CN107400151A - The screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root - Google Patents
The screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root Download PDFInfo
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- CN107400151A CN107400151A CN201611014271.8A CN201611014271A CN107400151A CN 107400151 A CN107400151 A CN 107400151A CN 201611014271 A CN201611014271 A CN 201611014271A CN 107400151 A CN107400151 A CN 107400151A
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- 235000009225 Stachys officinalis Nutrition 0.000 title claims abstract description 49
- 238000012216 screening Methods 0.000 title claims abstract description 42
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 38
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 38
- 239000004480 active ingredient Substances 0.000 title claims abstract description 29
- 238000000926 separation method Methods 0.000 title claims abstract description 26
- 241000519995 Stachys sylvatica Species 0.000 title claims abstract 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 136
- 238000000605 extraction Methods 0.000 claims abstract description 43
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 39
- 150000001875 compounds Chemical class 0.000 claims abstract description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000000287 crude extract Substances 0.000 claims abstract description 20
- 239000008367 deionised water Substances 0.000 claims abstract description 20
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 20
- 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 claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 15
- 239000000284 extract Substances 0.000 claims abstract description 12
- 238000010992 reflux Methods 0.000 claims abstract description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- 235000006708 antioxidants Nutrition 0.000 claims description 33
- 238000001514 detection method Methods 0.000 claims description 24
- 239000011347 resin Substances 0.000 claims description 23
- 229920005989 resin Polymers 0.000 claims description 23
- 238000010828 elution Methods 0.000 claims description 18
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical group CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- 239000003480 eluent Substances 0.000 claims description 16
- DOAOYJIOTYDTNV-UHFFFAOYSA-N butan-1-ol;ethyl acetate;hydrate Chemical compound O.CCCCO.CCOC(C)=O DOAOYJIOTYDTNV-UHFFFAOYSA-N 0.000 claims description 15
- 239000000178 monomer Substances 0.000 claims description 14
- 230000003064 anti-oxidating effect Effects 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000010829 isocratic elution Methods 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 239000003960 organic solvent Substances 0.000 abstract description 5
- 241001423752 Pedicularis canadensis Species 0.000 description 34
- 239000002034 butanolic fraction Substances 0.000 description 7
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 235000008216 herbs Nutrition 0.000 description 5
- 239000011148 porous material Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 239000013543 active substance Substances 0.000 description 3
- 230000001476 alcoholic effect Effects 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000010262 high-speed countercurrent chromatography Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- MRIFZKMKTDPBHR-XLOWEYQUSA-N 8-Epiiridotrial glucoside Chemical compound O([C@H]1[C@H]2[C@@H](C(=CO1)C=O)CC[C@H]2C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O MRIFZKMKTDPBHR-XLOWEYQUSA-N 0.000 description 1
- BVFLJHVBTFJPHJ-UHFFFAOYSA-N Ac-(3beta,5alpha)-Androst-16-en-3-ol Natural products C1=C(O)C(OC)=CC(C=CC(=O)OC2C(C(O)C(OCCC=3C=C(O)C(O)=CC=3)OC2COC2C(C(O)(CO)CO2)O)OC2C(C(O)C(O)C(C)O2)O)=C1 BVFLJHVBTFJPHJ-UHFFFAOYSA-N 0.000 description 1
- VVTVHDZQFUALQV-UHFFFAOYSA-N Alyssonoside Natural products C1=C(O)C(OC)=CC(C=CC(=O)NCCC=2C=CC(OC3C(C(O)C(O)C(CO)O3)O)=CC=2)=C1 VVTVHDZQFUALQV-UHFFFAOYSA-N 0.000 description 1
- 208000003643 Callosities Diseases 0.000 description 1
- 241001164374 Calyx Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- FNMHEHXNBNCPCI-QEOJJFGVSA-N Isoacteoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@H](OCCC=2C=C(O)C(O)=CC=2)O[C@H](COC(=O)\C=C\C=2C=C(O)C(O)=CC=2)[C@H]1O FNMHEHXNBNCPCI-QEOJJFGVSA-N 0.000 description 1
- WTPBQEGQIMLUIK-UHFFFAOYSA-N Leucosceptoside B Natural products C1=C(O)C(OC)=CC=C1CCOC1C(O)C(OC2C(C(O)C(O)C(C)O2)O)C(OC(=O)C=CC=2C=C(OC)C(O)=CC=2)C(COC2C(C(O)(CO)CO2)O)O1 WTPBQEGQIMLUIK-UHFFFAOYSA-N 0.000 description 1
- 241000167859 Pedicularis Species 0.000 description 1
- RMJYDWRKVAGFGZ-UHFFFAOYSA-N Phtheirospermoside Natural products C1=C(O)C(OC)=CC=C1C=CC(=O)OC1C(OC2C(C(O)C(O)C(C)O2)O)C(O)C(OCCC=2C=C(O)C(O)=CC=2)OC1CO RMJYDWRKVAGFGZ-UHFFFAOYSA-N 0.000 description 1
- 241000921313 Phyllopodium Species 0.000 description 1
- 241000207844 Scrophulariaceae Species 0.000 description 1
- BVFLJHVBTFJPHJ-VMPITWQZSA-N [2-[[3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxymethyl]-6-[2-(3,4-dihydroxyphenyl)ethoxy]-5-hydroxy-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-3-yl] (e)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)OC2C(C(O)C(OCCC=3C=C(O)C(O)=CC=3)OC2COC2C(C(O)(CO)CO2)O)OC2C(C(O)C(O)C(C)O2)O)=C1 BVFLJHVBTFJPHJ-VMPITWQZSA-N 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MRIFZKMKTDPBHR-UHFFFAOYSA-N boschnaloside Natural products CC1CCC(C(=CO2)C=O)C1C2OC1OC(CO)C(O)C(O)C1O MRIFZKMKTDPBHR-UHFFFAOYSA-N 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- FNMHEHXNBNCPCI-RYEKTNFUSA-N isoacteoside Natural products C[C@@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](COC(=O)C=Cc3ccc(O)c(O)c3)O[C@@H](OCCc4ccc(O)c(O)c4)[C@@H]2O)[C@H](O)[C@H](O)[C@H]1O FNMHEHXNBNCPCI-RYEKTNFUSA-N 0.000 description 1
- UMADJYFWGWTKSA-UHFFFAOYSA-N lamiophlomioside A Natural products C1=C(O)C(OC)=CC(CCOC2C(C(OC3C(C(O)C(O)C(C)O3)O)C(OC(=O)C=CC=3C=C(OC)C(O)=CC=3)C(COC3C(C(O)(CO)CO3)O)O2)O)=C1 UMADJYFWGWTKSA-UHFFFAOYSA-N 0.000 description 1
- ZMYQRHSOVRDQDL-NSWBACJASA-N leucosceptoside A Natural products COc1cc(C=CC(=O)O[C@@H]2[C@@H](CO)O[C@@H](OCCc3ccc(O)c(O)c3)[C@H](O)[C@H]2O[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)ccc1O ZMYQRHSOVRDQDL-NSWBACJASA-N 0.000 description 1
- ZMYQRHSOVRDQDL-CPPDSBOHSA-N leucosceptoside-a Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)O[C@H]2[C@@H]([C@@H](O)[C@H](OCCC=3C=C(O)C(O)=CC=3)O[C@@H]2CO)O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)=C1 ZMYQRHSOVRDQDL-CPPDSBOHSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229940023569 palmate Drugs 0.000 description 1
- OFNCUIXPAFLTJZ-UHFFFAOYSA-N plantainoside C Natural products C1=C(O)C(OC)=CC(C=CC(=O)OCC2C(C(OC3C(C(O)C(O)C(C)O3)O)C(O)C(OCCC=3C=C(O)C(O)=CC=3)O2)O)=C1 OFNCUIXPAFLTJZ-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- QFRYQWYZSQDFOS-UHFFFAOYSA-N verbascoside Natural products CC1OC(COC2C(O)C(COC3OC(C(O)C(O)C3O)C(=O)O)OC(Oc4cc(O)cc5OC(=CC(=O)c45)c6ccc(O)c(O)c6)C2O)C(O)C(O)C1O QFRYQWYZSQDFOS-UHFFFAOYSA-N 0.000 description 1
- KDSWDGKIENPKLB-QJDQKFITSA-N verbascoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC(=O)CCC=2C=C(O)C(O)=CC=2)[C@@H](CO)O[C@@H](OCCC=2C=C(O)C(O)=CC=2)[C@@H]1O KDSWDGKIENPKLB-QJDQKFITSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/80—Scrophulariaceae (Figwort family)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines Containing Plant Substances (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The screening of six kinds of antioxidant active ingredients and separation method, comprise the following steps in a kind of spot lip resupinate woodbetony leaf or root of disclosure of the invention：Extraction：By spot lip resupinate woodbetony leaf or root herb crushed after being dried, then screened, and ethanol solution refluxing extraction is used under the first predetermined temperature, crude extract is obtained after extract solution is concentrated under reduced pressure, be enriched with：Small polar compound is removed after the crude extract deionized water dissolving obtained in extraction step, then with ethyl acetate solution extraction, and remainder was subjected to column operation.The present invention is by using online DPPH HPLC antioxidant active ingredients screening technique and being used cooperatively using high-speed counter-current chromatograph, make in spot lip resupinate woodbetony leaf or root screening and the separation method of six kinds of antioxidant active ingredients very convenient and rapid, so as to shorten the time of screening and the separation of six kinds of antioxidant active ingredients in spot lip resupinate woodbetony leaf or root, improve production efficiency, and the amount of organic solvent used in technique is saved, so as to save production cost.
Description
Technical field
The present invention relates to material separation technology field, six kinds of antioxidant active ingredients in specially a kind of spot lip resupinate woodbetony leaf or root
Screening and separation method.
Background technology
Spot lip resupinate woodbetony leaf or root, the only short draft of Scrophulariaceae lousewort, accidentally rise, the few hair of whole body.Root Shu Sheng, it is several not increase
Slightly, for elder up to 15 centimetres, lower end is tapered into must shape.Phyllopodium goes out to go out with stem, often into thicket, there is long handle, and handle is longer in base leaf,
1-2 centimetres, shorter in cauline leaf, often how many film quality are expanded for lower half, when have thin long echinid, blade pinniform is shallow to be split to drastic crack, is had
When bottom leaf be full edge, lanceolar to long and narrow circle, two sides is hairless, and back side net vein is obvious and thin, often there is the white of evacuation
Color skin considers shape thing to be worth doing, and 5-9 pairs of sliver has a heavy sawtooth, and tooth often has a callosity and warp.Equal axillary is spent, there is short stalk；Calyx pipe shape, long 11-15
Millimeter, front cracking is about to 2/5, how many swollen, hairless, the only atomic echinid of breach, about 15, arteries and veins, wherein only 2 are relatively thick,
So also thin and delicate, slightly net is tied at only near pipe end, 2 pieces of tooth, there is short handle, how much palmate cracking, and sliver has the sawtooth of minority.
There are six kinds of anti-oxidation active substances, traditional side by this six kinds of anti-oxidation active substance separation in spot lip resupinate woodbetony leaf or root
Method process is complicated, and the time length needed, and substantial amounts of organic solvent is needed in technical process, and separation costs are high, efficiency
It is low.
The content of the invention
It is an object of the invention to provide a kind of screening of six kinds of antioxidant active ingredients in spot lip resupinate woodbetony leaf or root and separation side
Method, it is complicated to solve traditional procedure by this six kinds of anti-oxidation active substance separation, and the time length of needs, and in work
Need substantial amounts of organic solvent during skill, and the problem of separation costs are high, and efficiency is low.
To achieve the above object, the present invention provides following technical scheme：Six kinds of antioxidation activities in a kind of spot lip resupinate woodbetony leaf or root
The screening of composition and separation method, comprise the following steps：
S1, extraction：By spot lip resupinate woodbetony leaf or root herb crushed after being dried, then screened, and second is used under the first predetermined temperature
Alcoholic solution refluxing extraction, crude extract is obtained after extract solution is concentrated under reduced pressure.
S2, enrichment：Extracted after the crude extract deionized water dissolving obtained in extraction step, then with ethyl acetate solution
Small polar compound is removed, and remainder was subjected to column operation, splitter is washed with the ethanol solution of various concentrations
It is de-, eluent is obtained, the eluent is concentrated under reduced pressure, to obtain the enriched substance containing target component.
S3, isolate and purify：(1), using online DPPH-HPLC antioxidant active ingredients screening technique from spot lip resupinate woodbetony leaf or root just
Butanol fraction filters out 6 compounds with obvious antioxidation activity.
Wherein, online anti-oxidant screening conditions, using Agilent 1100 (250 × 4.6mmi.d., 5 μm) reverse-phase chromatography
Post, mobile phase are methanol solution, and the mass concentration of methanol is 45-50%, and isocratic elution, the gradient elution time is 0-45min, stream
Dynamic phase flow velocity be 1ml/min, Detection wavelength 330, and the μ g/ml methanol of DPPH solution concentrations 25 dissolves, flow rate of mobile phase 0.45ml/
Min, detection use Agilent 1260, DPPH reactions ring length 15m, Detection wavelength 517nm.
(2) this 6 kinds of high-purity monomer compounds, have been isolated and purified out using high-speed counter-current chromatograph.
Wherein, when being separated by high-speed counter-current chromatograph, ethyl acetate-positive fourth is used in the second predetermined temperature
Alcohol-water capacity system is eluted.
Preferably, it is 20-100 mesh sieves from sieve aperture scope, and spot lip resupinate woodbetony leaf or root is full during screening in the extraction step
The solid-liquid ratio of grass and ethanol solution is 1:3-1:30, and the use of the mass concentration of ethanol in ethanol solution is 0%-90%, and the
One predetermined temperature is 20 DEG C -80 DEG C, and extraction time is 1-5 times, extracts 0.5h-3h every time.
Preferably, in the enriching step, the number for making to be extracted with ethyl acetate is 3-10 times, and the splitter is using non-
Polar macroporous resin column, the non-polar macroporous resin post include D101 types macroreticular resin, AB-8 types macroreticular resin, D3520 types
Macroreticular resin and X-5 type macroreticular resins.
Preferably, in the enriching step, the volume of the ethanol solution is the 5 of the volume of the splitter center pillar bed
- 20 times again.
Preferably, in the enriching step, the mass concentration of ethanol is 50% in the ethanol solution.
Preferably, in the enriching step, splitter is eluted for 50% ethanol solution using mass concentration
Before, the ethanol solution that the ethanol solution and mass concentration that are successively 30% with deionized water, mass concentration are 40% is to splitter
Eluted, and the volume of the deionized water is 5 times -20 times of the volume of the splitter center pillar bed, the mass concentration
Volume for 30% ethanol solution is 5 times -20 times of the volume of the splitter center pillar bed, and the mass concentration is 40%
The volume of ethanol solution is 5 times -20 times of the volume of the splitter center pillar bed.
Preferably, in the purification procedures, chloroform, n-butanol, water in the ethyl acetate-n-butanol-water system
Volume ratio be 10-15：2-5：8-10, and the second preheating temperature is 20 DEG C -50 DEG C, and the rotating speed of high-speed counter-current chromatograph is
500-1000r/min。
Compared with prior art, the beneficial effects of the invention are as follows：The present invention is by using the anti-oxidant work of online DPPH-HPLC
Sexual element screening technique and being used cooperatively using high-speed counter-current chromatograph, make six kinds of antioxidant active ingredients in spot lip resupinate woodbetony leaf or root
Screening and separation method it is very convenient and rapid, so as to shorten the screening of six kinds of antioxidant active ingredients in spot lip resupinate woodbetony leaf or root
And the time of separation, production efficiency is improved, and the amount of organic solvent used in technique is saved, risen so as to save into production
This.
Using the inventive method obtain six kinds of monomeric compound boschnaloside, alyssonoside,
Leucosceptoside A, isoverbascoside, leucosceptoside B and verbascoside are used1H-NMR
With1C-NMR identification meets its compound structure (referring to Fig. 5), meanwhile, using HPLC detect purity more than 95% (referring to
Fig. 4).
Brief description of the drawings
Fig. 1 is DPPH-HPLC online equipments flow chart of the present invention；
Fig. 2 is spot lip resupinate woodbetony leaf or root crude extract HPLC chromatogram of the present invention and DPPH chromatograms；
Fig. 3 is spot lip resupinate woodbetony leaf or root crude extract HSCCC chromatographic fractionation figures of the present invention；
Fig. 4 is that HPLC of the present invention detects six isolated compound purity chromatograms of HSCCC；
Fig. 5 is the structure chart of six kinds of compounds of the invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
Fig. 1-5 are referred to, the present invention provides a kind of technical scheme：Six kinds of antioxidant active ingredients in a kind of spot lip resupinate woodbetony leaf or root
Screening and separation method, comprise the following steps：
S1, extraction：By spot lip resupinate woodbetony leaf or root herb crushed after being dried, then screened, and second is used under the first predetermined temperature
Alcoholic solution refluxing extraction, obtains crude extract after extract solution is concentrated under reduced pressure, in extraction step, during screening, be from sieve aperture scope
20-100 mesh sieves, and the solid-liquid ratio of spot lip resupinate woodbetony leaf or root herb and ethanol solution is 1:3-1:30, and use ethanol in ethanol solution
Mass concentration be 0%-90%, and the first predetermined temperature is 20 DEG C -80 DEG C, and extraction time is 1-5 time, is extracted every time
0.5h-3h。
S2, enrichment：Extracted after the crude extract deionized water dissolving obtained in extraction step, then with ethyl acetate solution
Small polar compound is removed, and remainder was subjected to column operation, splitter is washed with the ethanol solution of various concentrations
De-, in enriching step, the mass concentration of ethanol is 50% in ethanol solution, and in enriching step, the volume of ethanol solution is separation
5 times -20 times of the volume of post center pillar bed, in enriching step, splitter is entered for 50% ethanol solution using mass concentration
Go before eluting, the ethanol solution pair that the ethanol solution and mass concentration that are successively 30% with deionized water, mass concentration are 40%
Splitter is eluted, and the volume of deionized water is 5 times -20 times of the volume of splitter center pillar bed, mass concentration 30%
The volume of ethanol solution be 5 times -20 times of volume of splitter center pillar bed, mass concentration is the volume of 40% ethanol solution
It is 5 times -20 times of the volume of splitter center pillar bed, obtains eluent, eluent is concentrated under reduced pressure, obtains containing target component
Enriched substance, in enriching step, the number for making to be extracted with ethyl acetate is 3-10 times, and splitter uses non-polar macroporous resin
Post, it is big that non-polar macroporous resin post includes D101 types macroreticular resin, AB-8 types macroreticular resin, D3520 types macroreticular resin and X-5 types
Hole resin.
S3, isolate and purify：(1), using online DPPH-HPLC antioxidant active ingredients screening technique from spot lip resupinate woodbetony leaf or root just
Butanol fraction filters out 6 compounds with obvious antioxidation activity.
Wherein, online anti-oxidant screening conditions, using Agilent 1100 (250 × 4.6mmi.d., 5 μm) reverse-phase chromatography
Post, mobile phase are methanol solution, and the mass concentration of methanol is 45-50%, and isocratic elution, the gradient elution time is 0-45min, stream
Dynamic phase flow velocity be 1ml/min, Detection wavelength 330, and the μ g/ml methanol of DPPH solution concentrations 25 dissolves, flow rate of mobile phase 0.45ml/
Min, detection use Agilent 1260, DPPH reactions ring length 15m, Detection wavelength 517nm.
(2) this 6 kinds of high-purity monomer compounds, have been isolated and purified out using high-speed counter-current chromatograph, wherein, passing through height
When fast counter-current chromatograph is separated, eluted in the second predetermined temperature using ethyl acetate-n-butanol-water volume system,
In purification procedures, chloroform, n-butanol, the volume ratio of water are 10-15 in ethyl acetate-n-butanol-water system：2-5：8-
10, and the second preheating temperature is 20 DEG C -50 DEG C, and the rotating speed of high-speed counter-current chromatograph is 500-1000r/min.
Embodiment one
S1, extraction：Spot lip resupinate woodbetony leaf or root herb crushed after being dried is screened to 20 mesh, with 1:3 spot lip resupinate woodbetony leaf or root herbs with
The solid-liquid ratio of ethanol solution is simultaneously 30% ethanol solution refluxing extraction 1 time at a temperature of 20 DEG C with mass concentration, and during extraction
Between be 0.5h, obtain crude extract after extract solution is concentrated under reduced pressure.
S2, enrichment：Extracted after the crude extract deionized water dissolving obtained in extraction step, then with ethyl acetate solution
Remove small polar compound, and extraction times are 3 times, and by D101 types large pore resin absorption column on remainder, successively with 5 times
Ethanol that the deionized water of bed volume, the mass concentration of 5 times of bed volumes are 20%, the mass concentration of 5 times of bed volumes are
The mass concentration of 30% ethanol and 5 times of bed volumes be 40% ethanol elution, wherein 40% ethanol elution part be containing
There is the eluent of target component, by the eluent containing target component through being concentrated under reduced pressure into constant weight, produce containing target component
Enriched substance.
S3, isolate and purify：(1), using online DPPH-HPLC antioxidant active ingredients screening technique from spot lip resupinate woodbetony leaf or root just
Butanol fraction filters out 6 compounds with obvious antioxidation activity.
Wherein, online anti-oxidant screening conditions, using Agilent 1100 (250 × 4.6mmi.d., 5 μm) reverse-phase chromatography
Post, mobile phase are methanol solution, and the mass concentration of methanol is 45%, isocratic elution, and the gradient elution time is 10min, mobile phase
Flow velocity is 1ml/min, Detection wavelength 330, the dissolving of the μ g/ml methanol of DPPH solution concentrations 25, flow rate of mobile phase 0.45ml/min,
Detection uses Agilent 1260, DPPH reactions ring length 15m, Detection wavelength 517nm.
(2) 6 kinds of high-purity monomer compounds, are isolated and purified out using high-speed counter-current chromatograph, wherein, inverse by high speed
When flow chromatography instrument is separated, the solvent system that uses for ethyl acetate-n-butanol-water system in chloroform, n-butanol, the body of water
Product is than being 10：2：8, at a temperature of 20 DEG C, eluted using ethyl acetate-n-butanol-water volume system, and high-speed counter-current
Chromatographic rotating speed is 500r/min, so as to isolate and purify out 6 kinds of high-purity monomer compounds, and it is high-purity by 6 kinds of identification
The structural formula for spending monomeric compound is as shown in Figure 5.
Embodiment two
S1, extraction：Spot lip resupinate woodbetony leaf or root herb crushed after being dried is screened to 40 mesh, with 1：8 spot lip resupinate woodbetony leaf or root herbs with
The solid-liquid ratio of ethanol solution is simultaneously 40% ethanol solution refluxing extraction 2 times at a temperature of 30 DEG C with mass concentration, and during extraction
Between be 1h, obtain crude extract after extract solution is concentrated under reduced pressure.
S2, enrichment：Extracted after the crude extract deionized water dissolving obtained in extraction step, then with ethyl acetate solution
Remove small polar compound, and extraction times are 5 times, and by AB-8 types large pore resin absorption column on remainder, successively with 8 times
Ethanol that the deionized water of bed volume, the mass concentration of 8 times of bed volumes are 20%, the mass concentration of 8 times of bed volumes are
The mass concentration of 30% ethanol and 8 times of bed volumes be 40% ethanol elution, wherein 40% ethanol elution part be containing
There is the eluent of target component, by the eluent containing target component through being concentrated under reduced pressure into constant weight, produce containing target component
Enriched substance.
S3, isolate and purify：(1), using online DPPH-HPLC antioxidant active ingredients screening technique from spot lip resupinate woodbetony leaf or root just
Butanol fraction filters out 6 compounds with obvious antioxidation activity.
Wherein, online anti-oxidant screening conditions, using Agilent 1100 (250 × 4.6mmi.d., 5 μm) reverse-phase chromatography
Post, mobile phase are methanol solution, and the mass concentration of methanol is 46%, isocratic elution, and the gradient elution time is 20min, mobile phase
Flow velocity is 1ml/min, Detection wavelength 330, the dissolving of the μ g/ml methanol of DPPH solution concentrations 25, flow rate of mobile phase 0.45ml/min,
Detection uses Agilent 1260, DPPH reactions ring length 15m, Detection wavelength 517nm.
(2) 6 kinds of high-purity monomer compounds, are isolated and purified out using high-speed counter-current chromatograph, wherein, inverse by high speed
When flow chromatography instrument is separated, the solvent system that uses for ethyl acetate-n-butanol-water system in chloroform, n-butanol, the body of water
Product is than being 11：2：8, at a temperature of 25 DEG C, eluted using ethyl acetate-n-butanol-water volume system, and high-speed counter-current
Chromatographic rotating speed is 600r/min, so as to isolate and purify out 6 kinds of high-purity monomer compounds, and it is high-purity by 6 kinds of identification
The structural formula for spending monomeric compound is as shown in Figure 5.
Embodiment three
S1, extraction：Spot lip resupinate woodbetony leaf or root herb crushed after being dried is screened to 60 mesh, with 1：10 spot lip resupinate woodbetony leaf or root herbs with
The solid-liquid ratio of ethanol solution is simultaneously 50% ethanol solution refluxing extraction 3 times at a temperature of 40 DEG C with mass concentration, and during extraction
Between be 1.5h, obtain crude extract after extract solution is concentrated under reduced pressure.
S2, enrichment：Extracted after the crude extract deionized water dissolving obtained in extraction step, then with ethyl acetate solution
Remove small polar compound, and extraction times are 7 times, and by D3520 types large pore resin absorption column on remainder, successively with 10
Ethanol that the deionized water of times bed volume, the mass concentration of 10 times of bed volumes are 20%, the quality of 10 times of bed volumes are dense
The mass concentration spent for 30% ethanol and 10 times of bed volumes is 40% ethanol elution, wherein 40% ethanol elution part
For the eluent containing target component, by the eluent containing target component through being concentrated under reduced pressure into constant weight, produce containing target into
The enriched substance divided.
S3, isolate and purify：(1), using online DPPH-HPLC antioxidant active ingredients screening technique from spot lip resupinate woodbetony leaf or root just
Butanol fraction filters out 6 compounds with obvious antioxidation activity.
Wherein, online anti-oxidant screening conditions, using Agilent 1100 (250 × 4.6mmi.d., 5 μm) reverse-phase chromatography
Post, mobile phase are methanol solution, and the mass concentration of methanol is 47%, isocratic elution, and the gradient elution time is 30min, mobile phase
Flow velocity is 1ml/min, Detection wavelength 330, the dissolving of the μ g/ml methanol of DPPH solution concentrations 25, flow rate of mobile phase 0.45ml/min,
Detection uses Agilent 1260, DPPH reactions ring length 15m, Detection wavelength 517nm.
(2) 6 kinds of high-purity monomer compounds, are isolated and purified out using high-speed counter-current chromatograph, wherein, inverse by high speed
When flow chromatography instrument is separated, the solvent system that uses for ethyl acetate-n-butanol-water system in chloroform, n-butanol, the body of water
Product is than being 13：3：8, at a temperature of 30 DEG C, eluted using ethyl acetate-n-butanol-water volume system, and high-speed counter-current
Chromatographic rotating speed is 700r/min, so as to isolate and purify out 6 kinds of high-purity monomer compounds, and it is high-purity by 6 kinds of identification
The structural formula for spending monomeric compound is as shown in Figure 5.
Example IV
S1, extraction：Spot lip resupinate woodbetony leaf or root herb crushed after being dried is screened to 80 mesh, with 1：20 spot lip resupinate woodbetony leaf or root herbs with
The solid-liquid ratio of ethanol solution is simultaneously 70% ethanol solution refluxing extraction 4 times at a temperature of 60 DEG C with mass concentration, and during extraction
Between be 2h, obtain crude extract after extract solution is concentrated under reduced pressure.
S2, enrichment：Extracted after the crude extract deionized water dissolving obtained in extraction step, then with ethyl acetate solution
Remove small polar compound, and extraction times are 8 times, and by D3520 types large pore resin absorption column on remainder, successively with 15
Ethanol that the deionized water of times bed volume, the mass concentration of 15 times of bed volumes are 20%, the quality of 15 times of bed volumes are dense
The mass concentration spent for 30% ethanol and 15 times of bed volumes is 40% ethanol elution, wherein 40% ethanol elution part
For the eluent containing target component, by the eluent containing target component through being concentrated under reduced pressure into constant weight, produce containing target into
The enriched substance divided.
S3, isolate and purify：(1), using online DPPH-HPLC antioxidant active ingredients screening technique from spot lip resupinate woodbetony leaf or root just
Butanol fraction filters out 6 compounds with obvious antioxidation activity.
Wherein, online anti-oxidant screening conditions, using Agilent 1100 (250 × 4.6mmi.d., 5 μm) reverse-phase chromatography
Post, mobile phase are methanol solution, and the mass concentration of methanol is 48%, isocratic elution, and the gradient elution time is 40min, mobile phase
Flow velocity is 1ml/min, Detection wavelength 330, the dissolving of the μ g/ml methanol of DPPH solution concentrations 25, flow rate of mobile phase 0.45ml/min,
Detection uses Agilent 1260, DPPH reactions ring length 15m, Detection wavelength 517nm.
(2) 6 kinds of high-purity monomer compounds, are isolated and purified out using high-speed counter-current chromatograph, wherein, inverse by high speed
When flow chromatography instrument is separated, the solvent system that uses for ethyl acetate-n-butanol-water system in chloroform, n-butanol, the body of water
Product is than being 14：4：9, at a temperature of 40 DEG C, eluted using ethyl acetate-n-butanol-water volume system, and high-speed counter-current
Chromatographic rotating speed is 850r/min, so as to isolate and purify out 6 kinds of high-purity monomer compounds, and it is high-purity by 6 kinds of identification
The structural formula for spending monomeric compound is as shown in Figure 5.
Embodiment five
S1, extraction：Spot lip resupinate woodbetony leaf or root herb crushed after being dried is screened to 100 mesh, with 1：30 spot lip resupinate woodbetony leaf or root herbs
It is 90% ethanol solution refluxing extraction 5 times with the solid-liquid ratio of ethanol solution and with mass concentration at a temperature of 80 DEG C, and extracts
Time is 3h, and crude extract is obtained after extract solution is concentrated under reduced pressure.
S2, enrichment：Extracted after the crude extract deionized water dissolving obtained in extraction step, then with ethyl acetate solution
Remove small polar compound, and extraction times are 10 times, and by X-5 types large pore resin absorption column on remainder, successively with 20
Ethanol that the deionized water of times bed volume, the mass concentration of 20 times of bed volumes are 20%, the quality of 20 times of bed volumes are dense
The mass concentration spent for 30% ethanol and 20 times of bed volumes is 40% ethanol elution, wherein 40% ethanol elution part
For the eluent containing target component, by the eluent containing target component through being concentrated under reduced pressure into constant weight, produce containing target into
The enriched substance divided.
S3, isolate and purify：(1), using online DPPH-HPLC antioxidant active ingredients screening technique from spot lip resupinate woodbetony leaf or root just
Butanol fraction filters out 6 compounds with obvious antioxidation activity.
Wherein, online anti-oxidant screening conditions, using Agilent 1100 (250 × 4.6mmi.d., 5 μm) reverse-phase chromatography
Post, mobile phase are methanol solution, and the mass concentration of methanol is 50%, isocratic elution, and the gradient elution time is 45min, mobile phase
Flow velocity is 1ml/min, Detection wavelength 330, the dissolving of the μ g/ml methanol of DPPH solution concentrations 25, flow rate of mobile phase 0.45ml/min,
Detection uses Agilent 1260, DPPH reactions ring length 15m, Detection wavelength 517nm.
(2) 6 kinds of high-purity monomer compounds, are isolated and purified out using high-speed counter-current chromatograph, wherein, inverse by high speed
When flow chromatography instrument is separated, the solvent system that uses for ethyl acetate-n-butanol-water system in chloroform, n-butanol, the body of water
Product is than being 15：5：10, at a temperature of 50 DEG C, eluted using ethyl acetate-n-butanol-water volume system, and high speed is inverse
The rotating speed of flow chromatography instrument is 1000r/min, so as to isolate and purify out 6 kinds of high-purity monomer compounds, and by 6 kinds of height of identification
The structural formula of purity monomer compound is as shown in Figure 5.
In summary：The present invention is by using online DPPH-HPLC antioxidant active ingredients screening technique and using at a high speed
Counter-current chromatograph is used cooperatively, and makes in spot lip resupinate woodbetony leaf or root the screening of six kinds of antioxidant active ingredients and separation method very convenient
With it is rapid, so as to shorten the time of screening and the separation of six kinds of antioxidant active ingredients in spot lip resupinate woodbetony leaf or root, improve production
Efficiency, and the amount of organic solvent used in technique is saved, rise this so as to save into production.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of changes, modification can be carried out to these embodiments, replace without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (7)
1. the screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root, it is characterised in that including following
Step：
S1, extraction：By spot lip resupinate woodbetony leaf or root herb crushed after being dried, then screened, and it is molten with ethanol under the first predetermined temperature
Liquid refluxing extraction, crude extract is obtained after extract solution is concentrated under reduced pressure；
S2, enrichment：After the crude extract deionized water dissolving obtained in extraction step, then extracted and removed with ethyl acetate solution
Small polar compound, and remainder was subjected to column operation, splitter is eluted with the ethanol solution of various concentrations, obtained
To eluent, the eluent is concentrated under reduced pressure, to obtain the enriched substance containing target component；
S3, isolate and purify：(1), using online DPPH-HPLC antioxidant active ingredients screening technique from spot lip resupinate woodbetony leaf or root n-butanol
Part filters out 6 compounds with obvious antioxidation activity；
Wherein, online anti-oxidant screening conditions, using Agilent 1100 (250 × 4.6mmi.d., 5 μm) reverse-phase chromatographic column, stream
Dynamic is mutually methanol solution, and the mass concentration of methanol is 45-50%, isocratic elution, and the gradient elution time is 0-45min, mobile phase
Flow velocity is 1ml/min, Detection wavelength 330, the dissolving of the μ g/ml methanol of DPPH solution concentrations 25, flow rate of mobile phase 0.45ml/min,
Detection uses Agilent 1260, DPPH reactions ring length 15m, Detection wavelength 517nm；
(2) this 6 kinds of high-purity monomer compounds, have been isolated and purified out using high-speed counter-current chromatograph；
Wherein, when being separated by high-speed counter-current chromatograph, ethyl acetate-n-butanol-water is used in the second predetermined temperature
Volume system is eluted.
2. the screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root according to claim 1,
It is characterized in that：Be 20-100 mesh sieves from sieve aperture scope during screening in the extraction step, and spot lip resupinate woodbetony leaf or root herb with
The solid-liquid ratio of ethanol solution is 1:3-1:30, and the use of the mass concentration of ethanol in ethanol solution is 0%-90%, and first is pre-
Constant temperature degree is 20 DEG C -80 DEG C, and extraction time is 1-5 times, extracts 0.5h-3h every time.
3. the screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root according to claim 1,
It is characterized in that：In the enriching step, the number for making to be extracted with ethyl acetate is 3-10 times, and the splitter is using nonpolar
Macroporous resin column, the non-polar macroporous resin post include D101 types macroreticular resin, AB-8 types macroreticular resin, D3520 type macropores
Resin and X-5 type macroreticular resins.
4. the screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root according to claim 1,
It is characterized in that：In the enriching step, the volume of the ethanol solution is 5 times -20 of the volume of the splitter center pillar bed
Times.
5. the screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root according to claim 1,
It is characterized in that：In the enriching step, the mass concentration of ethanol is 50% in the ethanol solution.
6. the screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root according to claim 5,
It is characterized in that：In the enriching step, before using mass concentration to be eluted for 50% ethanol solution to splitter, according to
It is secondary be 30% with deionized water, mass concentration ethanol solution and mass concentration be 40% ethanol solution splitter is washed
It is de-, and the volume of the deionized water is 5 times -20 times of the volume of the splitter center pillar bed, the mass concentration is 30%
The volume of ethanol solution be 5 times -20 times of volume of the splitter center pillar bed, the ethanol that the mass concentration is 40% is molten
The volume of liquid is 5 times -20 times of the volume of the splitter center pillar bed.
7. the screening of six kinds of antioxidant active ingredients and separation method in a kind of spot lip resupinate woodbetony leaf or root according to claim 1,
It is characterized in that：In the purification procedures, chloroform, n-butanol, the body of water in the ethyl acetate-n-butanol-water system
Product ratio is 10-15：2-5：8-10, and the second preheating temperature is 20 DEG C -50 DEG C, and the rotating speed of high-speed counter-current chromatograph is 500-
1000r/min。
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| DE2609533B2 (en) * | 1975-03-07 | 1980-02-21 | Societe Civile De Recherches Et D' Etudes Nouvelles S.C.R.E.E.N., Paris | Process for the extraction of active substances, in particular of heteroside esters of caffeic acid, as well as medicaments containing these compounds |
| CN103613621A (en) * | 2013-12-04 | 2014-03-05 | 中国科学院西北高原生物研究所 | Method for preparing verbascoside and isoacteoside in pedicularis longiflora |
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| DE2609533B2 (en) * | 1975-03-07 | 1980-02-21 | Societe Civile De Recherches Et D' Etudes Nouvelles S.C.R.E.E.N., Paris | Process for the extraction of active substances, in particular of heteroside esters of caffeic acid, as well as medicaments containing these compounds |
| CN103613621A (en) * | 2013-12-04 | 2014-03-05 | 中国科学院西北高原生物研究所 | Method for preparing verbascoside and isoacteoside in pedicularis longiflora |
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