 flavonoid Natural products 0.000 description 4
- 150000002215 flavonoids Chemical class 0.000 description 4
- 235000017173 flavonoids Nutrition 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000003208 petroleum Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- 238000002211 ultraviolet spectrum Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical compound CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000009920 chelation Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000006837 decompression Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- HEILIGJNYTWOHU-UHFFFAOYSA-N ethanol 2-hydroxybenzoic acid Chemical compound CCO.OC(=O)C1=CC=CC=C1O HEILIGJNYTWOHU-UHFFFAOYSA-N 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 241000411851 herbal medicine Species 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000002798 polar solvent Substances 0.000 description 2
- 229940079877 pyrogallol Drugs 0.000 description 2
- 235000013599 spices Nutrition 0.000 description 2
- 150000003505 terpenes Chemical class 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 description 1
- BCHZICNRHXRCHY-UHFFFAOYSA-N 2h-oxazine Chemical compound N1OC=CC=C1 BCHZICNRHXRCHY-UHFFFAOYSA-N 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 240000008213 Brosimum alicastrum Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 240000000425 Chaenomeles speciosa Species 0.000 description 1
- 235000005078 Chaenomeles speciosa Nutrition 0.000 description 1
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 description 1
- 241000132446 Inula Species 0.000 description 1
- 229930183024 Kukoamine Natural products 0.000 description 1
- 244000297531 Lespedeza cuneata Species 0.000 description 1
- RINSJDWDXSWUOK-UHFFFAOYSA-N OC(=O)C1=CC=C(O)C(O)=C1.OC(=O)C1=CC=C(O)C(O)=C1 Chemical group OC(=O)C1=CC=C(O)C(O)=C1.OC(=O)C1=CC=C(O)C(O)=C1 RINSJDWDXSWUOK-UHFFFAOYSA-N 0.000 description 1
- 241001126796 Sphacelaria Species 0.000 description 1
- 241000234299 Zingiberaceae Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
- 150000004056 anthraquinones Chemical class 0.000 description 1
- 230000002391 anti-complement effect Effects 0.000 description 1
- 108010008730 anticomplement Proteins 0.000 description 1
- 238000002792 antioxidant assay Methods 0.000 description 1
- 239000012223 aqueous fraction Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 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
- 230000004071 biological effect Effects 0.000 description 1
- 239000002034 butanolic fraction Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 229940097217 cardiac glycoside Drugs 0.000 description 1
- 239000002368 cardiac glycoside Substances 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 150000004775 coumarins Chemical class 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000013461 design 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
- 235000011869 dried fruits Nutrition 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 description 1
- 239000002038 ethyl acetate fraction Substances 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229940068517 fruit extracts Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 235000005828 ramon Nutrition 0.000 description 1
- 239000012488 sample solution Substances 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
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 229930002534 steroid glycoside Natural products 0.000 description 1
- 150000008143 steroidal glycosides Chemical class 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for extracting and separating tsaoko amomum fruit extract, which can realize the extraction of antioxidant active monomer compounds in tsaoko amomum fruit medicinal materials, carry out structure identification on the active monomer compounds through modern spectrum technologies such as MS, NMR and the like and spectrum technologies, and confirm that the tsaoko amomum fruit extract is the tsaoko amomum fruit extract, thereby providing a material basis for researching the antioxidant activity of the tsaoko amomum fruit extract and being beneficial to the further comprehensive development and utilization of the tsaoko amomum fruit.
Description
Technical Field
The invention relates to a method for extracting and separating tsaoko amomum fruit extract, in particular to a method for extracting and separating antioxidant active substance tsaoko amomum fruit extract from tsaoko amomum fruit, belonging to the technical field of Chinese herbal medicine extraction.
Background
Tsaoko (academic name:Amomum tsaoko Crevost et Lemarie) Is a perennial herb of the genus Amomum of the family Zingiberaceae, which is mainly distributed in provinces such as Yunnan, Guangxi and Guizhou provinces of China. The tsaoko amomum fruit is one of a large variety of medicinal materials used as both medicine and food, is mainly used as spice in the field of food processing, and is also applied in the fields of traditional Chinese medicine and other fields. At present, with the continuous rise of the development of natural products, the tsaoko amomum fruit is taken as an important medicine-food dual-purpose plant, and the development and the utilization of the natural products of the tsaoko amomum fruit are further increased due to the wide biological activity and pharmacological action.
Existing studies have shown that extracts of tsaoko amomum have certain antioxidant activity, such as: inula lapidescens et al in research on antioxidant activity of fructus Tsaoko extract with different polar solvents (proceedings of medical institute of Youjiang Ming nationality, 2021, 2 months, 43 vol. 1, 37-40) by extracting fructus Tsaoko with ethanol to obtain extract, diluting, sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction, and measuring ABTS of fructus Tsaoko extract by ultraviolet spectrophotometry+Free radical, hydroxyl radical and Fe2+The chelating scavenging ability shows that the tsaoko amomum fruit extracts with different polar solvents have good antioxidant activity. However, the prior researches do not clarify the material basis of the antioxidant activity, so that the antioxidant activity of the tsaoko amomum fruit is further improvedThe comprehensive development and utilization of the antioxidant monomeric compound in the tsaoko amomum fruit extract are key to realize the extraction, separation and purification of the antioxidant monomeric compound.
Disclosure of Invention
The invention aims to provide a method for extracting and separating the tsaoko element from the tsaoko amomum fruits, which can realize the extraction of antioxidant active monomer compounds in the medicinal material of the tsaoko amomum fruits, and carry out structure identification on the active monomer compounds through modern spectrum technologies such as MS, NMR and the like and spectrum technologies to confirm the compounds as the tsaoko element, thereby providing a material basis for researching the antioxidant activity of water extracts of the tsaoko amomum fruits and being beneficial to the further comprehensive development and utilization of the tsaoko amomum fruits.
The invention is realized by the following technical scheme: a method for extracting and separating the tsaoko essence from the tsaoko amomum fruit comprises the following steps:
A. crushing a tsaoko medicinal material serving as a raw material, performing reflux extraction by using ethanol, and performing reduced pressure concentration to obtain a concentrated solution;
B. diluting the concentrated solution, extracting with ethyl acetate to obtain ethyl acetate extract, and concentrating to obtain ethyl acetate extract;
C. and taking the ethyl acetate extract, and performing silica gel column chromatography separation and C18 column reverse separation to obtain the tsaoko amomum fruit element.
In the step A, ethanol with the mass concentration of 95% is used for reflux extraction for 2-3 times, and the reflux extraction time is controlled to be 1.5-2 h each time.
And in the step A, concentrating the extracting solution obtained by reflux extraction at 50-55 ℃ under reduced pressure until no alcohol exists, and obtaining the concentrated solution.
And in the step B, diluting the concentrated solution by 2-3 times with water, and extracting the same volume of ethyl acetate for 2 times to obtain an ethyl acetate extract.
And in the step B, concentrating the ethyl acetate extract at 45 ℃ under reduced pressure to obtain an ethyl acetate extract.
And C, mixing the ethyl acetate extract with silica gel with the mass of 1-2 times of that of the ethyl acetate extract, blowing and drying the mixed sample at 40 ℃ to constant weight to obtain a dried silica gel mixed sample, and then carrying out silica gel column chromatography separation.
And in the step C, carrying out normal phase silica gel column chromatography on the silica gel mixed sample, carrying out wet column packing by using 10-20 times of silica gel as a filler, carrying out dry loading, carrying out normal pressure elution by using dichloromethane-methanol as an eluent with the volume ratio of 100: 0-30: 1, collecting by tubes, carrying out about 100mL per tube, carrying out thin layer chromatography analysis, carrying out detection on dark spot results by using dichloromethane-methanol with the volume ratio of 20: 1 as a developing agent under 254nm ultraviolet light, combining collected liquids containing target compounds, and concentrating under reduced pressure at 45 ℃ to obtain an extract.
In the step C, when the C18 column is reversely separated, methanol-water with the volume ratio of 50: 50 is used as a mobile phase, and the ultraviolet light is 254 nm.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention provides a method for extracting antioxidant active monomer compounds from tsaoko amomum fruits, which comprises the steps of extracting concentrated solution by ethanol reflux extraction, extracting ethyl acetate to obtain ethyl acetate extracts of antioxidant active substances of the tsaoko amomum fruits, and separating and purifying to obtain the active monomer compounds.
(2) The invention provides a material basis for researching the antioxidant activity of the tsaoko amomum fruit extract, and the active monomer compound extracted by the invention is subjected to structure identification by modern spectrum technologies such as MS, NMR and the like and spectrum technologies, so that the active monomer compound is determined to be the tsaoko amomum fruit element.
(3) The invention lays a foundation for further realizing the comprehensive development and utilization of tsaoko, and when the antioxidant activity experiment is carried out on the active monomer compound, namely, the tsaoko element extracted by the invention, the antioxidant activity experiment is carried out by sequentially carrying out DPPH free radical scavenging experiment, ABTS free radical scavenging experiment, superoxide anion free radical scavenging experiment, hydroxyl free radical scavenging experiment, ferrous ion chelation experiment and ferric ion reduction experiment, the tsaoko element has certain scavenging capacity on the free radicals and the ferric ions, so that the tsaoko element has certain antioxidant capacity, the antioxidant activity of the tsaoko element is related to the structure of the monomer compound, and the antioxidant action mechanism is mainly to avoid oxidative damage by scavenging the free radicals.
In conclusion, the invention provides a method for extracting an active monomer compound from tsaoko amomum fruits for the first time on the basis of the existing research on the antioxidant activity of the tsaoko amomum fruits, and identifies and experimentally verifies the extracted active monomer compound, thereby fully proving the antioxidant property of the antioxidant active substance of the tsaoko amomum fruits and providing a research basis for further comprehensive development and utilization of the tsaoko amomum fruits.
Drawings
FIG. 1 is a UV spectrum of a monomer compound I.
FIG. 2 is a mass spectrum of monomer compound I.
FIG. 3 is a graph showing the scavenging ability of monomeric compound I on DPPH radicals.
FIG. 4 is a graph showing the scavenging ability of monomeric compound I against ABTS radicals.
FIG. 5 is a graph showing the scavenging ability of the monomeric compound I for superoxide anion radicals.
FIG. 6 is a graph showing the scavenging ability of monomeric compound I for hydroxyl radicals.
FIG. 7 is a graph showing the ability of monomeric compound I to chelate ferrous ions.
FIG. 8 shows the monomer compounds I vs. Fe3+Is reduced.
Detailed Description
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is to be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention claimed, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The qualitative research of chemical components in the amomum tsao-ko of liu xiao ling and the like is carried out in the qualitative research of chemical components in the amomum tsao-ko of spice (Chinese seasoning, 2011, No. 1, volume 36, 104 and 106) through extraction methods of water extraction, ethanol extraction, petroleum ether extraction and the like, and the comprehensive qualitative research shows that the amomum tsao-ko contains various chemical components such as saccharides, proteins, amino acids, phenols, tannins, organic acids, saponins, flavones, anthraquinone, coumarins, lactones, cardiac glycosides, steroids, terpenes, volatile oil, grease, anthocyanin and the like. It can be known that tsaoko has rich chemical components, and the research on the antioxidant activity of the existing tsaoko extract is generally carried out on a certain type of extract in the tsaoko, but the material basis of the antioxidant activity of the tsaoko is not clarified in the existing research. For example, in the research of extraction of the total flavonoids of tsaoko amomum fruits and DPPH free radical scavenging activity, Yuan and the like, an ultrasonic-assisted method is adopted to extract the total flavonoids of tsaoko amomum fruits, and experiments prove that the DPPH free scavenging rate of the total flavonoids of tsaoko amomum fruits is 80.5%. For example, in the extraction of tsaoko polyphenol substances and LC-MS/MS analysis (294-plus 334 in 2017, the scientific and technical section of food industry), the extraction of polyphenol substances from oil-extracted tsaoko powder is carried out by Lespedeza cuneata, and tests prove that the tsaoko polyphenol has certain DPPH and ABTS free radical clear capability, and the concentration of the polyphenol is positively correlated with the antioxidant activity of the polyphenol.
Therefore, there is a need to design a new method for extracting antioxidant active monomeric compounds from tsaoko amomum fruits, which not only can separate and obtain the required antioxidant active substances, but also can determine the substance basis of the antioxidant activity of the tsaoko amomum fruits through structural identification, thereby laying the foundation for further comprehensive development and utilization of the tsaoko amomum fruits.
The following examples are provided to illustrate specific embodiments of the present invention, and it is understood that the scope of the present invention is not limited to the following examples. The percentages referred to in the following examples are in parts by mass, and the instruments, drugs and reagents used are as follows:
the instrument comprises the following steps:
BC-R501CA type 5L rotary evaporator set (Shanghai Bikai Biochemical Co., Ltd.); BC-R2012B model 20L rotary evaporator set (Shanghai Bikai Biochemical Co., Ltd.); DLSB-30/130 type cryogenic cooling circulation pump (Zhengzhou Jinyun Kongshu Co., Ltd.); BK-1000B ultrasonic cleaning machine (Jinnan Bake ultrasonic science and technology Co., Ltd.); an LPG-5 type centrifugal spray dryer (machinery equipment ltd, kawa); model VFD-1000 VDF freeze dryer (beijing bo doctor kang laboratory instruments ltd); BIOBASE BK-FD10P model lyophilizer (Boke Biochemical industries, Inc., Shandong); HC-5002S ultrasonic instrument (Kunshan Hui super automation equipment finite formula); X85-2S type constant temperature magnetic stirrer (shanghai meipu instruments manufacturing company); HH-Z4 model digital display constant temperature water bath (east Guangmong instrument factory, gold Tan city); 100 x 200mm silica gel thin layer plates (Qingdao sea silica gel desiccant, Inc.); 200-300 mesh silica gel (Qingdao sea silica gel desiccant Co., Ltd.); waters model 2695 preparative liquid chromatographs (Waters); analytical liquid chromatograph model agilent 1100 (agilent); preparing a liquid-phase static compression column 50MM (Happy New Yonghou district Ramon pharmaceutical equipment factory); 2500mL filter flask (Chengdu Vibration Source glass Instrument Co., Ltd.).
Drugs and reagents:
the fructus Tsaoko is purchased from Yulin city of Guangxi, and is identified as fructus Tsaoko (Amomum tsao-ko ) of ZingiberaceaeA. tsao- ko Crevost et Lemarie) The dried fruit of (1). Pulverizing into coarse powder, and storing under ventilation drying condition. Dichloromethane, acetonitrile, 95% ethanol, methanol, absolute ethanol, phosphoric acid, n-butanol (all from Dourbaton Chemicals, Inc. AR); 2, 6-di-tert-butyl-4-methylphenol (BHT, Shanghai Crystal pure reagent Co., Ltd., premium grade purity); DPPH, ABTS (Shanghai gold ear Biotech Co., Ltd. AR); FeCl3·6H2O (Shanghai Productivity chemical Co., Ltd.).
Example 1: extracting and separating tsaoko essence from tsaoko amomum fruit
38kg of tsaoko medicinal material is selected for crushing, 95% ethanol is used for reflux extraction for 3 times, each time is 2 hours, the concentrated solution is obtained after being concentrated under reduced pressure at 55 ℃ until no alcohol exists, water is added for dilution by 3 times, the equal volume of ethyl acetate is used for extraction for 2 times, the extracted ethyl acetate extract is concentrated under reduced pressure at 45 ℃ to obtain ethyl acetate extract, silica gel with the mass being 2 times of the extract is used for sample mixing, and the sample mixing is dried by blowing at 40 ℃ to have constant weight, so that the dried silica gel mixed sample is obtained. Mixing silica gel with a sample, performing normal phase silica gel column chromatography with 10 times of silica gel as filler, performing wet column packing, performing dry loading, performing normal pressure elution with dichloromethane-methanol as eluent at a volume ratio of 30: 1, collecting by tubes, performing thin layer chromatography with dichloromethane-methanol at a volume ratio of 20: 1 as developing agent, detecting dark spot under 254nm ultraviolet light, combining collected liquids containing target compounds, and concentrating under reduced pressure at 45 deg.C to obtain extract. Then methanol-water (50: 50) is used as a mobile phase, lambda =254nm, C18 reversed phase chromatographic packing is used for high pressure preparation and separation to obtain a target component, and the target component is concentrated and dried at 45 ℃ and is dried under reduced pressure at 40 ℃ to obtain a monomer compound I (200 mg), wherein the yield is 0.53% (mg/g).
Example 2: extracting and separating tsaoko essence from tsaoko amomum fruit
38kg of tsaoko medicinal material is selected for crushing, 95% ethanol is used for reflux extraction for 3 times, each time is 1.5h, the concentrated solution is obtained by vacuum concentration at 55 ℃ until no alcohol exists, water is added for dilution by 2 times, the equal volume of ethyl acetate is used for extraction for 2 times, the ethyl acetate extract obtained by extraction is vacuum concentrated at 45 ℃ to obtain an ethyl acetate extract, equal mass of silica gel is used for sample mixing, and the sample mixing is dried by blowing at 40 ℃ to constant weight, so that the dry silica gel mixed sample is obtained. Mixing silica gel with a sample, performing normal phase silica gel column chromatography with 20 times of silica gel as filler, performing wet column packing, performing dry loading, performing normal pressure elution with dichloromethane-methanol as eluent at a volume ratio of 50: 1, collecting by tubes, performing thin layer chromatography with dichloromethane-methanol at a volume ratio of 20: 1 as developing agent, detecting dark spot under 254nm ultraviolet light, combining collected liquids containing target compounds, and concentrating under reduced pressure at 45 deg.C to obtain extract. Then methanol-water (50: 50) is used as a mobile phase, lambda =254nm, C18 reversed phase chromatographic packing is used for high pressure preparation and separation to obtain a target component, and the target component is concentrated and dried at 45 ℃ and is dried under reduced pressure at 40 ℃ to obtain a monomer compound II (210 mg), wherein the yield is 0.55% (mg/g).
Example 3: extracting and separating tsaoko essence from tsaoko amomum fruit
38kg of tsaoko medicinal material is selected for crushing, 95% ethanol is used for reflux extraction for 2 times, each time is 1.5 hours, the concentrated solution is obtained after decompression concentration at 50 ℃ until no alcohol exists, water is added for dilution by 2 times, equal volume of ethyl acetate is used for extraction for 2 times, the extracted ethyl acetate extract is decompressed and concentrated at 45 ℃ to obtain an ethyl acetate extract, silica gel with the mass of 2 times of the extract is used for sample mixing, and the sample mixing is dried by blowing at 40 ℃ to constant weight, so that the dried silica gel mixed sample is obtained. Mixing silica gel with a sample, performing normal phase silica gel column chromatography with 15 times of silica gel as filler, performing wet column packing, performing dry loading, performing normal pressure elution with dichloromethane-methanol as eluent at a volume ratio of 30: 1, collecting by tubes, performing thin layer chromatography with dichloromethane-methanol at a volume ratio of 20: 1 as developing agent, detecting dark spot under 254nm ultraviolet light, combining collected liquids containing target compounds, and concentrating under reduced pressure at 45 deg.C to obtain extract. Then methanol-water (50: 50) is used as a mobile phase, lambda =254nm, C18 reversed phase chromatographic packing is used for high pressure preparation and separation to obtain a target component, and the target component is concentrated and dried at 45 ℃ and is dried under reduced pressure at 40 ℃ to obtain a monomer compound III (198 mg), wherein the yield is 0.52% (mg/g).
Example 4: extracting and separating tsaoko essence from tsaoko amomum fruit
38kg of tsaoko medicinal material is selected for crushing, 95% ethanol is used for reflux extraction for 3 times, each time is 1.5 hours, the concentrated solution is obtained after decompression concentration at 50 ℃ until no alcohol exists, water is added for dilution by 3 times, equal volume of ethyl acetate is used for extraction for 2 times, the extracted ethyl acetate extract is decompressed and concentrated at 45 ℃ to obtain an ethyl acetate extract, silica gel with the mass of 2 times of the extract is used for sample mixing, and the sample mixing is dried by blowing at 40 ℃ to constant weight, so that the dried silica gel mixed sample is obtained. Mixing silica gel with a sample, performing normal phase silica gel column chromatography with 20 times of silica gel as filler, performing wet column packing, performing dry loading, performing normal pressure elution with dichloromethane-methanol as eluent at a volume ratio of 30: 1, collecting by tubes, performing thin layer chromatography with dichloromethane-methanol at a volume ratio of 20: 1 as developing agent, detecting dark spot under 254nm ultraviolet light, combining collected liquids containing target compounds, and concentrating under reduced pressure at 45 deg.C to obtain extract. And separating with methanol-water (50: 50) as mobile phase and lambda =254nm C18 reversed phase chromatographic packing under high pressure to obtain target component, concentrating and drying at 45 deg.C, and drying under reduced pressure at 40 deg.C to obtain a monomer compound IV (202 mg) with yield of 0.53% (mg/g).
Example 5: extracting and separating tsaoko essence from tsaoko amomum fruit
38kg of tsaoko medicinal material is selected for crushing, 95% ethanol is used for reflux extraction for 2 times, each time is 2 hours, the concentrated solution is obtained after being concentrated under the reduced pressure at 55 ℃ until no alcohol exists, water is added for dilution by 2 times, the equal volume of ethyl acetate is used for extraction for 2 times, the extracted ethyl acetate extract is concentrated under the reduced pressure at 45 ℃ to obtain an ethyl acetate extract, the sample is mixed with equal mass of silica gel, and the sample is dried by blowing at 40 ℃ to constant weight to obtain a dried silica gel mixed sample. Mixing silica gel with a sample, performing normal phase silica gel column chromatography with 15 times of silica gel as filler, performing wet column packing, performing dry loading, performing normal pressure elution with dichloromethane-methanol as eluent at a volume ratio of 30: 1, collecting by tubes, performing thin layer chromatography with dichloromethane-methanol at a volume ratio of 20: 1 as developing agent, detecting dark spot under 254nm ultraviolet light, combining collected liquids containing target compounds, and concentrating under reduced pressure at 45 deg.C to obtain extract. Then methanol-water (50: 50) is used as a mobile phase, lambda =254nm, C18 reversed phase chromatographic packing is used for high pressure preparation and separation to obtain a target component, and the target component is concentrated and dried at 45 ℃ and is dried under reduced pressure at 40 ℃ to obtain a monomer compound V (201 mg), wherein the yield is 0.53% (mg/g).
Example 6: structural identification of monomeric Compound I
Subjecting the monomer compound I obtained above to ultraviolet spectroscopy (UV) and nuclear magnetic resonance (1H-NMR and13C-NMR), Mass Spectrum (MS), etc.
(1) Ultraviolet spectrum
The uv absorption curve of the monomeric compound shows a maximum absorption peak at a wavelength of 234.3nm, indicating the possible presence of two conjugated double bonds, and a low intensity absorption peak (n → pi transition) at a wavelength of 306.8nm, indicating the possible presence of a carbonyl group. See fig. 1.
(2) Nuclear magnetic resonance hydrogen spectrum (1H NMR):
the signals of the nuclear magnetic resonance hydrogen spectrum of the compound are assigned as follows:1H NMR（DMSO-d6, 400 MHz）δ： 9.37（1H, s, H-10），6.88- 6.75（1H, m, H-3），3.79（1H, dt, J=9.2, 4.7Hz, H-5），2.75-2.94（1H, m, H-1），2.39（1H, dt, J=18.7, 5.2Hz, Hβ -4），2.29（1H, dt, J=18.7, 3.2Hz H-6），2.21-2.31（1H, m, Hα -4），1.90（1H, ddd, J=13.2, 10.3, 6.6Hz, Hβ -9），1.68-1.56（1H, m, H-7a）1.54-1.43 (1H, m, H-7 b), 1.43-1.37 (2H, m, H-8), 1.32 (1H, dt, J =12.9, 7.0Hz, H-9). See table 1 below.
TABLE 1 assignment of signals in NMR spectra (1H NMR）
(3) Nuclear magnetic resonance carbon spectrum (13C NMR）：
The nuclear magnetic resonance carbon spectrum signals of the compound are assigned as follows:13C NMR（DMSO-d 6100 MHz) δ: a total of 10 carbon signals occurred at 37.06 (C-1), 144.29 (C-2), 149.22 (C-3), 31.5 (C-4), 66.71 (C-5), 42.82 (C-6), 25.28 (C-7), 24.7 (C-8), 32.2 (C-9), 194.67 (C-10). See table 2 below.
TABLE 2 signal assignment of NMR carbon spectra (13C NMR）
(4) Mass spectrum:
excimer ion peak given in Mass Spectrum ESI + [2M + Li +]+: m/z 339.34. The compound molecular weight M =166.22 is indicated. See fig. 2.
(4) Structural formula (xvi):
in summary, according to NMR1H NMR、13C NMR) and mass spectrum, and comparing with the existing literature (Wangjiali et al, "separation and structure identification of peanut coat chemical components", Tianjin Chinese medicine university Proc., 2019, 38(2): 175-plus 179), determining that the structure is protocatechuic acid (3, 4-dihydroxy benzoic acid), belonging to phenolic acid compounds. White powder with molecular formula C7H6O4And the molecular weight is 154.12.
From the above, according to the ultraviolet spectrum, nuclear magnetic resonance (1H NMR、13C NMR) and mass spectrum, and then compared with the existing literature (Kinghong et al, "anti-complement active ingredients of four medicinal plants"; "FudanUniversity, 2011: 103-. Light yellow liquid with molecular formula of C10H12O2And the molecular weight is 166.22. The specific structural formula is shown as the following formula (1).
Example 7: in vitro antioxidant Activity assay of monomeric Compound I
Taking the monomer compound I and a reference BHT, and preparing the concentrations of the monomer compound I and the reference BHT: 5. 10, 20, 30 and 40 mu g/mL of the working solution series, and storing at low temperature of 4 ℃ for later use.
(1) Determination of DPPH radical scavenging Capacity
The monomer compound I and the BHT working solution are respectively taken to carry out a DPPH free radical scavenging test. Monomer compound and BHT all set up 11 and have stopper colour comparison tube, compile as 0 ~ 10 No.: adding 2mL of 0.1 mmol/L DPPH working solution and 2mL of absolute ethyl alcohol into a No. 0 colorimetric tube to serve as a blank tube; respectively adding 2mL of 0.1 mmol/L DPPH working solution and 2mL of series working solutions (5, 10, 20, 30 and 40 mu g/mL) of corresponding medicines into a No. 1-5 colorimetric tube to serve as test tubes; 2mL of absolute ethyl alcohol and 2mL of serial working solutions (5, 10, 20, 30 and 40 mu g/mL) of corresponding medicines are respectively added into a No. 6-10 tube to serve as a control tube. After mixing the colorimetric tubes uniformly, the reaction is carried out for 30 min in water bath at 30 ℃ in the dark. Absolute ethyl alcohol is used for zero setting, an ultraviolet-visible spectrophotometer is adopted to measure the absorbance (A) value of each reaction solution at the wavelength of 517 nm, and the DPPH free radical clearance rate is calculated according to the formula: DPPH free radical clearance (%) [ 1- (assay-a control)/a blank ] × 100%.
Blank A, 2mL of DPPH working solution and 2mL of absolute ethyl alcohol;
in the A test, 2mL of DPPH working solution and 2mL of series working solutions of corresponding medicines are added;
control A is a series of working solutions of 2mL of absolute ethyl alcohol and 2mL of corresponding drugs.
As shown in fig. 3, the control BHT and the tsaoko element gradually increase in the removal capacity of DPPH radicals with increasing sample concentration, and the control BHT has a significantly higher removal capacity of DPPH radicals than the tsaoko element.
(2) Determination of ABTS free radical scavenging Capacity
And taking the monomer compound I and the BHT working solution to respectively carry out a superoxide anion free radical scavenging test. Monomer compound and BHT all set up 11 and have stopper colour comparison tube, compile as 0 ~ 10 No.: adding 6 mL of ABTS working solution and 0.6 mL of absolute ethyl alcohol into a No. 0 colorimetric tube as a blank tube, and adding 0.6 mL of each of 6 mL of ABTS working solution and series working solutions (5, 10, 20, 30 and 40 mu g/mL) of corresponding medicines into a No. 1-5 colorimetric tube as a test tube; 6 mL of absolute ethyl alcohol and 0.6 mL of a series of working solutions (5, 10, 20, 30 and 40 mu g/mL) of corresponding medicines are added into a No. 6-10 colorimetric tube to serve as control tubes. After mixing the colorimetric tubes uniformly, reacting for 1h in a dark place at room temperature, zeroing with absolute ethyl alcohol, measuring the absorbance (A) value of each reaction solution at the wavelength of 734 nm by using an ultraviolet-visible spectrophotometer, and calculating the ABTS free radical clearance according to the formula: ABTS free radical clearance (%) [ 1- (a trial-a control)/a blank ] × 100%.
Blank A: 6 mL of ABTS working solution and 0.6 mL of absolute ethyl alcohol;
a test: 6 mL ABTS working solution +0.6 mL serial working solution of corresponding medicine;
control A: 6 mL of absolute ethyl alcohol and 0.6 mL of a series of working solutions of corresponding medicines.
As shown in FIG. 4, as the concentration of the sample increases, the scavenging ability of the Amoxicillin to ABTS free radicals is not obvious, the increasing trend shows a leveling phenomenon, and the control BHT has a stronger scavenging ability to ABTS free radicals.
(3) Determination of superoxide anion radical scavenging ability
And taking the monomer compound I and the BHT working solution to respectively carry out a superoxide anion free radical scavenging test. Monomer compound and BHT all set up 11 and have stopper colour comparison tube, compile as 0 ~ 10 No.: adding 4 mL of 1 mol/L Tris-HCl buffer solution (pH 7.4), 0.4 mL of 2.5mmol/L pyrogallol solution and 1 mL of absolute ethyl alcohol into a No. 0 colorimetric tube to serve as a blank tube; adding 4 mL of 1 mol/L Tris-HCl buffer solution, 0.4 mL of 2.5mmol/L pyrogallol solution and 1 mL of series working solutions (5, 10, 20, 30 and 40 mu g/mL) of corresponding medicines into a No. 1-5 colorimetric tube to serve as test tubes; a6-10 colorimetric tube is added with 4 mL of 1 mol/L Tris-HCl buffer solution, 0.4 mL of distilled water and 1 mL of series working solutions (5, 10, 20, 30 and 40 mu g/mL) of corresponding medicines respectively to serve as a control tube. After mixing the color tubes, the reaction was carried out at room temperature for 5 min, and then 1 mL of 8 mmol/L HCl solution was added to terminate the reaction. And (3) carrying out zero setting by using distilled water, measuring the A value of each reaction solution at the wavelength of 320 nm by using an ultraviolet-visible spectrophotometer, and calculating the superoxide anion removal rate according to a formula: superoxide anion clearance (%) [ 1- (a test-a control)/a blank ] × 100%.
Blank A: absorbance values of 4 mL Tris-HCl +1 mL absolute ethanol + 0.4 mL pyrogallol +1 mL 8 mmol/L HCl;
a test: 4 mL Tris-HCl +1 mL serial working solution of corresponding drug + 0.4 mL pyrogallol +1 mL absorbance value of 8 mmol/L HCl;
control A: absorbance values of 4 mL Tris-HCl +1 mL of the corresponding drug's serial working solution + 0.4 mL distilled water +1 mL 8 mmol/L HCl.
As shown in FIG. 5, the scavenging ability of the tsaoko element and BHT for superoxide anions was less significantly changed with the change of the concentration in the set concentration range, but the scavenging ability of the tsaoko element for superoxide anion radicals was significantly stronger than that of the control BHT.
(4) Determination of hydroxyl radical scavenging Capacity
And taking the monomer compound I and the BHT working solution to perform a hydroxyl radical scavenging test respectively. Monomer compound and BHT all set up 6 and have stopper colour comparison tube, compile as No. 0 ~ 5: adding 1 mL of FeSO into a No. 0 colorimetric tube4Solution, 1 mL salicylic acid-ethanol solution, 1 mL distilled water, 1 mL H2O2Adding 1 mL of FeSO into the solution as a blank tube and a No. 1-5 colorimetric tube4Solution, 1 mL salicylic acid-ethanol solution, 1 mL H2O2The solution and the corresponding drug working solution (5, 10, 20, 30, 40 μ g/mL) are each 1 mL, and are used as test tubes, and are adjusted to zero with distilled water, and purple is usedThe A value of each reaction solution was measured at a wavelength of 510 nm by an external-visible spectrophotometer, and the hydroxyl radical clearance was calculated according to the formula: hydroxyl radical clearance (%) ([ A blank-A test/A blank)]×100％。
Blank A: 1 mL FeSO4Solution +1 mL salicylic acid-ethanol solution +1 mL distilled water +1 mL H2O2A solution;
a test: 1 mL FeSO4Solution +1 mL salicylic acid-ethanol solution +1 mL H2O2Solution +1 mL of the corresponding drug in the working solution.
As shown in fig. 6, the measurement results show that the tsaoko element and the BHT as a control have a certain ability to remove hydroxyl radicals, and the removal ability increases with increasing concentration, but the BHT as a control has a relatively strong ability to remove hydroxyl radicals.
(5) With metal ions (Fe)2+) Determination of chelating Capacity
Taking the monomer compound I and BHT working solution to carry out Fe respectively2+And (4) testing the chelating ability. Monomer compound and BHT all set up 6 and have stopper colour comparison tube, compile as No. 0 ~ 5: 0.1 mL of FeCl is added into the No. 0 colorimetric tube20.2 mL of 5mmol/L phenanthroline and 5mL of distilled water are used as blank tubes, and 0.1 mL of FeCl is added into a No. 1-5 colorimetric tube20.2 mL of 5mmol/L phenanthroline oxazine, 3 mL of distilled water and 2mL of series working solutions (5, 10, 20, 30 and 40 mu g/mL) of corresponding medicines are used as test tubes, the distilled water is used for zero adjustment, after reaction for 10 min, an ultraviolet-visible spectrophotometer is used for measuring the A value of each reaction solution at the wavelength of 562 nm, and the lower the absorbance, the higher the metal chelating capacity is. Calculating the chelating capacity of ferrous ions according to the formula: ferrous ion chelating capacity (%) ([ A blank-A test/A blank)]×100％。
Blank A: 5mL of distilled water +0.1 mL of FeCl2+0.2 mL of 5mmol/L phenanthroline;
a test: 3 mL of distilled water +0.1 mL of FeCl2+0.2 mL of 5mmol/L phenanthroline and 2mL of a series of working solutions of corresponding medicines.
The measurement result is shown in FIG. 7, and when the concentration of the Amomum tsao-ko element and BHT of the control product is 5 mug/mL, the result is for Fe2+Chelating capacity is nearly equal, but followsWith increasing concentration, the tsaoko element is in pairs of Fe2+The change in chelating capacity is greatest.
（6） Fe3+Determination of the reducing ability
Taking the monomer compound I and BHT working solution to carry out Fe respectively3+And (4) carrying out reduction test. Both the monomer compound and BHT were placed in 5 15 mL centrifuge tubes, and 1 mL each of the corresponding drug-containing working solutions (5, 10, 20, 30, 40. mu.g/mL), 2.5 mL of 0.2 mol/L phosphate buffer (pH 6.6), and 2.5 mL of 1% potassium ferricyanide solution were added. Heating each centrifuge tube in a water bath at 50 ℃ for 20 min, adding 2.5 mL of 10% trichloroacetic acid solution to terminate the reaction, and then centrifuging at 3000 r/min for 10 min; taking 2.5 mL of supernatant fluid to a colorimetric tube with a plug, adding 2.5 mL of distilled water and 0.1% FeCl30.5 mL of the solution is mixed evenly and kept stand for reaction for 10 min. The A value of each reaction solution was measured at a wavelength of 700 nm using an ultraviolet-visible spectrophotometer by zeroing with distilled water. The value of A represents the Fe of the drug pair to be detected3+The larger the value of A, the stronger the reducing ability of the drug.
Sample A: [1 mL of sample +2.5 mL of phosphate buffer solution at pH 6.6 +2.5 mL of 1% potassium ferricyanide solution]Water bath for 20 min and trichloroacetic acid solution 2.5 mL to terminate the reaction (centrifugation), and supernatant 2.5 mL, 2.5 mL distilled water and 0.5 mL 0.1% FeCl is taken3Solutions of
Control A: [1 mL of BHT +2.5 mL of phosphate buffer solution having a pH of 6.6 +2.5 mL of 1% potassium ferricyanide solution]Water bath for 20 min and trichloroacetic acid solution 2.5 mL to terminate the reaction (centrifugation), and supernatant 2.5 mL, 2.5 mL distilled water and 0.5 mL 0.1% FeCl is taken3And (3) solution.
The results of the measurement are shown in FIG. 8. The greater the absorbance value, the greater the reduction ability, i.e., the greater the reduction ability, and the greater the oxidation resistance, as shown in fig. 8, the greater the absorbance values of the control BHT and the tsaoko element, but the most significant change in the absorbance values of the control BHT.
Based on the above embodiments, the present invention provides a method for extracting single compound, tsaoko element, from separated grass. The kukoamine belongs to terpenoids, which refer to hydrocarbons with isoprene as skeleton and oxygen-containing derivatives thereofBiologically, the olefinic bond in the structure is active in chemical property, has strong reducibility (namely oxidation resistance), and can react with an oxidant, thereby protecting the organism from oxidative damage[1]. Monomeric compounds differ in their ability to scavenge different free radicals, and may be related to the structural characteristics of the compound, including: spatial structure, position of hydroxyl group, number of hydroxyl groups, phenolic hydroxyl group on benzene ring, etc[2]。
DPPH free radical elimination experiment, ABTS free radical elimination experiment, superoxide anion free radical elimination experiment, hydroxyl free radical elimination experiment, ferrous ion chelation experiment and ferric ion reduction experiment show that the tsaoko element has certain elimination capacity to the free radicals and the ferric ions, which indicates that the tsaoko element has certain oxidation resistance, the oxidation resistance activity of the tsaoko element is related to the structure of a monomer compound, and the oxidation resistance action mechanism is mainly to avoid oxidation damage by eliminating the free radicals.
[1] The antioxidant effect of the extract of Sphacelaria gracilis (Hokko Swinhonis, Lianghui, Shidayong, etc..) J. China public health, 2005, 21(9):1082-1083.
[2] Zhaowei, Liu Bei Yu, Liu Dan, etc. the research on the structure-activity relationship of flavonoids has been advanced [ J ] Chinese herbal medicine, 2015, 46(21):3264-3271.
Example 8:
considering the method for extracting and separating the tsaoko element from the tsaoko amomum fruits in the above examples 1 to 5, the methodological verification process is as follows:
(1) and (3) precision test: precisely absorbing 10 muL of the tsaoko agent solution for sample introduction for 1 time, and recording a chromatogram. As a result: the RSD value of the peak area was 0.13%, indicating good precision of the instrument.
(2) And (3) stability test: precisely sucking the tsaoko element solution, injecting samples for 0, 2, 4, 8, 12 and 24 hours respectively, and determining peak area. As a result: the RSD value of the area of the tsaoko element peak is 0.43 percent, which indicates that the tsaoko element solution is stable within 24 hours.
(3) And (3) repeatability test: precisely weighing 6 parts of tsaoko sample, each part is about 1mg, preparing a test solution according to the preparation method of the test solution, and measuring according to chromatographic conditions. As a result: the content of the tsaoko amomin is 0.53 percent (mg/g), the RSD value is 0.83 percent, and the repeatability of the method is good.
(4) Sample recovery rate test: precisely weighing about 0.5mg of the determined tsaoko element, weighing 5 parts in parallel, placing into a conical flask with a plug, adding standard substance at a ratio of 0.5: 1, 1:1 and 1.5: 1, and preparing 3 parts at each ratio to obtain test solution. The sample injection volume of each sample solution is 10 mu L, the peak area is measured, and the recovery rate is calculated. As a result: the average recovery rate of the tsaoko element is 100.18 percent, the RSD is 1.37 percent, and the result shows that the method has good recovery rate.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
Claims (8)
1. A method for extracting and separating the tsaoko essence from the tsaoko amomum fruit is characterized by comprising the following steps: the method comprises the following steps:
A. crushing a tsaoko medicinal material serving as a raw material, performing reflux extraction by using ethanol, and performing reduced pressure concentration to obtain a concentrated solution;
B. diluting the concentrated solution, extracting with ethyl acetate to obtain ethyl acetate extract, and concentrating to obtain ethyl acetate extract;
C. and taking the ethyl acetate extract, and performing silica gel column chromatography separation and C18 column reverse separation to obtain the tsaoko amomum fruit element.
2. The method for extracting and separating the tsaoko element from the tsaoko amomum fruit as claimed in claim 1, wherein: in the step A, ethanol with the mass concentration of 95% is used for reflux extraction for 2-3 times, and the reflux extraction time is controlled to be 1.5-2 h each time.
3. The method for extracting and separating the tsaoko element from the tsaoko amomum fruit as claimed in claim 1, wherein: and in the step A, concentrating the extracting solution obtained by reflux extraction at 50-55 ℃ under reduced pressure until no alcohol exists, and obtaining the concentrated solution.
4. The method for extracting and separating the tsaoko element from the tsaoko amomum fruit as claimed in claim 1, wherein: and in the step B, diluting the concentrated solution by 2-3 times with water, and extracting the same volume of ethyl acetate for 2 times to obtain an ethyl acetate extract.
5. The method for extracting and separating the tsaoko element from the tsaoko amomum fruit as claimed in claim 1, wherein: and in the step B, concentrating the ethyl acetate extract at 45 ℃ under reduced pressure to obtain an ethyl acetate extract.
6. The method for extracting and separating the tsaoko element from the tsaoko amomum fruit as claimed in claim 1, wherein: and C, mixing the ethyl acetate extract with silica gel with the mass of 1-2 times of that of the ethyl acetate extract, blowing and drying the mixed sample at 40 ℃ to constant weight to obtain a dried silica gel mixed sample, and then carrying out silica gel column chromatography separation.
7. The method for extracting and separating the tsaoko element from the tsaoko amomum fruit as claimed in claim 6, wherein: and in the step C, carrying out normal phase silica gel column chromatography on the silica gel mixed sample, carrying out wet column packing by using 10-20 times of silica gel as a filler, carrying out dry loading, carrying out normal pressure elution by using dichloromethane-methanol as an eluent with the volume ratio of 100: 0-30: 1, collecting by tubes, carrying out about 100mL per tube, carrying out thin layer chromatography analysis, carrying out detection on dark spot results by using dichloromethane-methanol with the volume ratio of 20: 1 as a developing agent under 254nm ultraviolet light, combining collected liquids containing target compounds, and concentrating under reduced pressure at 45 ℃ to obtain an extract.
8. The method for extracting and separating the tsaoko element from the tsaoko amomum fruit as claimed in claim 1, wherein: in the step C, when the C18 column is reversely separated, methanol-water with the volume ratio of 50: 50 is used as a mobile phase, and the ultraviolet light is 254 nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111127531.3A CN113636923A (en) | 2021-09-26 | 2021-09-26 | Method for extracting and separating tsaoko essence from tsaoko amomum fruits |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111127531.3A CN113636923A (en) | 2021-09-26 | 2021-09-26 | Method for extracting and separating tsaoko essence from tsaoko amomum fruits |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113636923A true CN113636923A (en) | 2021-11-12 |
Family
ID=78426228
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111127531.3A Pending CN113636923A (en) | 2021-09-26 | 2021-09-26 | Method for extracting and separating tsaoko essence from tsaoko amomum fruits |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113636923A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116898938A (en) * | 2023-08-16 | 2023-10-20 | 河北瑞龙生物科技有限公司 | Method for extracting polyphenol substances from fructus tsaoko |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107019776A (en) * | 2016-01-26 | 2017-08-08 | 财团法人工业技术研究院 | Treat or slow down autoimmunity correlation disease medical composition and its purposes of active ingredient |
-
2021
- 2021-09-26 CN CN202111127531.3A patent/CN113636923A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107019776A (en) * | 2016-01-26 | 2017-08-08 | 财团法人工业技术研究院 | Treat or slow down autoimmunity correlation disease medical composition and its purposes of active ingredient |
Non-Patent Citations (3)
| Title |
|---|
| SURK-SIK MOON ET AL.: "Isotsaokoin, an Antifungal Agent from Amomum tsao-ko" * |
| 陈石梅等: "草果醇提物不同极性部位的体外抗氧化活性研究" * |
| 黄比翼等: "不同极性溶剂的草果提取物抗氧化活性研究" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116898938A (en) * | 2023-08-16 | 2023-10-20 | 河北瑞龙生物科技有限公司 | Method for extracting polyphenol substances from fructus tsaoko |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hazekamp et al. | Quantitative analysis of cannabinoids from Cannabis sativa using 1H-NMR | |
| Ajila et al. | Extraction and analysis of polyphenols: recent trends | |
| Li et al. | Chemical profiling of Radix Paeoniae evaluated by ultra-performance liquid chromatography/photo-diode-array/quadrupole time-of-flight mass spectrometry | |
| Chen et al. | Analysis of alkaloids in Coptis chinensis Franch by accelerated solvent extraction combined with ultra performance liquid chromatographic analysis with photodiode array and tandem mass spectrometry detections | |
| Tang et al. | Rapid and simple method for screening of natural antioxidants from Chinese herb Flos Lonicerae Japonicae by DPPH‐HPLC‐DAD‐TOF/MS | |
| Horvath et al. | Identification and quantification of eight flavones in root and shoot tissues of the medicinal plant Huang-qin (Scutellaria baicalensis Georgi) using high-performance liquid chromatography with diode array and mass spectrometric detection | |
| Koivikko et al. | High‐performance liquid chromatographic analysis of phlorotannins from the brown alga Fucus vesiculosus | |
| Svedström et al. | Fractionation of polyphenols in hawthorn into polymeric procyanidins, phenolic acids and flavonoids prior to high-performance liquid chromatographic analysis | |
| Jiao et al. | Ultrasonic extraction and HPLC determination of anthraquinones, aloe‐emodine, emodine, rheine, chrysophanol and physcione, in roots of Polygoni multiflori | |
| Wu et al. | Simultaneous qualitative and quantitative determination of phenolic compounds in Aloe barbadensis Mill by liquid chromatography–mass spectrometry-ion trap-time-of-flight and high performance liquid chromatography-diode array detector | |
| Blazics et al. | Identification and LC-MS-MS determination of acteoside, the main antioxidant compound of Euphrasia rostkoviana, using the isolated target analyte as external standard | |
| CN107505405B (en) | Method for efficiently and rapidly extracting and measuring flavonoid pigment in Chinese rose petals | |
| CN113651687B (en) | Method for extracting antioxidant active substances from fructus Tsaoko | |
| Wang et al. | Screening, separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi‐mode microplate reader | |
| Stalikas | Phenolic acids and flavonoids: occurrence and analytical methods | |
| Zhang et al. | Screening and isolation of potential neuraminidase inhibitors from leaves of Ligustrum lucidum Ait. based on ultrafiltration, LC/MS, and online extraction-separation methods | |
| Sun et al. | Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by HPLC-DAD-IT-TOF-MS | |
| Kulić et al. | A detailed view on the proanthocyanidins in Ginkgo extract EGb 761 | |
| CN113636923A (en) | Method for extracting and separating tsaoko essence from tsaoko amomum fruits | |
| Li et al. | Screening and isolation of cyclooxygenase‐2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry, and complex chromatography | |
| Fu et al. | A practical method for the rapid detection and structural characterization of major constituents from traditional Chinese medical formulas: analysis of multiple constituents in Yinchenhao Decoction | |
| Ye et al. | Enrichment and isolation of barbigerone from Millettia pachycarpa Benth. using high‐speed counter‐current chromatography and preparative HPLC | |
| CN115368359A (en) | Alkaloid antioxidant and preparation method and application thereof | |
| Shao et al. | Screening and identifying antioxidants from Oplopanax elatus using 2, 2ʹ‐diphenyl‐1‐picrylhydrazyl with off‐line two‐dimensional HPLC coupled with diode array detection and tandem time‐of‐flight mass spectrometry | |
| CN112843124A (en) | Preparation method and quality standard of salvia miltiorrhiza formula granules |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211112 |