CN105717229A - Method for simultaneously measuring 11 flavonoid constituents in vine tea through HPLC - Google Patents
Method for simultaneously measuring 11 flavonoid constituents in vine tea through HPLC Download PDFInfo
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- CN105717229A CN105717229A CN201610079019.9A CN201610079019A CN105717229A CN 105717229 A CN105717229 A CN 105717229A CN 201610079019 A CN201610079019 A CN 201610079019A CN 105717229 A CN105717229 A CN 105717229A
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- absolute methanol
- ampelopsis grossedentata
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 17
- 239000000470 constituent Substances 0.000 title claims abstract description 7
- 229930003935 flavonoid Natural products 0.000 title abstract description 7
- 150000002215 flavonoids Chemical class 0.000 title abstract description 7
- 235000017173 flavonoids Nutrition 0.000 title abstract description 7
- 241001122767 Theaceae Species 0.000 title abstract 5
- KJXSIXMJHKAJOD-LSDHHAIUSA-N (+)-dihydromyricetin Chemical compound C1([C@@H]2[C@H](C(C3=C(O)C=C(O)C=C3O2)=O)O)=CC(O)=C(O)C(O)=C1 KJXSIXMJHKAJOD-LSDHHAIUSA-N 0.000 claims abstract description 54
- KQILIWXGGKGKNX-UHFFFAOYSA-N dihydromyricetin Natural products OC1C(=C(Oc2cc(O)cc(O)c12)c3cc(O)c(O)c(O)c3)O KQILIWXGGKGKNX-UHFFFAOYSA-N 0.000 claims abstract description 27
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims abstract description 26
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 claims abstract description 26
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000012360 testing method Methods 0.000 claims abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 22
- CXQWRCVTCMQVQX-LSDHHAIUSA-N (+)-taxifolin Chemical compound C1([C@@H]2[C@H](C(C3=C(O)C=C(O)C=C3O2)=O)O)=CC=C(O)C(O)=C1 CXQWRCVTCMQVQX-LSDHHAIUSA-N 0.000 claims abstract description 15
- 239000012488 sample solution Substances 0.000 claims abstract description 15
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 claims abstract description 14
- 229940117893 apigenin Drugs 0.000 claims abstract description 14
- 229960001587 hesperetin Drugs 0.000 claims abstract description 14
- DCYOADKBABEMIQ-OWMUPTOHSA-N myricitrin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=C(O)C=2)OC2=CC(O)=CC(O)=C2C1=O DCYOADKBABEMIQ-OWMUPTOHSA-N 0.000 claims abstract description 14
- DCYOADKBABEMIQ-FLCVNNLFSA-N myricitrin Natural products O([C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O1)C1=C(c2cc(O)c(O)c(O)c2)Oc2c(c(O)cc(O)c2)C1=O DCYOADKBABEMIQ-FLCVNNLFSA-N 0.000 claims abstract description 14
- 229960001285 quercetin Drugs 0.000 claims abstract description 14
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 claims abstract description 13
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims abstract description 13
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims abstract description 13
- KZNIFHPLKGYRTM-UHFFFAOYSA-N apigenin Chemical compound C1=CC(O)=CC=C1C1=CC(=O)C2=C(O)C=C(O)C=C2O1 KZNIFHPLKGYRTM-UHFFFAOYSA-N 0.000 claims abstract description 13
- XADJWCRESPGUTB-UHFFFAOYSA-N apigenin Natural products C1=CC(O)=CC=C1C1=CC(=O)C2=CC(O)=C(O)C=C2O1 XADJWCRESPGUTB-UHFFFAOYSA-N 0.000 claims abstract description 13
- 235000008714 apigenin Nutrition 0.000 claims abstract description 13
- XCGZWJIXHMSSQC-UHFFFAOYSA-N dihydroquercetin Natural products OC1=CC2OC(=C(O)C(=O)C2C(O)=C1)c1ccc(O)c(O)c1 XCGZWJIXHMSSQC-UHFFFAOYSA-N 0.000 claims abstract description 13
- AIONOLUJZLIMTK-AWEZNQCLSA-N hesperetin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 AIONOLUJZLIMTK-AWEZNQCLSA-N 0.000 claims abstract description 13
- AIONOLUJZLIMTK-UHFFFAOYSA-N hesperetin Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(O)=CC(O)=C2C(=O)C1 AIONOLUJZLIMTK-UHFFFAOYSA-N 0.000 claims abstract description 13
- 235000010209 hesperetin Nutrition 0.000 claims abstract description 13
- FTODBIPDTXRIGS-UHFFFAOYSA-N homoeriodictyol Natural products C1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 FTODBIPDTXRIGS-UHFFFAOYSA-N 0.000 claims abstract description 13
- 235000008777 kaempferol Nutrition 0.000 claims abstract description 13
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229940117954 naringenin Drugs 0.000 claims abstract description 13
- 235000005875 quercetin Nutrition 0.000 claims abstract description 13
- 229960004555 rutoside Drugs 0.000 claims abstract description 13
- PADQINQHPQKXNL-LSDHHAIUSA-N (+)-dihydrokaempferol Chemical compound C1([C@@H]2[C@H](C(C3=C(O)C=C(O)C=C3O2)=O)O)=CC=C(O)C=C1 PADQINQHPQKXNL-LSDHHAIUSA-N 0.000 claims abstract description 12
- FTVWIRXFELQLPI-ZDUSSCGKSA-N (S)-naringenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 FTVWIRXFELQLPI-ZDUSSCGKSA-N 0.000 claims abstract description 12
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 claims abstract description 12
- UIDUJXXQMGYOIN-UHFFFAOYSA-N aromadendrin Natural products CC1(C)C2C1CCC(C)C1C2C(C)CC1 UIDUJXXQMGYOIN-UHFFFAOYSA-N 0.000 claims abstract description 12
- RAYJUFCFJUVJBB-UHFFFAOYSA-N dihydrokaempferol Natural products OC1Oc2c(O)cc(O)cc2C(=O)C1c3ccc(O)cc3 RAYJUFCFJUVJBB-UHFFFAOYSA-N 0.000 claims abstract description 12
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims abstract description 12
- WGEYAGZBLYNDFV-UHFFFAOYSA-N naringenin Natural products C1(=O)C2=C(O)C=C(O)C=C2OC(C1)C1=CC=C(CC1)O WGEYAGZBLYNDFV-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000007625 naringenin Nutrition 0.000 claims abstract description 12
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000005493 rutin Nutrition 0.000 claims abstract description 12
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 claims abstract description 12
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- 241001018563 Nekemias grossedentata Species 0.000 claims description 41
- 229930003944 flavone Natural products 0.000 claims description 38
- 235000011949 flavones Nutrition 0.000 claims description 38
- 150000002213 flavones Chemical class 0.000 claims description 34
- 239000004615 ingredient Substances 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 239000012982 microporous membrane Substances 0.000 claims description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 abstract description 6
- 241000196324 Embryophyta Species 0.000 abstract description 5
- 238000005259 measurement Methods 0.000 abstract description 2
- IKMDFBPHZNJCSN-UHFFFAOYSA-N Myricetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC(O)=C(O)C(O)=C1 IKMDFBPHZNJCSN-UHFFFAOYSA-N 0.000 abstract 1
- PCOBUQBNVYZTBU-UHFFFAOYSA-N myricetin Natural products OC1=C(O)C(O)=CC(C=2OC3=CC(O)=C(O)C(O)=C3C(=O)C=2)=C1 PCOBUQBNVYZTBU-UHFFFAOYSA-N 0.000 abstract 1
- 235000007743 myricetin Nutrition 0.000 abstract 1
- 229940116852 myricetin Drugs 0.000 abstract 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 4
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 150000002212 flavone derivatives Chemical class 0.000 description 3
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- 230000014759 maintenance of location Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 244000269722 Thea sinensis Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
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- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 241000563984 Ampelopsis Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 230000004531 blood pressure lowering effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- KQNGHARGJDXHKF-UHFFFAOYSA-N dihydrotamarixetin Natural products C1=C(O)C(OC)=CC=C1C1C(O)C(=O)C2=C(O)C=C(O)C=C2O1 KQNGHARGJDXHKF-UHFFFAOYSA-N 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- -1 flavone compound Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002443 hepatoprotective effect Effects 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 210000003026 hypopharynx Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Pyrane Compounds (AREA)
Abstract
The invention discloses a method for simultaneously measuring 11 flavonoid constituents in vine tea through HPLC and belongs to the technical field of medicinal plant constituent analysis.The method includes the steps that firstly, a mixed standard substance solution and a vine tea test sample solution are prepared respectively, then the mixed standard substance solution and the vine tea test sample solution are detected respectively through high performance liquid chromatography, and the contents of dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, myricetin, myricetrin, kaempferol, quercetin, rutin and apigenin in a test sample are obtained through calculation.The measuring method is good in stability and reproducibility, easy to operate, accurate and reliable in result and capable of being applied to simultaneous measurement of multiple flavonoid constituents in the vine tea.
Description
Technical field
The invention belongs to medicinal plants component analysis technical field, be specifically related to a kind of HPLC of utilization and measure the method for 11 kinds of flavones ingredients in Ampelopsis grossedentata simultaneously.
Background technology
Ampelopsis grossedentata is a kind of Wild Liane in Vitaceae Ampelopsis, formal name used at school be Ampelopsis grossedentata (AmpelopsisgrossedentataW.T.Wan), it is the distinctive a kind of medicine food dual purpose plant of China, the mountain area of provinces and regions height above sea level 200 ~ 1300m such as is distributed mainly on the south China Yangtze river basin.Ampelopsis grossedentata sweet in the mouth, light, cool in nature, there is the effect such as heat-clearing and toxic substances removing, wind-damp dispelling, among the people be usually used in the diseases such as treatment cold, fever, laryngopharynx swelling and pain, icterohepatitis, bleb furuncle, existing history of more than one thousand years.Modern pharmacological research shows, Ampelopsis grossedentata has antioxidation, inhibiting bacteria and diminishing inflammation, blood sugar lowering, blood fat reducing, blood pressure lowering and hepatoprotective effect, and the diseases such as the pharyngolaryngitis caused because tobacco and wine are excessive etc., digestive functional disturbance are had unique effects.Ampelopsis grossedentata (Ampelopsis grossedentata) leaf in 2013 is approved as new raw-food material [2013 No. 16] by national health State Family Planning Commission.Existing substantial amounts of Ampelopsis grossedentata drink tea product and deep processed product are commercially sold at present.
Research shows, the main secondary metabolites in Ampelopsis grossedentata, based on flavone compound, is a kind of natural plants that the flavones content found in nature so far is the highest, therefore is described as " king of plant flavone ".Flavonoid substances wide variety in Ampelopsis grossedentata, has had now been found that tens kinds of flavones ingredients, including dihydromyricetin, Quercetin, Taxifolin, kaempferol, Ampelopsis grossedentata element, myricetrin, hesperetin, apigenin etc..
The analyzing detecting method of current flavonoid content mainly has ultraviolet spectrophotometry and high performance liquid chromatography (HPLC) etc., and wherein spectrophotometry is the content of total flavones, it is impossible to reflect the situation of each flavones ingredient.HPLC method has the features such as easy operation, highly sensitive and good stability, it has also become analyze the main method measuring flavone.At present, the content that HPLC method measures vine tea flavone class material is utilized to have certain bibliographical information, but mainly measure the content of indivedual flavones ingredient such as the dihydromyricetin of Ampelopsis grossedentata, ampelopsin and myricetrin, the mensuration of other flavones ingredients is seldom related to, the report more simultaneously not measured.But Ampelopsis grossedentata is due to the difference of the aspects such as kind, ecological environment and cultivation management condition, and its flavones ingredient content exists significant difference.Therefore, setting up a kind of simplicity, analyze the method for multiple flavones ingredient in Ampelopsis grossedentata that measures accurately, control and scientific evaluation to Ampelopsis grossedentata quality are significant.
Summary of the invention
It is an object of the invention to provide a kind of HPLC of utilization and measure the method for 11 kinds of flavones ingredients in Ampelopsis grossedentata simultaneously, the method is simple, and degree of accuracy is high, favorable reproducibility, can provide reliable basis for scientific evaluation and effective inherent quality controlling Ampelopsis grossedentata.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of HPLC of utilization measures the method for 11 kinds of flavones ingredients in Ampelopsis grossedentata simultaneously, comprises the following steps:
1) preparation of hybrid standard product solution: accurate appropriate dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin and the apigenin standard substance of weighing are in the brown volumetric flask of 10mL respectively, dissolve with absolute methanol and be settled to scale, obtain each constituent concentration respectively 112.5,135,150,125,165,125,210,138,99,300,100mg L-1Hybrid standard product solution；
2) preparation of test sample solution: Ampelopsis grossedentata sample liquid nitrogen grinding is become powder, lyophilization, then pulverized to water content less than 5%, sieve after, weigh obtained freeze-drying powder 0.1g in triangular flask, add 10mL absolute methanol, shake up, 37 DEG C of mechanical shaking extraction 12h, 4500r/min is centrifuged 15min, residue adds 10mL absolute methanol again, repeats to extract 1 time, and wash residue with a small amount of absolute methanol under the same terms, merge 3 supernatant and be settled to 25mL, obtaining test sample solution；
3) by gained hybrid standard product solution and test sample solution respectively after 0.45 μm of organic filtering with microporous membrane, adopt high performance liquid chromatography to detect, be computed obtaining the content of dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin and apigenin in test sample；
The condition of its high performance liquid chromatography is: chromatographic column: VisionHTC18 post, 250mm × 4.6mm, 5 μm；Mobile phase: absolute methanol-0.4wt% phosphate aqueous solution；Flow velocity: 1.0mL min-1；Column temperature: 30 DEG C；Sampling volume: 5 μ L；Detection wavelength: 290nm, 360nm；Gradient elution program is: 0 ~ 20min, and the volumetric concentration of absolute methanol is 30%；20 ~ 30min, the volumetric concentration of absolute methanol is increased to 40% by 30%；30 ~ 45min, the volumetric concentration of absolute methanol is increased to 42% by 40%；45 ~ 60min, the volumetric concentration of absolute methanol is increased to 50% by 42%.
Described Ampelopsis grossedentata sample is the young stem and leaf of Ampelopsis grossedentata.
It is an advantage of the current invention that:
1, under the chromatographic condition that present invention determine that, just can completing to analyze measuring while in Ampelopsis grossedentata 11 kinds of flavones ingredients, and the chromatographic peak peak shape of each flavones ingredient is sharp-pointed in 60min, symmetry is good, and its separating degree is all higher than 1.5, it is possible to reach to be kept completely separate.
2, to measure the analysis method of 11 kinds of flavones ingredients in Ampelopsis grossedentata while setting up simple, reproducible for the present invention, and result accurately and reliably, can provide foundation for the quality control of Ampelopsis grossedentata.
Accompanying drawing explanation
Fig. 1 is dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin abosrption spectrogram in 200-600nm wave-length coverage；
Fig. 2 is ampelopsin, myricetrin, kaempferol, Quercetin, rutin, apigenin abosrption spectrogram in 200-600nm wave-length coverage；
Fig. 3 is that hybrid standard product solution of the present invention is at 290nm(A) and the 360nm(B) liquid chromatogram under wavelength；
Fig. 4 is that Youxi Ampelopsis grossedentata sample 1 is at 290nm(A) and the 360nm(B) liquid chromatogram under wavelength；
Wherein, each peak mark in figure: 1-dihydromyricetin；2-dihydroquercetin；3-aromadendrin；4-myricetrin；5-rutin；6-ampelopsin；7-naringenin；8-Quercetin；9-hesperetin；10-kaempferol；11-apigenin.
Detailed description of the invention
In order to make content of the present invention easily facilitate understanding, below in conjunction with detailed description of the invention, technical solutions according to the invention are described further, but the present invention is not limited only to this.
1 experimental section
1.1 instruments and reagent
ThermoFisherLCQFleet HPLC-MS instrument, is furnished with quaternary gradient pump, diode array detector, automatic sampler；SartoriusBT125D type analysis balance (Sai Duolisi scientific instrument (Beijing) company limited)；High speed centrifuge (Sigma3K-30)；Ultraviolet, visible spectrophotometer (ThermoScientificEvolution201)；Freezer dryer (TelstarLyoQuest-55).
Dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin and apigenin standard substance are all purchased from Shanghai Tongtian Biotechnology Co., Ltd. (purity is more than 98%), absolute methanol (chromatographically pure, Fisher company), water is ultra-pure water, and all the other reagent are analytical pure.
Ampelopsis grossedentata sample gathers from Youxi County, Fujian Province county respectively, Jiangkou, Guizhou Province county, through Life Science College botany teaching and research room of University Of Agriculture and Forestry In Fujian be accredited as Ampelopsis grossedentata (AmpelopsisgrossedentataW.T.Wan)。
1.2 high-efficient liquid phase chromatogram conditions:
Chromatographic column: VisionHTC18 post (250mm × 4.6mm, 5 μm)；Mobile phase: absolute methanol (A)-0.4wt% phosphate aqueous solution (B)；Condition of gradient elution: 0 ~ 20min, 30%A；20 ~ 30min, 30%A-40%A；30 ~ 45min, 40%A-42%A；45 ~ 60min, 42%A-50%A；Flow velocity: 1.0mL min-1；Column temperature: 30 DEG C；Sampling volume: 5 μ L；Detection wavelength: 290nm, 360nm.
The preparation of 1.3 hybrid standard product solution:
Accurate appropriate dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin and the apigenin standard substance of weighing are in the brown volumetric flask of 10mL respectively, dissolve with absolute methanol and be settled to scale, obtain each constituent concentration respectively 112.5,135,150,125,165,125,210,138,99,300,100mg L-1Hybrid standard product solution, it is stored for liquid phase analysis under-20 DEG C of conditions.
The preparation of 1.4 test sample solution:
Ampelopsis grossedentata sample liquid nitrogen grinding is become powder, and lyophilization less than 5% to water content, is then pulverized and is sealed after 40 mesh sieves, be stored in-80 DEG C standby；Weigh obtained freeze-drying powder 0.1g in triangular flask, add 10mL absolute methanol, shake up, 37 DEG C of centrifugal 15min of mechanical shaking extraction 12h, 4500r/min, residue adds 10mL absolute methanol again, repeat to extract 1 time under the same terms, and wash residue with a small amount of absolute methanol, merge 3 supernatant and be settled to 25mL, obtain test sample solution, it is stored for liquid phase analysis under-20 DEG C of conditions.
1.5 high-performance liquid chromatogram determinations
By gained hybrid standard product solution and test sample solution respectively after 0.45 μm of organic filtering with microporous membrane, after adopting high performance liquid chromatography to detect, it is computed obtaining the content of dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin and apigenin in test sample.
2 results and discussion
The selection of 2.1 detection wavelength
Utilizing ultraviolet-uisible spectrophotometer that 11 kinds of flavonoid standard substance carry out all-wave length (within the scope of 200 ~ 600nm) scanning, to determine the maximum absorption wavelength of different flavones ingredient, result is shown in Fig. 1, Fig. 2.As seen from Figure 1, dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin have maximum absorption band at about 290nm；Ampelopsin, myricetrin, kaempferol, Quercetin, rutin, apigenin have maximum absorption band near 360nm as seen from Figure 2.
The selection of 2.2 gradient
Set different gradient programs, according to the peak shape of chromatographic peak under each gradient program and separating degree, it is determined that best gradient is 0 ~ 20min, 30%A-30%A；20 ~ 30min, 30%A-40%A；30 ~ 45min, 40%A-42%A；45 ~ 60min, 42%A-50%A.Adopting above-mentioned gradient elution program, 11 kinds of flavones ingredients can reach to be kept completely separate in 60min, and its separating degree is all higher than 1.5, and peak shape is sharp-pointed, symmetry good (such as Fig. 3, wherein A is the liquid chromatogram under 290nm, and B is the liquid chromatogram under 360nm).
The methodological study of 2.3HPLC quantitative analysis
2.3.1 linear relationship is investigated
Accurate draw hybrid standard product solution 0.25,0.5,1,2,4,8mL, dilute with absolute methanol respectively and be settled to 10mL, chromatographic condition by 1.2 by each for gained concentration standards solution after 0.45 μm of organic filtering with microporous membrane, sample introduction 5 μ L is measured, with peak area for vertical coordinate (Y), mass concentration (X) is abscissa, drawing standard curve, calculating regression equation and the correlation coefficient of standard curve, result is in Table 1.
Table 1HPLC measures the regression equation of 11 kinds of flavonoid standard substance, correlation coefficient, the range of linearity
As shown in Table 1, the mass concentration of 11 kinds of flavones ingredients such as dihydromyricetin, dihydroquercetin and respective peaks area are good linear relation.
2.3.2 precision test
Accurate draw hybrid standard product solution 5mL, add absolute methanol be diluted to the concentration of dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin and apigenin respectively 22.5,27.0,30.0,25.0,33.0,25.0,42.0,27.6,19.8,60.0,20.0mg L-1, by the chromatographic condition under 1.2 by gained solution after 0.45 μm of organic filtering with microporous membrane, continuous sample introduction 6 times, each 5 μ L are measured, and record peak area also calculates the content of each flavones ingredient, then calculates relative standard deviation RSD according to measurement result.It is computed, the corresponding RSD value of dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin, apigenin respectively 0.41%, 0.21%, 0.16%, 0.25%, 0.11%, 0.17%, 0.23%, 0.35%, 0.28%, 0.24%, 0.35%, result shows that the method precision is good.
2.3.3 replica test
Precision weighs with 6 parts of a collection of Ampelopsis grossedentata sample, method by 1.4 is parallel prepares into 6 parts of test sample solution, gained solution sample introduction 5 μ L after 0.45 μm of organic filtering with microporous membrane is measured by the chromatographic condition by 1.2, record peak area and calculate the content of each flavones ingredient, then calculating RSD value.Being computed, the retention time of 11 kinds of flavones ingredients and the RSD value of content are respectively less than 3%, it was shown that the repeatability of this assay method is good.
2.3.4 stability test
Precision weighs a Ampelopsis grossedentata sample, test sample solution is prepared into by the method for 1.4, gained solution after 0.45 μm of organic filtering with microporous membrane, is measured respectively at 0,2,4,6,8,12,24 hours sample introduction 5 μ L, and calculates the content of each flavones ingredient by the chromatographic condition by 1.2.Being computed, the retention time of 11 kinds of flavones ingredients and the RSD value of content are respectively less than 3%, it was shown that test sample solution is stable in 24h.
2.3.5 average recovery test
Precision weighs 6 parts of the same Ampelopsis grossedentata sample of known 11 kinds of flavones ingredient content, it is separately added into appropriate 11 flavones ingredient standard substance, and prepare test sample solution according to the method for 1.4, gained solution sample introduction 5 μ L after 0.45 μm of organic filtering with microporous membrane is measured by the chromatographic condition by 1.2, record peak area also calculates average recovery, and result is in Table 2.
The recovery test result of 211 kinds of flavones ingredients of table
By table 2 it is shown that the response rate of the method is in the scope of 85.0% ~ 101.21%, relative standard deviation RSD≤2.04%, it was shown that the method has good reliability and accuracy.
The assay of 11 kinds of flavones ingredients in 2.4 Ampelopsis grossedentata samples
Method according to the present invention, 11 kinds of flavones ingredients in 4 Ampelopsis grossedentata samples in Youxi County, Fujian Province and two places of production of Jiangkou, Guizhou Province are measured simultaneously, and by confirming 11 kinds of flavones ingredients in chromatogram compared with the uv-spectrogram and retention time of standard substance, result is shown in Fig. 4 and Biao 3.
In table 3 Ampelopsis grossedentata sample, (by dry weight basis, unit is mg g to the content of 11 kinds of flavones ingredients-1)
11 kinds of flavones ingredients from Fig. 4 and Biao 3 it can be seen that in Ampelopsis grossedentata sample can be good separation, the flavones ingredient content of different sample rooms is different.
The foregoing is only presently preferred embodiments of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to the covering scope of the present invention.
Claims (2)
1. one kind measures the method for 11 kinds of flavones ingredients in Ampelopsis grossedentata with HPLC simultaneously, it is characterised in that comprise the following steps:
1) preparation of hybrid standard product solution: accurate appropriate dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin and the apigenin standard substance of weighing are in the brown volumetric flask of 10mL respectively, dissolve with absolute methanol and be settled to scale, obtain each constituent concentration respectively 112.5,135,150,125,165,125,210,138,99,300,100mg L-1Hybrid standard product solution；
2) preparation of test sample solution: Ampelopsis grossedentata sample liquid nitrogen grinding is become powder, lyophilization, then pulverized to water content less than 5%, sieve after, weigh obtained freeze-drying powder 0.1g in triangular flask, add 10mL absolute methanol, shake up, 37 DEG C of mechanical shaking extraction 12h, 4500r/min is centrifuged 15min, residue adds 10mL absolute methanol again, repeats to extract 1 time, and wash residue with a small amount of absolute methanol under the same terms, merge 3 supernatant and be settled to 25mL, obtaining test sample solution；
3) by gained hybrid standard product solution and test sample solution respectively after 0.45 μm of organic filtering with microporous membrane, adopt high performance liquid chromatography to detect, be computed obtaining the content of dihydromyricetin, dihydroquercetin, aromadendrin, naringenin, hesperetin, ampelopsin, myricetrin, kaempferol, Quercetin, rutin and apigenin in test sample；
The condition of its high performance liquid chromatography is: chromatographic column: VisionHTC18 post, 250mm × 4.6mm, 5 μm；Mobile phase: absolute methanol-0.4wt% phosphate aqueous solution；Flow velocity: 1.0mL min-1；Column temperature: 30 DEG C；Sampling volume: 5 μ L；Detection wavelength: 290nm, 360nm；Gradient elution program is: 0 ~ 20min, and the volumetric concentration of absolute methanol is 30%；20 ~ 30min, the volumetric concentration of absolute methanol is increased to 40% by 30%；30 ~ 45min, the volumetric concentration of absolute methanol is increased to 42% by 40%；45 ~ 60min, the volumetric concentration of absolute methanol is increased to 50% by 42%.
2. measure the method for 11 kinds of flavones ingredients in Ampelopsis grossedentata according to claim 1 with HPLC, it is characterised in that described Ampelopsis grossedentata sample is the young stem and leaf of Ampelopsis grossedentata simultaneously.
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