CN104341487A - Lobed kudzuvine root protein and preparation method of nanoparticles of lobed kudzuvine root protein - Google Patents
Lobed kudzuvine root protein and preparation method of nanoparticles of lobed kudzuvine root protein Download PDFInfo
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- CN104341487A CN104341487A CN201410541087.3A CN201410541087A CN104341487A CN 104341487 A CN104341487 A CN 104341487A CN 201410541087 A CN201410541087 A CN 201410541087A CN 104341487 A CN104341487 A CN 104341487A
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Abstract
The invention discloses a lobed kudzuvine root protein and a preparation method of nanoparticles of the lobed kudzuvine root protein. The lobed kudzuvine root protein with the sequence of DFVYDMCGNVLN is obtained by extraction with a Tris-HCl buffer solution, graded sedimentation with ethanol and ion exchange chromatographic separation. The nanoparticles of the lobed kudzuvine root protein are prepared by heating a water solution of the lobed kudzuvine root protein at the temperature of 100 DEG C for 60min under the conditions that the protein concentration is 1mg/mL and the pH is 6.05, and the nanoparticles of the lobed kudzuvine root protein show relatively low cytotoxicity in vitro. A lyophilized product of the nanoparticles of the lobed kudzuvine root protein has excellent flowability and is easy to store stably. The lobed kudzuvine root protein disclosed by the invention is from foods and has high safety. According to the preparation method of the nanoparticles, no crosslinking agent is not used, finished products widely exist in various decoctions using lobed kudzuvine root, and the nanoparticles can be taken by the majority of people for many years and have high safety. The nanoparticles of the lobed kudzuvine root protein disclosed by the invention are expected to be applied to in vivo delivery of a variety of medicaments.
Description
Technical field
The present invention relates to the protein drug novel form in medical art and preparation technique, be specifically related to a kind of root of kudzu vine albumen and nanometer grain preparation method thereof.
Background technology
Start the definition in plan (National Nanotechnology Initiative, NNI) according to American National nanosecond science and technology, the particle of particle diameter in 1-100 nm range scale is called nano particle.In recent decades, nano particle is widely used in the development research of medicament transport system, comprising nano-medicament carrier prepared by the various differing materials such as metal aggregate, inorganic particle, hyperbranched polymer, polymer micelle.At various nano-medicament carrier while representing the advantage such as targeting and penetrance gradually, also expose the shortcoming in biocompatibility, cytotoxicity and degradability.After the nanometer of material, the physico-chemical property (optics, electricity, surfactivity etc.) of nano material all can present with starting materials distinct character.The biomacromolecule (as serum albumin, silk-protein etc.) that current application and nano-medicament carrier are studied all does not have can the natural research model of reference, the physico-chemical property and the associated biomolecule security thereof that obtain nano particle also need long-term observation, just can obtain comprehensive assessment.The potential risk that nano-medicament carrier development brings causes scientific circles and worries widely.
" soup " provides abundant research model to biomacromolecule self-assembling nanoparticles: from food to Chinese medicine, all there is colloidal dispersion in much soup, and the colloidal solid of Corbicula fluminea soup is comparatively homogeneous, and median size is 78 nm; Colloidal solid composition in balsam pear soup is comparatively complicated, and average particle size distribution scope is comparatively large, and its intermediate value is 347 nm; The extensive existence of the particle of nanoscale in traditional Chinese herbal decoction is also shown to the colloid science investigation of tens kinds of traditional Chinese herbal decoction.The self-assembly behavior of the natural nano particle that these exist in soup and the protein of protein, particularly nonenzymatic glycosylation is closely related.The length of the time that various soup is drunk in human history, audient are wide, can be considered to safe (Generally Recognized as Safe, GRAS) from the angle of risk management.Present stage nano-medicament carrier research can be solved to the research of the nano particle that self-assembly in the decoction process of soup is formed and lack learning prototype and the problem to security concern aspect.
The root of kudzu vine is a kind of food raw material and Chinese medicinal material of our people's life-time service, in traditional Chinese herbal decoction, particularly has very important purposes in Chinese medicine compound prescription decoction.The root of kudzu vine has a liter positive antidiarrheal, expelling pathogenic factors from muscles for reducing heat, the effect of promoting eruption of promoting the production of body fluid, modern medicine has hypoglycemic, resisting oxidation free radical, hypotensive, antitumor, the effectiveness such as anti-arrhythmia, reduction blood fat, in prescription " GEGEN QINLIAN TANG ", the root of kudzu vine plays main pharmacodynamics in prescription as " monarch drug in a prescription ".But up to the present, about the research of root of kudzu vine protein nano particle there is not yet bibliographical information both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of root of kudzu vine albumen and nanometer grain preparation method thereof, present stage nano-medicament carrier research can be solved to the research of the nano particle that self-assembly in the decoction process of soup is formed and lack learning prototype and the problem to security concern aspect.
For achieving the above object, the present invention adopts following technical scheme:
A kind of root of kudzu vine albumen, aminoacid sequence is DFVYDMCGNVLN.
The preparation method of described albumen is through the extracting of Tris-HCl damping fluid aqueous phase, ion-exchange chromatography Macro-Prep High-Q, is separated and obtains an electrophoretically pure root of kudzu vine albumen from the root of kudzu vine prepared slices of Chinese crude drugs.
A kind of root of kudzu vine albumen is preparing the application on root of kudzu vine protein nano particle.
The preparation method of described nano particle is the Tris-HCI solution this root of kudzu vine albumen being formulated as 1 mg/mL, through 60-100 DEG C of heating 1 hour, through 6000 g ultrafiltration 5 min, namely obtains root of kudzu vine protein nano particle solution; And apply dynamic light scattering technique dynamic light scattering, multi-angle laser light scattering and scanning electron microscope character research is carried out to root of kudzu vine protein nano particle.
The preparation of root of kudzu vine protein crude extract: root of kudzu vine medicine materical crude slice is pulverized through miniature high-speed pulverizer.Mix by root of kudzu vine powder with containing 0.1 mol/L Tris-hydrochloride buffer (0.02 mol/L, pH7.2) with mass ratio 1:5, be placed in 4 DEG C, stirring and leaching 12 h.The root of kudzu vine vat liquor obtained by two layers of filtered through gauze, discards filter residue.By filtrate at 4 DEG C through 10, centrifugal 15 min of 000 g, collect supernatant liquor.At supernatant liquor being placed in 4 DEG C, the dehydrated alcohol of slowly precooling while stirring, makes ethanol final concentration reach 20%.Leave standstill 4 h, with 10, centrifugal 15 min of 000 g, collecting precipitation.With same concentrations Tris-hydrochloride buffer dialysed overnight after precipitation uses Tris-hydrochloride buffer (0.02 mol/L, pH8.1) fully to dissolve, the solution obtained is root of kudzu vine protein crude extract.
The ion exchange mechansim of root of kudzu vine albumen: 100 mL root of kudzu vine protein crude extract streams are added to Tris-hydrochloride buffer (0.02 mol/L, pH8.1) Macro-Prep High-Q chromatographic column (Bio-RAD company is balanced, column volume: 10*250 mm), after continuing balance 3 times of column volumes with same concentration Tris-hydrochloride buffer, again with Tris-hydrochloride buffer (0.02 mol/L containing 0 ~ 0.3 mol/L NaCl, pH8.1) linear gradient elution 200 mL, finally with Tris-hydrochloride buffer (0.02 mol/L containing 0.3 mol/L NaCl, pH8.1) wash-out 1 column volume.Flow velocity 0.8 mL/min, ultraviolet spectrophotometer measure wavelength be 280 nm, through chromatographic separation component with automatically distribute collect instrument collect, after carry out protein ingredient qualification with SDS-PAGE.
The essential property of root of kudzu vine albumen characterizes: with the molecular size range of this purified root of kudzu vine albumen obtained of MALDI-TOF mass spectroscopy, with etc. point focusing measure the iso-electric point of this root of kudzu vine albumen, the N-measuring this root of kudzu vine albumen with Edman degradation holds primary structural sequence.
The preparation of root of kudzu vine protein nano particle: the Tris-HCI solution this root of kudzu vine albumen being formulated as 1 mg/mL, through 60-100 DEG C of heating 1 hour, through 6000 g ultrafiltration 5 min, can obtain root of kudzu vine protein nano particle solution.And apply dynamic light scattering technique dynamic light scattering, multi-angle laser light scattering and scanning electron microscope character research is carried out to root of kudzu vine protein nano particle, the root of kudzu vine protein nano mean particle size that the present invention prepares is 150 ± 1.0 nm, surface charge is negativity, and zeta potential value is-23 ± 0.5 mV.
The Berberine prepared for carrier with this root of kudzu vine albumen-root of kudzu vine protein nano particle, charging ratio is 44.2%, and median size is 150 ± 1.0 nm.Root of kudzu vine protein nano particle all shows lower cytotoxicity in vitro.
The invention has the advantages that: the root of kudzu vine dietary protein origin that the present invention relates to is in food, and security is high.The preparation method of its nano particle does not relate to any linking agent, and its finished product is extensively present in the decoction of all kinds of use root of kudzu vine, and for many people takes for many years, security is high.Send in the body that the root of kudzu vine protein nano particle that the present invention relates to is expected to be applied to multi-medicament.
Accompanying drawing explanation
Accompanying drawing 1 root of kudzu vine protein crude extract ion-exchange chromatography.Wherein OD280 is the OD value under 280nm, and Time is the time, the concentration that C (NaCl) is NaCl.
Accompanying drawing 2 root of kudzu vine protein SDS-PAGE electrophorogram.Wherein M is Maker.
Accompanying drawing 3 root of kudzu vine protein nano particle size distribution figure.
Accompanying drawing 4 root of kudzu vine protein nano granulosa cell toxicity.Wherein L02 is normal liver cell, and Hep-G2 is human liver cancer cell.
Embodiment
embodiment 1: the extraction of root of kudzu vine albumen
Root of kudzu vine medicine materical crude slice is pulverized through miniature high-speed pulverizer.Mix by root of kudzu vine powder with containing 0.1 mol/L Tris-hydrochloride buffer (0.02 mol/L, pH7.2) with mass ratio 1:5, be placed in 4 DEG C, stirring and leaching 12 h.The root of kudzu vine vat liquor obtained by two layers of filtered through gauze, discards filter residue.By filtrate at 4 DEG C through 10, centrifugal 15 min of 000 g, collect supernatant liquor.At supernatant liquor being placed in 4 DEG C, the dehydrated alcohol of slowly precooling while stirring, makes ethanol final concentration reach 20%.Leave standstill 4 h, with 10, centrifugal 15 min of 000 g, collecting precipitation.With same concentrations Tris-hydrochloride buffer dialysed overnight after precipitation uses Tris-hydrochloride buffer (0.02 mol/L, pH8.1) fully to dissolve, the solution obtained is root of kudzu vine protein crude extract.
100 mL root of kudzu vine protein crude extract streams are added to and balances Macro-Prep High-Q chromatographic column (Bio-RAD company with Tris-hydrochloride buffer (0.02 mol/L, pH8.1), column volume: 10 × 250 mm), after continuing balance 3 times of column volumes with same concentration Tris-hydrochloride buffer, again with Tris-hydrochloride buffer (0.02 mol/L, pH8.1) linear gradient elution 200 mL containing 0 ~ 0.3 mol/L NaCl, finally with Tris-hydrochloride buffer (0.02 mol/L, pH8.1) wash-out 1 column volume containing 0.3 mol/L NaCl.Root of kudzu vine protein crude extract High-Q color atlas and each component S DS-PAGE electrophorogram are shown in accompanying drawing 1.
The basic biochemical property of root of kudzu vine albumen, is characterized by that molecular weight is 20731.6 Da, iso-electric point is that 6.34, N-holds primary structural sequence to be DFVYDMCGNVLN.
embodiment 2: the preparation of root of kudzu vine protein nano particle
This root of kudzu vine albumen is formulated as the Tris-HCI solution of 1 mg/mL, through 60-100 DEG C of heating 1 hour, after 6000 g ultrafiltration 5min, root of kudzu vine protein nano particle can be obtained.
Measure its granularity and surface potential with laser particle analyzer, recording its particle diameter is 150 ± 1.0 nm, and surface potential is-23 ± 0.5 mV.Root of kudzu vine protein nano particle size distribution figure is shown in accompanying drawing 3.
embodiment 3: the vitro cytotoxicity of root of kudzu vine protein nano particle measures
Have employed the vitro cytotoxicity of normal liver cell (L-02), human liver cancer cell (Hep-G2), mensuration root of kudzu vine protein nano particle.Cell all uses containing 20% calf serum RPMI1640 nutrient solution, in 37 DEG C, cultivates in 5% CO2 culture environment.During mensuration, cell is by 4 × 10
4individual/mL is connected to 96 orifice plates, and 200 μ L/holes, often group establishes 6 parallel holes.Cultivate 24 hours, discard substratum, the sample after doubling dilution is added with every hole 100 μ L amount.Add not containing the substratum 100 μ L of serum simultaneously, be used as blank.Measure cell proliferation rate with mtt assay after continuing cultivation 24 h, calculation formula is as follows:
Survival rate=(the blank group of A590 application of sample group-A590) blank group × 100% of/A590
Root of kudzu vine protein nano granulosa cell toxicity test shows, concentrations of nanoparticles lower than 89 ug/mL to can't suppress can't the increment of T suppression cell, and more obvious to the nontoxicity of Human normal hepatocyte L-02.The vitro cytotoxicity of root of kudzu vine protein nano particle is shown in accompanying drawing 4.
Embodiment 5: root of kudzu vine protein nano particle loads Berberine ability and measures
The Berberine reference liquid (being dissolved in chromatographically pure methyl alcohol) of accurate formulation 10 mg/mL, gets 50 μ L reference liquids and adds in 1.995 mL root of kudzu vine protein samples, make Berberine final concentration be 0.25 mg/mL.Get containing 0.25 mg/mL Berberine with not containing each 1.6 mL of root of kudzu vine protein liquid of Berberine, at 100 DEG C, be incubated 60 min.Get root of kudzu vine albumen 1 mL containing Berberine (0.25 mg/mL), add 100 kDa super filter tube 25 DEG C, centrifugal 60 min of 5000 g.Add Tris-hydrochloric acid (0.02 mol/L, pH8.1) 1 mL, after repeatedly rinsing super filter tube upper sample pond, the centrifugal 60min of 5000 g, after liquid is completely through ultra-filtration membrane, measures content of berberine, is free content of berberine.Drug loading efficiency calculation formula is as follows:
Charging ratio=(total AST content-free AST content)/total AST content
After measured, root of kudzu vine albumen is 44.8% to Berberine charging ratio under these experimental conditions.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> University of Fuzhou
<120> root of kudzu vine albumen and nanometer grain preparation method thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> root of kudzu vine albumen
<400> 1
Asp Phe Val Tyr Asp Met Cys Gly Asn Val Leu Asn
1 5 10
Claims (4)
1. a root of kudzu vine albumen, is characterized in that: the aminoacid sequence of described albumen is DFVYDMCGNVLN.
2. a kind of root of kudzu vine albumen according to claim 1, it is characterized in that: the preparation method of described albumen is through the extracting of Tris-HCl damping fluid aqueous phase, ion-exchange chromatography Macro-Prep High-Q, is separated and obtains an electrophoretically pure root of kudzu vine albumen from the root of kudzu vine prepared slices of Chinese crude drugs.
3. a kind of root of kudzu vine albumen as claimed in claim 1 is preparing the application on root of kudzu vine protein nano particle.
4. the application of a kind of root of kudzu vine albumen according to claim 3, it is characterized in that: the preparation method of described nano particle is the Tris-HCI solution this root of kudzu vine albumen being formulated as 1 mg/mL, through 60-100 DEG C of heating 1 hour, through 6000 g ultrafiltration 5 min, namely obtain root of kudzu vine protein nano particle solution; And apply dynamic light scattering technique dynamic light scattering, multi-angle laser light scattering and scanning electron microscope character research is carried out to root of kudzu vine protein nano particle.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107412730A (en) * | 2017-04-27 | 2017-12-01 | 浙江工商大学 | A kind of glutinous rehmannia protein nano particle and preparation method thereof |
| CN108607100A (en) * | 2018-05-04 | 2018-10-02 | 福州大学 | A kind of Radix Angelicae Sinensis albumen self assembly particle and application |
| CN109956995A (en) * | 2017-12-14 | 2019-07-02 | 浙江工商大学 | A kind of paclitaxel loaded Almond Proteins nano-carrier and preparation method thereof |
| CN109966780A (en) * | 2019-03-21 | 2019-07-05 | 浙江工商大学 | The separation method of functionalized nanoparticles in a kind of aquatic shellfish boiling soup |
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| CN1765923A (en) * | 2004-10-29 | 2006-05-03 | 浙江工业大学 | Method for extracting and separating protein from the root of kudzu vine |
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| CN1765923A (en) * | 2004-10-29 | 2006-05-03 | 浙江工业大学 | Method for extracting and separating protein from the root of kudzu vine |
| CN101513389A (en) * | 2009-03-20 | 2009-08-26 | 中国药科大学 | Oral puerarin nano suspension for improving bioavailability of puerarin |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107412730A (en) * | 2017-04-27 | 2017-12-01 | 浙江工商大学 | A kind of glutinous rehmannia protein nano particle and preparation method thereof |
| CN107412730B (en) * | 2017-04-27 | 2020-08-14 | 浙江工商大学 | Dihuang protein nano-particles and preparation method thereof |
| CN109956995A (en) * | 2017-12-14 | 2019-07-02 | 浙江工商大学 | A kind of paclitaxel loaded Almond Proteins nano-carrier and preparation method thereof |
| CN109956995B (en) * | 2017-12-14 | 2023-07-28 | 浙江工商大学 | Paclitaxel-loaded bitter almond protein nano-carrier and preparation method thereof |
| CN108607100A (en) * | 2018-05-04 | 2018-10-02 | 福州大学 | A kind of Radix Angelicae Sinensis albumen self assembly particle and application |
| CN108607100B (en) * | 2018-05-04 | 2020-12-25 | 福州大学 | Angelica sinensis protein self-assembly particle and application |
| CN109966780A (en) * | 2019-03-21 | 2019-07-05 | 浙江工商大学 | The separation method of functionalized nanoparticles in a kind of aquatic shellfish boiling soup |
| CN109966780B (en) * | 2019-03-21 | 2021-03-09 | 浙江工商大学 | Method for separating functional nanoparticles from aquatic shellfish cooking soup |
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