CN108191804B - The method of purification of Flavonoid substances Quercetin in a kind of Guava Leaf - Google Patents
The method of purification of Flavonoid substances Quercetin in a kind of Guava Leaf Download PDFInfo
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- CN108191804B CN108191804B CN201711466673.6A CN201711466673A CN108191804B CN 108191804 B CN108191804 B CN 108191804B CN 201711466673 A CN201711466673 A CN 201711466673A CN 108191804 B CN108191804 B CN 108191804B
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- guava leaf
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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 60
- 241000508269 Psidium Species 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000000746 purification Methods 0.000 title claims abstract description 34
- 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 30
- 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 30
- 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 30
- 229960001285 quercetin Drugs 0.000 title claims abstract description 30
- 235000005875 quercetin Nutrition 0.000 title claims abstract description 30
- 239000000126 substance Substances 0.000 title claims abstract description 20
- 229930003935 flavonoid Natural products 0.000 title claims abstract description 19
- 150000002215 flavonoids Chemical class 0.000 title claims abstract description 19
- 235000017173 flavonoids Nutrition 0.000 title claims abstract description 19
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229930003944 flavone Natural products 0.000 claims abstract description 19
- 235000011949 flavones Nutrition 0.000 claims abstract description 19
- 239000000178 monomer Substances 0.000 claims abstract description 18
- 229920000344 molecularly imprinted polymer Polymers 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 150000002213 flavones Chemical class 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 10
- 239000012043 crude product Substances 0.000 claims abstract description 10
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 10
- 238000005119 centrifugation Methods 0.000 claims abstract description 9
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- 239000000284 extract Substances 0.000 claims abstract description 7
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- 239000006228 supernatant Substances 0.000 claims abstract description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 26
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- 239000012528 membrane Substances 0.000 claims description 16
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- 239000003431 cross linking reagent Substances 0.000 claims description 12
- 239000003999 initiator Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 8
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 7
- 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 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 claims description 7
- ZCHPKWUIAASXPV-UHFFFAOYSA-N acetic acid;methanol Chemical compound OC.CC(O)=O ZCHPKWUIAASXPV-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
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- 238000012719 thermal polymerization Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 4
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- 230000008676 import Effects 0.000 claims description 4
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- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 238000007873 sieving Methods 0.000 claims description 2
- ICBJBNAUJWZPBY-UHFFFAOYSA-N 2-hydroxyethyl 3-methylbut-2-enoate Chemical group CC(=CC(=O)OCCO)C ICBJBNAUJWZPBY-UHFFFAOYSA-N 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 9
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- 241000219926 Myrtaceae Species 0.000 description 1
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- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 244000236580 Psidium pyriferum Species 0.000 description 1
- 235000013929 Psidium pyriferum Nutrition 0.000 description 1
- 244000294611 Punica granatum Species 0.000 description 1
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- 239000002253 acid Substances 0.000 description 1
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- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 125000004356 hydroxy functional group Chemical group O* 0.000 description 1
- 230000001631 hypertensive effect Effects 0.000 description 1
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- 150000002632 lipids Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
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- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
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- 239000002994 raw material Substances 0.000 description 1
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- STECJAGHUSJQJN-FWXGHANASA-N scopolamine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-FWXGHANASA-N 0.000 description 1
- 229960002646 scopolamine Drugs 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000003883 substance clean up Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 1
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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
- 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)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of method of purification of Flavonoid substances Quercetin in Guava Leaf, include the following steps: that water is added after (1) crushes Guava Leaf, heat, filtering obtains guava leaf extract flavones crude product；(2) it is dissolved in ethanol solution, takes supernatant to carry out ultrafiltration circulation after filtering, obtains ultrafiltration membrance filter solution；(3) it is dissolved in acetonitrile using Quercetin monomer as template molecule, function monomer 4-vinylpridine is added and prepares molecularly imprinted polymer；(4) ultrafiltration membrance filter solution is added in molecularly imprinted polymer, closed concussion is carried out in constant-temperature table, the Flavonoid substances Quercetin is obtained after centrifugation.Method of purification of the invention, the feed liquid separated further refine purification with the imprinted polymer of synthesis again, obtain the Guava Leaf Flavonoid substances Quercetin of purifying, simple process environmental protection, income at low cost is high, and refining effect is good.
Description
Technical field
The invention belongs to Flavonoid substances quercitrins in Guava Leaf compound purification field more particularly to a kind of Guava Leaf
The method of purification of element.
Background technique
Guava (Psidium guajava Linn.) Myrtaceae arbor originates in South America.In South China various regions plant
Training, common to have ease for wild species, north reaches the Anning Valley in Sichuan Province of South-west Sichuan, is born on wasteland or haugh；Fruit is edible；Ye Han
Volatile oil and tannin etc., hyoscine have stop dysentery, hemostasis, stomach invigorating and other effects；Ye Jing, which boils, removes tannin, dries and is used as tealeaves, taste
It is sweet, there is heat-clearing effect.The chemical constitution study of Guava Leaf is shown: containing protein, polysaccharide, polyphenol, Huang in Guava Leaf
Ketone compounds and triterpene compound etc..
Flavone compound (flavonoids) is that one kind is present in nature, has 2- phenyl chromone
(flavone) compound of structure.Have a ketone carbonyl group in their molecules, oxygen atom on first has alkalinity, can with it is strong
Acid is at salt, and hydroxy derivatives have a yellow more, therefore also known as flavone or flavones.Flavone compound in plant usually with sugar
It is combined into glycoside, fraction exists in the form of free state (aglycon).All contain flavone compound in most plants,
It plant growth, develop, bloom, result and antibacterial in terms of play an important role.
Quercetin also known as quercetin are one kind of flavone compound, and Quercetin is dissolved in glacial acetic acid, and alkaline aqueous solution is in
Yellow, is practically insoluble in water, and ethanol solution taste is very bitter.It can be used as drug, there is preferable eliminating the phlegm, antitussive action, and have certain
Antiasthmatic effect.In addition there are reduce blood pressure, enhance capillary resistance, reduction capillary fragility, reducing blood lipid, expansion hat
The effects of shape artery, increase coronary blood flow.For treating chronic bronchitis, also there is auxiliary to coronary heart disease and hypertensive patient
Therapeutic effect.
The separation of traditional guava leaf extract mainly uses solvent method, macroreticular resin absorbing method, microwave loss mechanisms, surpasses
Sound wave extraction method, supercritical extraction, but these method and processes are complicated, refining effect is poor, energy-saving and environment-friendly requirement is not met, at
This height and income is low.And capillary electrophoresis, enzyme process are generally used for purifying micro or trace substance, to purification condition require compared with
Height is not suitable for industrial purification.Therefore it researches and develops new purification technique and is used for industry, to obtain higher general flavone quality point
Several guava leaf extract products is particularly significant.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the shortcomings of to mention in background above technology and defect, provide one
The method of purification of Flavonoid substances Quercetin in kind Guava Leaf.
In order to solve the above technical problems, technical solution proposed by the present invention is to provide Flavonoid substances in a kind of Guava Leaf
The method of purification of Quercetin, includes the following steps:
(1) by Guava Leaf grinding and sieving, water is added in obtained Guava Leaf powder, heats, filtering obtains a kind stone
Pomegranate leaf extract flavones crude product；
(2) the flavones crude product obtained after the step (1) is dissolved in ethanol solution, takes supernatant to import after filtering super
It filters and carries out ultrafiltration circulation in device, obtain ultrafiltration membrance filter solution；
(3) it is dissolved in acetonitrile, is added after function monomer 4-vinylpridine in room using Quercetin monomer as template molecule
The lower stirring of temperature, acts on template molecule sufficiently with function monomer, crosslinking agent and initiator is then added, is surpassed after mixing well
Sound is passed through nitrogen and forms oxygen-free environment to remove the oxygen of dissolution, and sealing, which is placed in thermostatical oil bath, carries out thermal polymerization, polymerize
Product is eluted in Soxhlet extractor with methanol-acetic acid solution, and to remove template molecule, again with methanol washes away excessive acetic acid,
It is dried in vacuo in vacuum drying oven, the molecularly imprinted polymer after being synthesized；
(4) molecularly imprinted polymer obtained after the step (3) synthesis is accurately weighed, is obtained after the step (2) are added
The ultrafiltration membrance filter solution arrived, carries out closed concussion in constant-temperature table, obtains the molecularly imprinted polymer for being adsorbed with Quercetin,
The Flavonoid substances Quercetin is obtained after centrifugation, the molecularly imprinted polymer after centrifugation may be reused.
Above-mentioned method of purification, it is preferred that in the step (1), be sieved for 40-60 mesh, the water volume of addition be
38-42 times of Guava Leaf powder product.
Preferably, in the step (1), heating temperature is 50-70 DEG C, heating time 25-30min.
Preferably, in the step (2), ultrafiltration recycles the film that the film used is 10000 for retention relative molecular mass.
Preferably, in the step (2), the time of ultrafiltration circulation is 2-3h, pressure 0.3-0.9MPa, temperature 30-
60℃。
Preferably, in the step (3), acetonitrile is no less than 40mL, and mixing time is no less than 4h, ultrasonic time 10-
20min, being passed through the nitrogen time is 20-30min.
Preferably, in the step (3), crosslinking agent is ethylene glycol dimethacrylate (EDMA), and initiator is azo
Bis-isobutyronitrile (AIBN), the dosage of the initiator are the 1-2% of function monomer and crosslinking agent gross mass.
Preferably, in the step (3), oil bath pot temperature is 50-70 DEG C, and the thermal polymerization time is 22-26h, methanol-acetic acid
The volume ratio of solution is methanol: acetic acid=9: 1, elution time 46-50h, and vacuum drying temperature is 30-50 DEG C, vacuum drying
Time is 22-26h.
Preferably, in the step (4), ultrafiltration membrance filter solution 10mL is added in every 30mg molecularly imprinted polymer.
Preferably, in the step (4), the temperature of constant-temperature table is 30-50 DEG C, and the concussion time is 22-26h.
Membrane separation technique and molecular imprinting technology are the purification new technologies researched and developed in recent years.Membrane separation technique has normal
Temperature operation, selectivity is high, adaptable, simple process, pollutes the advantages that few.Molecular imprinting technology be according to antibody and antigen,
Enzyme-to-substrate etc. is principle there are the Molecular Recognization of nature, has identification target point using target molecule as templated synthesis
A kind of technology of the macromolecule imprinted polymer of son.Since the impurity contained in flavones crude product is more, the present invention is by UF membrane
Technology is combined with molecular imprinting technology, is separated, is separated to Guava Leaf crude extract using membrane separation technique
Feed liquid further refines purification with the imprinted polymer of synthesis again, obtains the Guava Leaf Flavonoid substances Quercetin of purifying, from
Molecularly imprinted polymer after the heart may be reused.
Compared with prior art, the invention has the benefit that
1, method of purification of the invention combines membrane separation technique with molecular imprinting technology, using membrane separation technique pair
Guava Leaf crude extract is separated, and the feed liquid separated further refines purification with the imprinted polymer of synthesis again, is obtained
The Guava Leaf Flavonoid substances Quercetin that must be purified, simple process environmental protection, income at low cost is high, and refining effect is good.
2, method of purification of the invention, membrane separation technique have normal-temperature operation, high, the adaptable, simple process of selectivity,
The advantages that pollution is few, molecular imprinting technology identify that the accuracy of target molecule is high, and selectivity is high, pollution-free, high reliablity, and
The molecularly imprinted polymer obtained after centrifugation may be reused, and be effectively saved process costs.
Specific embodiment
To facilitate the understanding of the present invention, the present invention is done below in conjunction with preferred embodiment and more comprehensively, is meticulously described,
But protection scope of the present invention is not limited to following specific embodiments.
Unless otherwise defined, all technical terms used hereinafter are generally understood meaning phase with those skilled in the art
Together.Technical term used herein is intended merely to the purpose of description specific embodiment, and it is of the invention to be not intended to limitation
Protection scope.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through city
Field is commercially available or can be prepared by existing method.
Embodiment 1:
The method of purification of Flavonoid substances Quercetin, includes the following steps: in a kind of Guava Leaf of the invention
(1) Guava Leaf is smashed it through into 40 meshes, water is added in obtained Guava Leaf powder, the water volume of addition is
40 times of Guava Leaf powder product, are heated to 60 DEG C and maintain 25min, filter, obtain guava leaf extract flavones crude product；
(2) the flavones crude product obtained after the step (1) is dissolved in ethanol solution, takes supernatant to import after filtering super
It filters and carries out ultrafiltration circulation 2h in device, the film used is the film of retention relative molecular mass 10000, operating pressure 0.3MPa,
The temperature of supernatant is 30 DEG C, obtains ultrafiltration membrance filter solution；
(3) it is dissolved in Quercetin monomer as template molecule in acetonitrile (no less than 40mL), function monomer 4- ethylene is added
Stirring is no less than 4h at room temperature after yl pyridines, acts on template molecule sufficiently with function monomer, and crosslinking agent is then added and draws
Agent is sent out, crosslinking agent is ethylene glycol dimethacrylate, and initiator is azodiisobutyronitrile, and the dosage of the initiator is function
The 1.5% of monomer and crosslinking agent gross mass carries out ultrasound 10min to remove the oxygen of dissolution, is passed through nitrogen after mixing well
20min forms oxygen-free environment, and sealing is placed in 60 DEG C of thermostatical oil baths and carries out thermal polymerization for 24 hours, and polymerizate is with volume ratio
Methanol: acetic acid=9: 1 methanol-acetic acid solution elutes 48h in Soxhlet extractor, and again with methanol washes away excessive acetic acid, in
It is dried in vacuo for 24 hours for 40 DEG C in vacuum drying oven, the molecularly imprinted polymer after being synthesized；
(4) the molecularly imprinted polymer 30mg obtained after the step (3) synthesis is accurately weighed, the step (2) are added
The ultrafiltration membrance filter solution 10mL obtained afterwards carries out closed concussion in 40 DEG C of constant-temperature tables and obtains the Huang for 24 hours, after centrifugation
Letones Quercetin.
It is detected, the rejection after measuring the step (2) is 64.69%, and the flavones mass fraction obtained after purification is
72% in ultrafiltration membrance filter solution, Quercetin maximum apparent adsorption quantity QmaxFor 3.675mg/g.
The test method of rejection: protein content in solution is tested with spectrophotometry, calculates rejection, step is such as
Under:
1. the configuration of standard solution: bovine serum albumin(BSA) is dried under vacuum to constant weight at 105 DEG C of temperature, accurately weighs ox blood
Pure albumen 1.000g is dissolved in 1000ml volumetric flask, draw respectively bovine serum albumin solution 0.5,1.0,1.5,2.0,2.5,
3.0,3.5,4.0,4.5,5.0,6.0,7.0,8.0,9.0,10.0ml is in 10ml volumetric flask plus distilled water is diluted to scale, matches
It is set to the ox that concentration is 50,100,150,200,250,300,350,400,450,500,600,700,800,900,1000mg/l
Seralbumin standard solution.
2. the production of standard curve: protein has absorption maximum to the ultraviolet light of 280nm, and protein solution 280nm absorbs
It is worth directly proportional to its concentration.By 7.3.1 standard solution under wavelength 280nm, with 1cm cuvette, on ultraviolet specrophotometer
Optical density is measured, distilled water is blank.Using protein concentration C as abscissa, absorbance A is ordinate drawing, produces standard song
Line.
3. the preparation of sample solution: selecting the protein of a certain molecular weight, being configured to concentration is 1000mg/l -3000mg/
The protein solution of l uses after ovalbumin solution centrifugation or filtering, and the protein solution of preparation is commented as ultrafiltration film properties
The solution of valence uses.
4. the evaluation of ultrafiltration membrane: configured sample solution is operated under 0.3MPa, normal temperature condition by ultrafiltration membrane,
Ultrafiltrate is collected after 20min, stoste and ultrafiltrate measure optical density in 280nm ultraviolet region respectively, check in from standard curve
Corresponding concentration.
5. the calculating of rejection: rejection=(1-C1/C2) × 100%, wherein C1For ultrafiltrate concentration, C2It is dense for stoste
Degree.
Maximum apparent adsorption quantity calculation method:
It can get the bond type of absorption point, in conjunction with the equilibrium constant and Bmax etc. by Scatchard molecule
Important information.Scatchard equation are as follows:
In formula, Q and QmaxFor balance binding capacity and maximum performance binding capacity；CeFor equilibrium concentration in solution；KDFor bound site
The equilibrium dissociation constant of point.It, can be in the hope of K according to linear relationship slope and intercept when being mapped with Q/C to QDAnd Qmax, two
Parameter.
The method of purification of the present embodiment combines membrane separation technique with molecular imprinting technology, using membrane separation technique pair
Guava Leaf crude extract is separated, and the feed liquid separated further refines purification with the imprinted polymer of synthesis again, is obtained
The Guava Leaf Flavonoid substances Quercetin that must be purified, simple process environmental protection, income at low cost is high, and refining effect is good.
Embodiment 2:
The method of purification of Flavonoid substances Quercetin, includes the following steps: in a kind of Guava Leaf of the invention
(1) Guava Leaf is smashed it through into 60 meshes, water is added in obtained Guava Leaf powder, the water volume of addition is
40 times of Guava Leaf powder product, are heated to 60 DEG C and maintain 30min, filter, obtain guava leaf extract flavones crude product；
(2) the flavones crude product obtained after the step (1) is dissolved in ethanol solution, takes supernatant to import after filtering super
It filters and carries out ultrafiltration circulation 3h in device, the film used is the film of retention relative molecular mass 10000, operating pressure 0.4MPa,
The temperature of supernatant is 50 DEG C, obtains ultrafiltration membrance filter solution；
(3) it is dissolved in Quercetin monomer as template molecule in acetonitrile (no less than 40mL), function monomer 4- ethylene is added
Stirring is no less than 4h at room temperature after yl pyridines, acts on template molecule sufficiently with function monomer, and crosslinking agent is then added and draws
Agent is sent out, crosslinking agent is ethylene glycol dimethacrylate, and initiator is azodiisobutyronitrile, and the dosage of the initiator is function
The 1.5% of monomer and crosslinking agent gross mass carries out ultrasound 20min to remove the oxygen of dissolution, is passed through nitrogen after mixing well
30min forms oxygen-free environment, and sealing is placed in 60 DEG C of thermostatical oil baths and carries out thermal polymerization for 24 hours, and polymerizate is with volume ratio
Methanol: acetic acid=9: 1 methanol-acetic acid solution elutes 48h in Soxhlet extractor, and again with methanol washes away excessive acetic acid, in
It is dried in vacuo for 24 hours for 30 DEG C in vacuum drying oven, the molecularly imprinted polymer after being synthesized；Blank polymerization is prepared in the same way
Object；
(4) the molecularly imprinted polymer 30mg obtained after the step (3) synthesis is accurately weighed, the step (2) are added
The ultrafiltration membrance filter solution 10mL obtained afterwards carries out closed concussion in 40 DEG C of constant-temperature tables and obtains the Huang for 24 hours, after centrifugation
Letones Quercetin.
It is detected, the rejection after measuring the step (2) is 64.37%, and the flavones mass fraction obtained after purification is
71% in ultrafiltration membrance filter solution.Quercetin maximum apparent adsorption quantity QmaxFor 3.038mg/g.
The test method of rejection: protein content in solution is tested with spectrophotometry, calculates rejection, step is such as
Under:
1. the configuration of standard solution: bovine serum albumin(BSA) is dried under vacuum to constant weight at 105 DEG C of temperature, accurately weighs ox blood
Pure albumen 1.000g is dissolved in 1000ml volumetric flask, draw respectively bovine serum albumin solution 0.5,1.0,1.5,2.0,2.5,
3.0,3.5,4.0,4.5,5.0,6.0,7.0,8.0,9.0,10.0ml is in 10ml volumetric flask plus distilled water is diluted to scale, matches
It is set to the ox that concentration is 50,100,150,200,250,300,350,400,450,500,600,700,800,900,1000mg/l
Seralbumin standard solution.
2. the production of standard curve: protein has absorption maximum to the ultraviolet light of 280nm, and protein solution 280nm absorbs
It is worth directly proportional to its concentration.By 7.3.1 standard solution under wavelength 280nm, with 1cm cuvette, on ultraviolet specrophotometer
Optical density is measured, distilled water is blank.Using protein concentration C as abscissa, absorbance A is ordinate drawing, produces standard song
Line.
3. the preparation of sample solution: selecting the protein of a certain molecular weight, being configured to concentration is 1000mg/l -3000mg/
The protein solution of l uses after ovalbumin solution centrifugation or filtering, and the protein solution of preparation is commented as ultrafiltration film properties
The solution of valence uses.
4. the evaluation of ultrafiltration membrane: configured sample solution is operated under 0.3MPa, normal temperature condition by ultrafiltration membrane,
Ultrafiltrate is collected after 20min, stoste and ultrafiltrate measure optical density in 280nm ultraviolet region respectively, check in from standard curve
Corresponding concentration.
5. the calculating of rejection: rejection=(1-C1/C2) × 100%, wherein C1For ultrafiltrate concentration, C2It is dense for stoste
Degree.
Maximum apparent adsorption quantity calculation method:
It can get the bond type of absorption point, in conjunction with the equilibrium constant and Bmax etc. by Scatchard molecule
Important information.Scatchard equation are as follows:
In formula, Q and QmaxFor balance binding capacity and maximum performance binding capacity；CeFor equilibrium concentration in solution；KDFor bound site
The equilibrium dissociation constant of point.It, can be in the hope of K according to linear relationship slope and intercept when being mapped with Q/C to QDAnd QmaxTwo ginsengs
Number.
The method of purification of the present embodiment combines membrane separation technique with molecular imprinting technology, using membrane separation technique pair
Guava Leaf crude extract is separated, and the feed liquid separated further refines purification with the imprinted polymer of synthesis again, is obtained
The Guava Leaf Flavonoid substances Quercetin that must be purified, simple process environmental protection, income at low cost is high, and refining effect is good.
Claims (9)
1. the method for purification of Flavonoid substances Quercetin, includes the following steps: in a kind of Guava Leaf
(1) by Guava Leaf grinding and sieving, water is added in obtained Guava Leaf powder, heats, filtering obtains Guava Leaf
Extract flavone crude product；
(2) the flavones crude product obtained after the step (1) is dissolved in ethanol solution, supernatant is taken to import ultrafiltration dress after filtering
Middle progress ultrafiltration circulation is set, the film that the film that the ultrafiltration circulation uses is 10000 for retention relative molecular mass obtains ultrafiltration membrane
Filtering solution；
(3) it is dissolved in acetonitrile, is added after function monomer 4-vinylpridine at room temperature using Quercetin monomer as template molecule
Stirring, acts on template molecule sufficiently with function monomer, and crosslinking agent and initiator is then added, carried out after mixing well it is ultrasonic with
The oxygen for removing dissolution is passed through nitrogen and forms oxygen-free environment, and sealing, which is placed in thermostatical oil bath, carries out thermal polymerization, polymerizate
It is eluted in Soxhlet extractor with methanol-acetic acid solution, to remove template molecule, again with methanol washes away excessive acetic acid, Yu Zhen
It is dried in vacuo in empty baking oven, the molecularly imprinted polymer after being synthesized；
(4) obtained molecularly imprinted polymer after the step (3) synthesis is accurately weighed, is added after the step (2) and to obtain
Ultrafiltration membrance filter solution carries out closed concussion in constant-temperature table, obtains the molecular engram for being adsorbed with Flavonoid substances Quercetin
Polymer obtains the Flavonoid substances Quercetin after centrifugation.
2. method of purification according to claim 1, which is characterized in that in the step (1), be sieved for 40-60 mesh,
The water volume of addition is 38-42 times of Guava Leaf powder product.
3. method of purification according to claim 1 or 2, which is characterized in that in the step (1), heating temperature 50-70
DEG C, heating time 25-30min.
4. method of purification according to claim 1, which is characterized in that in the step (2), the time of ultrafiltration circulation is 2-
3h, pressure 0.3-0.9MPa, temperature are 30-60 DEG C.
5. method of purification according to claim 1, which is characterized in that in the step (3), acetonitrile is no less than 40mL, stirs
Mixing the time is no less than 4h, ultrasonic time 10-20min, and being passed through the nitrogen time is 20-30min.
6. method of purification according to claim 1 or 5, which is characterized in that in the step (3), crosslinking agent is ethylene glycol
Dimethylacrylate, initiator are azodiisobutyronitrile, and the dosage of the initiator is function monomer and crosslinking agent gross mass
1-2%.
7. method of purification according to claim 1 or 5, which is characterized in that in the step (3), oil bath pot temperature is 50-
70 DEG C, the thermal polymerization time is 22-26h, and the volume ratio of methanol-acetic acid solution is methanol: acetic acid=9: 1, elution time 46-
50h, vacuum drying temperature are 30-50 DEG C, vacuum drying time 22-26h.
8. method of purification according to claim 1, which is characterized in that in the step (4), every 30mg molecular engram polymerization
Ultrafiltration membrance filter solution 10mL is added in object.
9. method of purification according to claim 1, which is characterized in that in the step (4), the temperature of constant-temperature table is
30-50 DEG C, the concussion time is 22-26h.
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