CN105924420B - The method that Quercetin and phloretin are extracted from Camellia Leaves - Google Patents
The method that Quercetin and phloretin are extracted from Camellia Leaves Download PDFInfo
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- CN105924420B CN105924420B CN201610279409.0A CN201610279409A CN105924420B CN 105924420 B CN105924420 B CN 105924420B CN 201610279409 A CN201610279409 A CN 201610279409A CN 105924420 B CN105924420 B CN 105924420B
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- phloretin
- quercetin
- camellia leaves
- methanol
- silica gel
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- 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 title claims abstract description 100
- VGEREEWJJVICBM-UHFFFAOYSA-N phloretin Chemical compound C1=CC(O)=CC=C1CCC(=O)C1=C(O)C=C(O)C=C1O VGEREEWJJVICBM-UHFFFAOYSA-N 0.000 title claims abstract description 96
- 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 title claims abstract description 50
- 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 title claims abstract description 50
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 229960001285 quercetin Drugs 0.000 title claims abstract description 50
- 235000005875 quercetin Nutrition 0.000 title claims abstract description 50
- ZWTDXYUDJYDHJR-UHFFFAOYSA-N (E)-1-(2,4-dihydroxyphenyl)-3-(2,4-dihydroxyphenyl)-2-propen-1-one Natural products OC1=CC(O)=CC=C1C=CC(=O)C1=CC=C(O)C=C1O ZWTDXYUDJYDHJR-UHFFFAOYSA-N 0.000 title claims abstract description 48
- YQHMWTPYORBCMF-UHFFFAOYSA-N Naringenin chalcone Natural products C1=CC(O)=CC=C1C=CC(=O)C1=C(O)C=C(O)C=C1O YQHMWTPYORBCMF-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 100
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 17
- 239000000287 crude extract Substances 0.000 claims abstract description 11
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 9
- 239000002904 solvent Substances 0.000 claims abstract description 7
- 239000011347 resin Substances 0.000 claims abstract description 6
- 229920005989 resin Polymers 0.000 claims abstract description 6
- 238000010828 elution Methods 0.000 claims description 43
- 235000019441 ethanol Nutrition 0.000 claims description 42
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 21
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 15
- 239000000741 silica gel Substances 0.000 claims description 15
- 229910002027 silica gel Inorganic materials 0.000 claims description 15
- 239000003480 eluent Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 10
- 230000002745 absorbent Effects 0.000 claims description 5
- 239000002250 absorbent Substances 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 10
- 239000000284 extract Substances 0.000 abstract description 8
- 238000000746 purification Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 13
- 239000012071 phase Substances 0.000 description 13
- 229960001866 silicon dioxide Drugs 0.000 description 12
- 238000004809 thin layer chromatography Methods 0.000 description 11
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 241001122767 Theaceae Species 0.000 description 7
- 239000003921 oil Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- -1 flavone compound Chemical class 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000007445 Chromatographic isolation Methods 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000377 silicon dioxide Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229930003944 flavone Natural products 0.000 description 2
- 235000011949 flavones Nutrition 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 2
- 241000526900 Camellia oleifera Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 238000011106 process-scale chromatography Methods 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The method that the present invention provides a kind of to extract Quercetin and phloretin from Camellia Leaves.Using ethanol solution as solvent, Camellia Leaves are extracted, obtain crude extract；It is detached with D101 macroporous adsorbing resin for purification, then is detached through the different silica gel column chromatography of mobile phase used twice, most afterwards through DAC chromatography post separations, you can obtain the mixture for containing only phloretin and Quercetin.The present invention has successfully extracted Quercetin and phloretin from Camellia Leaves for the first time, the former extracts from Camellia Plants for the first time.The extracting method has many advantages, such as that high income, product purity are high.
Description
Technical field
The present invention relates to medicine extractive technique fields, and Quercetin and phloretin are extracted from Camellia Leaves more particularly, to one kind
Method.
Background technology
Camellia Leaves are the leaf of plant of theaceae oil tea, rich in a variety of effective components, especially flavone compound, glycoside
Compound.Flavone compound has a multiple biological activities, such as cardiovascular system activity, antibacterial and antiviral activity, anti-swollen
Tumor activity, resisting oxidation free radical activity, analgesic activities, liver-protecting activity etc..Glycosides compound has expelling phlegm and arresting coughing, antitumor, anti-
The bioactivity such as fungi, antibacterial and norcholesterol.
How the bioactive ingredients such as Quercetin are extracted from Camellia Leaves, it is of crucial importance to deep development oil tea crop.
Invention content
The purpose of the present invention is to provide the methods of extraction Quercetin and phloretin, and the extracting method is for the first time from oil tea
Quercetin and phloretin are successfully extracted in leaf, and the extracting method has many advantages, such as that high income, product purity are high.
In order to solve the above technical problems, the present invention provides following technical schemes：
The method that Quercetin and phloretin are extracted from Camellia Leaves, includes the following steps：
A：Using ethanol solution as solvent, Camellia Leaves are extracted, obtain crude extract；
B：With D101 macroporous absorbent resin adsorption and enrichments, water, 15-25wt% ethanol solutions and 75-85wt% second are used successively
Alcoholic solution carries out constant elution to the crude extract；
C：The eluent that the 75-85wt% ethanol solutions are afforded carries out silica gel column chromatography separation, with 9-11: 1 to 1
: the methylene chloride-methanol of 2.5-3.5 gradients carries out gradient elution, and Fractional Collections eluent, through chromatography, merging contains root
The fraction section of Pi Su, Quercetin are named as the first sequence component；
D：The first sequence component is subjected to silica gel column chromatography separation, with the dichloromethane-of 45-55: 1 to 9-11: 1 gradient
Methanol carries out gradient elution, and Fractional Collections eluent merges the fraction section containing phloretin, Quercetin, name through chromatography
For the second sequence component；
E：Using DAC chromatographic columns, the methanol-water with 55-65% to 85-95% gradients is mobile phase to second sequence
Component carries out gradient elution, and Fractional Collections eluent merges the fraction section containing phloretin, Quercetin through chromatography.
Said extracted method is gradually isolated the mixture of Quercetin and phloretin by the thick mode to essence from Camellia Leaves.
On the basis of drier oil tealeaves, the present invention to total recovery rate of Quercetin and phloretin in 78.4mg/kg or more, and
The purity of product is 90% or more.
The ratio of mobile phase (eluting liquid used) of the present invention refers both to volume ratio, and " % " in mobile phase refers to
Percentage by volume shared by non-water substance, ethanol solution refer both to the aqueous solution of ethyl alcohol, for example, 15-25% ethanol solutions refer to ethyl alcohol
Percent by volume is 15-25% aqueous solutions.The aqueous solution that 55% methanol-water nail alcohol percent by volume is 55%.
Constant elution of the present invention refers to that the composition ratio of mobile phase is fixed.
Gradient elution of the present invention refers to constantly changing the concentration proportioning of mobile phase in elution process, but originate dense
Degree and endpoint concentration are fixed, such as the gradient elution in step C refers to：Methylene chloride-methanol is 9- according to initial concentration
11: 1, the mode that elution endpoint concentration is 1: 2.5-3.5 carries out gradient elution.
DAC chromatographic columns spindle of the present invention is to dynamic compression process-scale chromatography post.
Extracting method of the present invention is suitable for the Camellia Leaves of different genera, is especially suitable for C. olelfera (Camellia
Oleifera Abel), recovery rate is high.
Each step of extracting method of the present invention can be further improved, such as：
In step A, used solvent, solid-liquid ratio, temperature are extracted to the impurity content and active ingredient in crude extract
Recovery rate all has an impact.Solvent preferably uses the ethanol-water solution of 40-60%, more preferable 50% ethanol-water solution.Solid-liquid ratio
Preferably 1：2.5-3.5 (1g solids：2.5-3.5mL solvent), more preferable 1：2.5-3.Extraction temperature is preferably 70-80 DEG C, and
And it is best to be extracted with reflux.The impurity content in crude extract can be reduced using the above extracting condition, improve Quercetin, phloretin
Recovery rate.
In addition, in order to reduce the difficulty of later stage separation, it can be concentrated and dried crude extract after leaching, remove ethyl alcohol.
In step B, the volume of mobile phase used in each gradient elution is preferably 3.5-4.5 times of column volume, more preferably
4-4.5 times, ensure that ingredient to be extracted can be fully eluted out.The preferred water of concentration, the 15-20% ethyl alcohol of mobile phase used are molten
Liquid, 75-80% ethanol solutions.
In step C, silica gel column chromatography is separated into separation means, and chromatography is to evaluate the means of separating resulting, and the two is mutual
Cooperation, filters out the eluent containing two Quercetin, phloretin Objective extraction objects.Screening is usually coarse sizing, is usually passed through
The similar eluent of chromatogram is first merged, then compares with the standard items chromatogram of Objective extraction object, therefrom sieve by chromatography
It selects containing there are two the eluent of one of Objective extraction object, is merged.
In the step C, the grain size of silica gel used is preferably 200-300 mesh, and the gradient elution is preferably：Use 9-10
: the methylene chloride-methanol of 1 to 1: 2.5-3 gradient carries out gradient elution can be improved using the condition advanced optimized above
Separating degree and column effect.
In the step C, further include before the silica gel column chromatography separation：The 75-85% ethanol solutions are eluted
The eluent that arrives is concentrated and dried, interference that can be to avoid ethyl alcohol to follow-up silica gel column chromatography.
It can arbitrarily be used in addition, the chromatography in the step C can be thin-layer chromatography (TLC), high performance liquid chromatography etc.
In qualitative method, latter of which is more acurrate.
In addition, when carrying out silica gel column chromatography, among ensureing that material to be separated is dispersed in silica gel, preferably by dichloromethane
Alkane-methanol dilution material to be separated, while admixing silica gel, later water bath method again.By treated above, substance is reloaded into silicon
It is chromatographed in rubber column gel column.
Equally, in the step D, can be through chromatography：Efficient liquid phase chromatographic analysis and/or tlc analysis etc..
In the step D, mobile phase preferably carries out gradient with the methylene chloride-methanol of 45-50: 1 to 9-10: 1 gradient and washes
De-
In the step E, when gradient elution, the initial concentration of mobile phase is preferably 55-60%, and emphasis concentration is preferably
85-90% is imitated with improving separating degree and column.Used chromatography can be efficient liquid phase chromatographic analysis, tlc analysis etc..
Compared with prior art, the present invention can reach following technique effect：
(1) Quercetin of high-purity, the mixture of phloretin are extracted out from Camellia Leaves for the first time；
(2) by slightly to the extracting mode of essence conducive to the large-scale promotion of extracting method；
(3) extract yield is high.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, other drawings may also be obtained based on these drawings.
The liquid chromatogram for the sample collected after 80% ethanol elution that Fig. 1 provides for the embodiment of the present invention 1；
Fig. 2 is CO-7's1H-NMR spectrum；
Fig. 3 is CO-7's13C-NMR spectrograms.
Specific implementation mode
Technical scheme of the present invention is clearly and completely described below in conjunction with the drawings and specific embodiments, but
Be it will be understood to those of skill in the art that it is following described embodiments are some of the embodiments of the present invention, rather than it is whole
Embodiment is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.Based on the embodiments of the present invention, ability
The every other embodiment that domain those of ordinary skill is obtained without making creative work, belongs to guarantor of the present invention
The range of shield.The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same
Or production firm person is not specified in instrument, is the conventional products that can be obtained by commercially available purchase.
The ratio of heretofore described mobile phase refers both to volume ratio.
Embodiment 1
The first step：
Naturally the oil tea dried leaf dried in the shade be crushed into 60 mesh sieve, take 20kg in two batches with 50% ethyl alcohol by solid-liquid ratio 1: 3
It is heated to reflux at 70-80 DEG C, repeats extraction 2 times, extract 2h every time, merge gained supernatant, filtering obtains supernatant, and decompression is dense
It is reduced to no alcohol taste, obtains the original volume crude extract sample of about half, it is spare.
Second step：
With the D101 type macroporous absorbent resins of industrial ethanol postincubation about 10L, no alcohol taste is washed with water, the first step is obtained
Extracting solution loading at twice uses water, 20% ethyl alcohol, 80% ethanol elution, 4 times of column volumes of each gradient elution, according to TLC successively
With HPLC analysis results, each position merges concentration.
(about 150g, HPLC spectrogram are as shown in Figure 1, elution program for the sample concentration drying collected after 80% ethanol elution：
50% methanol aqueous solutions of 0-10min, 80% methanol aqueous solutions of 10-20min, 100% methanol of 20-21min；Detection wavelength
254nm, 10 μ L of sample size), it is dissolved in 10: 1 methylene chloride-methanol, admixes 300g tlc silica gels (200~300
Mesh), water-bath volatilizes, and fills column at twice, wherein each blanket layer filling silica gel about 800g.
Flow phase system is methylene chloride-methanol (10: 1 to 1: 3 gradient elution), is collected as once per 500mL, is passed through
TLC combining data detections are that 1-2+2 ' (is indicated：By first fraction, second fraction and second of silica gel of first time silicagel column
Second fraction of column merges), 3-7+3 ' -7 ', 8-10+8 ' -10 ', 11-13+11 ', 14-16+12 ' -13 ', 17-19+14 ',
20-22+15 ' -18 ', 19 ' -21 ', 23-26+22 ' -27 ', 27-40,28 ' -34 ', 35 ' -44 ', 41-47,48-64,65,66-
67,68-70,71,72-78,45 ' -51 ', 52 ' -63 ', 64 ' -67 ', 68 ' -71 ', 72 ' -84 ' amount to 24 component (silicon twice
The fraction of rubber column gel column with the subsequent symbol of serial number " ' " be distinguish, part fraction carries out cross-combining, with plus sige linking (with primary
Fraction use-linking of silicagel column), for example, for the first time the extracting solution of dress column after elution, the liquid of collection number is respectively 1,
2 ... 78, second of extracting solution for filling column is after elution, the liquid number respectively 1 ', 2 ' ... 84 ' of collection).
Third walks：
3-7+3 ' -7 ' utilize silica gel (200-300 mesh) chromatography, 50：1 to 10：1 methanol-water carries out gradient elution,
It is analyzed through TLC, merges the similar fraction of chromatogram, and the fraction at least containing one of Quercetin and phloretin is merged, according to
The sequencing of elution, number 12-14.
4th step：
During third is walked 12-14 components using DAC prepare chromatographic isolation, 60% to 90% methanol-water gradient elution, in conjunction with
HPLC is analyzed, obtain containing only two monomer compound of quercetin, phloretin mixture (abbreviation CO-7), total 8.4mg.
5th step：Characterize extract
CO-7 is subjected to Mass Spectrometer Method.
CO-7 is subjected to nuclear magnetic resonance map detection, CO-7's1H-NMR、13C-NMR difference is as shown in Figures 2 and 3.
1In H-NMR, it is seen that one group of phenyl ring meta position proton signal δ 6.15 (1H, brs) and δ 6.35 (1H, brs) return respectively
Belong to the H-6 and H-8 of Quercetin, the aromatic signal δ 6.86 (1H, d, J=8.3Hz) of one group of ABX Coupling System, δ 7.60
(1H, d, J=8.3Hz) and δ 7.71 (1H, s) are respectively belonging to the H-2 ', H-3 ' and H-6 ' of Quercetin；Between one group of symmetrical phenyl ring
Position proton signal δ 5.79 (2H, brs) belongs to the H-6 and H-8 of phloretin, the aromatic letter of one group of AA ' BB ' Coupling System
Number δ 6.67 (2H, d, J=8.3Hz), δ 7.01 (2H, d, J=8.3Hz) are respectively belonging to the H-2 ' of phloretin, 6 ' and H-3 ',
6 ', the proton signal δ 3.21 (1H, m) and δ 2.81 (1H, m) of one group of A2B2 Coupling System be respectively belonging to phloretin H- α and
H-β.13In C-NMR, Quercetin and the complete carbon signal of phloretin can be found, the two ratio is about 2:1, by carbon spectrum and hydrogen
Modal data is compareed with the chromatogram of the Quercetin of existing literature report, phloretin respectively, almost the same.Therefore, it is possible to identify CO-7
For the mixture of Quercetin and phloretin.
As a result prove that CO-7 contains two kinds of compounds of Quercetin and phloretin.
After testing, the purity of CO-7 is 97.5%.
The molecular formula such as following formula (one) of Quercetin, the molecular formula such as following formula of phloretin can be determined in conjunction with above-mentioned characterization result
(2).
Embodiment 2
The first step：
Naturally the oil tea dried leaf dried in the shade be crushed into 60 mesh sieve, take 20kg in two batches with 40% ethyl alcohol by solid-liquid ratio 1:
2.5 are heated to reflux at 70-80 DEG C, repeat extraction 2 times, extract 2h every time, merge gained supernatant, and filtering obtains supernatant, subtracts
Pressure is concentrated into no alcohol taste, obtains the original volume crude extract sample of about half, spare.
Second step：
With the D101 type macroporous absorbent resins of industrial ethanol postincubation about 10L, no alcohol taste is washed with water, the first step is obtained
Extracting solution loading at twice, uses water, 15% ethyl alcohol, 75% ethanol elution successively, 4.5 times of column volumes of each gradient elution, according to
TLC and HPLC analysis results, each position merge concentration.
The sample concentration drying (about 150g) collected after 80% ethanol elution, is dissolved in 10: 1 methylene chloride-methanol,
300g tlc silica gels (200~300 mesh) are admixed, water-bath volatilizes, and fills column at twice, wherein each blanket layer filling silica gel is about
800g。
Flow phase system is methylene chloride-methanol (9: 1 to 1: 2.5 gradient elution), is collected as once per 500mL, is passed through
TLC combining data detections contain the fraction section of at least one of Quercetin, phloretin, are named as the first sequence component.
Third walks：
First sequence component utilize silica gel (200-300 mesh) chromatography, 45：1 to 9：1 methanol-water carries out gradient and washes
It is de-, it is analyzed through TLC, merges the similar fraction of chromatogram, and the fraction at least containing one of Quercetin and phloretin merged, life
Entitled second sequence component.
4th step：
During third is walked the second sequence component using DAC prepare chromatographic isolation, 55% to 85% methanol-water gradient elution,
Analyzed in conjunction with HPLC, obtain containing only two monomer compound of quercetin, phloretin mixture (abbreviation CO-7), total 8.7mg.
5th step：Characterize extract
Equally, using mass spectrum and1H-NMR、13C-NMR is characterized, as a result same as Example 1, CO-7 be Quercetin,
The mixture of phloretin.
Embodiment 3
The first step：
Naturally the oil tea dried leaf dried in the shade be crushed into 60 mesh sieve, take 20kg in two batches with 60% ethyl alcohol by solid-liquid ratio 1:
3.5 are heated to reflux at 70-80 DEG C, repeat extraction 2 times, extract 2h every time, merge gained supernatant, and filtering obtains supernatant, subtracts
Pressure is concentrated into no alcohol taste, obtains the original volume crude extract sample of about half, spare.
Second step：
With the D101 type macroporous absorbent resins of industrial ethanol postincubation about 10L, no alcohol taste is washed with water, the first step is obtained
Extracting solution loading at twice, uses water, 25% ethyl alcohol, 85% ethanol elution successively, 3.5 times of column volumes of each gradient elution, according to
TLC and HPLC analysis results, each position merge concentration.
The sample concentration drying (about 150g) collected after 80% ethanol elution, is dissolved in 10: 1 methylene chloride-methanol,
300g tlc silica gels (200~300 mesh) are admixed, water-bath volatilizes, and fills column at twice, wherein each blanket layer filling silica gel is about
800g。
Flow phase system is methylene chloride-methanol (11: 1 to 1: 3.5 gradient elution), is collected as once per 500mL, is passed through
TLC combining data detections contain the fraction section of at least one of Quercetin, phloretin, are named as the first sequence component.
Third walks：
First sequence component utilize silica gel (200-300 mesh) chromatography, 55：1 to 11：1 methanol-water carries out gradient and washes
It is de-, it is analyzed through TLC, merges the similar fraction of chromatogram, and the fraction at least containing one of Quercetin and phloretin merged, life
Entitled second sequence component.
4th step：
During third is walked the second sequence component using DAC prepare chromatographic isolation, 65% to 95% methanol-water gradient elution,
Analyzed in conjunction with HPLC, obtain containing only two monomer compound of quercetin, phloretin mixture (abbreviation CO-7), total 8.1mg.
5th step：Characterize extract
Equally, using mass spectrum and1H-NMR、13C-NMR is characterized, as a result same as Example 1, CO-7 be Quercetin,
The mixture of phloretin.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, it will be understood by those of ordinary skill in the art that：Its according to
So can with technical scheme described in the above embodiments is modified, either to which part or all technical features into
Row equivalent replacement；And these modifications or replacements, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (12)
1. the method for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that include the following steps：
A：Using ethanol solution as solvent, Camellia Leaves are extracted, obtain crude extract；
B：With D101 macroporous absorbent resin adsorption and enrichments, use water, 15-25wt% ethanol solutions and 75-85wt% ethyl alcohol molten successively
Liquid carries out constant elution to the crude extract；
C：The eluent that the 75-85wt% ethanol solutions are afforded carries out silica gel column chromatography separation, with 9-11: 1 to 1:
The methylene chloride-methanol of 2.5-3.5 gradients carries out gradient elution, and Fractional Collections eluent, through chromatography, merging contains root skin
The fraction section of element, Quercetin, is named as the first sequence component；
D：The first sequence component is subjected to silica gel column chromatography separation, with the methylene chloride-methanol of 45-55: 1 to 9-11: 1 gradient
Gradient elution is carried out, Fractional Collections eluent merges the fraction section containing phloretin, Quercetin through chromatography, is named as the
Two sequence components；
E：Using DAC chromatographic columns, the methanol-water with 55-65% to 85-95% gradients is mobile phase to the second sequence component
Gradient elution is carried out, Fractional Collections eluent merges the fraction section containing phloretin, Quercetin through chromatography.
2. the method according to claim 1 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that the step
In rapid A, the method for extraction is：Solid-liquid ratio is 1：3, the refluxing extraction at 70-80 DEG C.
3. the method according to claim 2 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that the step
In rapid A, ethanol solution is the ethanol-water solution of 40-60%.
4. the method according to claim 1 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that the step
In rapid B, the volume of mobile phase used in each gradient elution is 3.5-4.5 times of column volume.
5. the method according to claim 1 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that the step
In rapid C, the grain size of silica gel used is 200-300 mesh.
6. the method according to claim 5 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that the ladder
Degree elutes：Gradient elution is carried out with the methylene chloride-methanol of 9-10: 1 to 1: 2.5-3 gradient.
7. the method according to claim 1 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that the step
In rapid D, the grain size of silica gel used is 200-300 mesh.
8. the method according to claim 1 or claim 7 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that institute
It states in step D, the gradient elution is：Gradient elution is carried out with 50: 1~10: 1 methylene chloride-methanol.
9. the method according to claim 1 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that the step
In rapid E, the methanol-water with 60% to 90% gradient is that mobile phase carries out gradient elution to the second sequence component.
10. the method according to claim 1 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that in institute
It states in step C, further includes before the silica gel column chromatography separation：The eluent that the 75-85wt% ethanol solutions are afforded
It is concentrated and dried.
11. the method according to claim 10 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that in institute
It states in step C, further includes before being detached with the silica gel column chromatography after the concentrate drying：It is dissolved in methylene chloride-methanol
In, and silica gel is admixed, water-bath later removes solvent.
12. the method according to claim 1 for extracting Quercetin and phloretin from Camellia Leaves, which is characterized in that in institute
It states in step A, further includes after the extraction：Concentration removes alcohol.
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| CN103336069A (en) * | 2013-06-13 | 2013-10-02 | 江苏省农业科学院 | HPLC (High Performance Liquid Chromatography) determination method of phenolic compounds in peach fruit |
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| CN103336069A (en) * | 2013-06-13 | 2013-10-02 | 江苏省农业科学院 | HPLC (High Performance Liquid Chromatography) determination method of phenolic compounds in peach fruit |
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