CN103739688A - Liquorice protein nanoparticles and preparation method thereof - Google Patents
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- CN103739688A CN103739688A CN201310738014.9A CN201310738014A CN103739688A CN 103739688 A CN103739688 A CN 103739688A CN 201310738014 A CN201310738014 A CN 201310738014A CN 103739688 A CN103739688 A CN 103739688A
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Abstract
The invention discloses preparation methods of liquorice protein and nanoparticles of the liquorice protein. The liquorice protein with molecular weight of 31.0 kDa and isoelectric point of 4.5 is obtained through separation and purification. A primary structure sequence of N-end of the liquorice protein is NPDGLIACYCGQYCW. The liquorice protein nanoparticles are prepared by heating a saline solution of the liquorice protein at 100 DEG C under the conditions that the protein concentration is 1 mg/mL, and pH is 7.9 for 60 minutes, and the average particle size of the liquorice protein nanoparticles is 74.09+/-0.69 nm. Freeze-dried products of the liquorice protein nanoparticles are good in liquidity and re-solubility, and easy to stably store. The liquorice protein disclosed by the invention derives from food, and is high in safety. The preparation method of the liquorice protein nanoparticles does not involve any crosslinking agent, finished products of the liquorice protein nanoparticles widely exist in various decoction using liquorice, are taken by the majority of people for many years, and are high in safety. The liquorice protein nanoparticles disclosed by the invention are expected to be applied to in-vivo delivery of various medicines.
Description
Technical field
The present invention relates to protein drug novel form and preparation technique in medical technical field, be specifically related to a kind ofly there is drug disposition and send Radix Glycyrrhizae protein nano particle of effect and preparation method thereof.
Background technology
According to American National nanosecond science and technology, start the definition in plan (National Nanotechnology Initiative, NNI), 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, the nano-medicament carrier of preparing comprising various differing materials such as metal aggregate, inorganic particle, hyperbranched polymer, polymer micelles.At various nano-medicament carriers, when representing gradually the advantages such as targeting and penetrance, also expose the shortcoming aspect biocompatibility, cytotoxicity and degradability.After the nanometer of material, the physico-chemical property of nano material (optics, electricity, surfactivity etc.) all can present and the distinct character of starting materials.The biomacromolecule (as serum albumin, silk-protein etc.) of at present application and nano-medicament carrier research does not all have natural research model that can 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 has caused that scientific circles worry widely.
" soup " provides abundant research model to biomacromolecule self-assembling nanoparticles: from food to Chinese medicine, all have colloidal dispersion in much soup, the colloidal solid of Corbicula fluminea soup is homogeneous comparatively, and median size is 78 nm; Colloidal solid in balsam pear soup forms comparatively complicated, and average particle size distribution scope is larger, and its intermediate value is 347 nm; The extensive existence of the particle that the colloid science investigation of tens kinds of traditional Chinese herbal decoction is also shown to nanoscale in traditional Chinese herbal decoction.The self-assembly behavior of the protein of natural nano particle and protein, particularly nonenzymatic glycosylation that these exist in soup is closely related.The length of the time that various soup is drunk in human history, audient are wide, from the angle of risk management, can be considered to safe (Generally Recognized as Safe, GRAS).The research of the nano particle that self-assembly in the decoction process of soup is formed can solve present stage nano-medicament carrier research and lack learning prototype and the problem to security concern aspect.
Radix Glycyrrhizae is a kind of food raw material and Chinese medicinal material of China people life-time service, in the notice > > of the < < announcing about further standard healthy food material management, is put in the raw material of " integration of drinking and medicinal herbs " in the Ministry of Health.Radix Glycyrrhizae, in traditional Chinese herbal decoction, particularly has very important purposes in Chinese medicine compound prescription decoction.Radix Glycyrrhizae can " coordinating the actions of various ingredients in a prescription ", as in MAXINGSHIGAN TANG, " beneficial gas and in, close with gypsum and promote the production of body fluid, and can be in harmonious proportion between cold Wen Xuan falls, be to make it use for assistant ".Radix Glycyrrhizae is as making medicine, or is called " guiding drug ", and with the language expression of modern science, the effect that it may play is the various effective constituents in set side's medicine, realizes the effect of sending in body.
But up to the present, the research of relevant Radix Glycyrrhizae 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 Radix Glycyrrhizae protein nano particle and preparation method thereof.For compound after nanometer, uncertainty in security, the design of the Radix Glycyrrhizae protein nano particle the present invention relates to is from two " natural " systems: the one, and the natural protein component being present in food, the 2nd, the nanometer process of this protein occurs in hot procedure, and its nano particle is present in various " soup " extensively to be taken.The Radix Glycyrrhizae protein nano particle In vitro cell experiment that the present invention obtains has shown utmost point hypotoxicity, and medicine stowage capacity is strong, rule of surface, and size distribution is even, good stability.
For achieving the above object, the present invention adopts following technical scheme:
A Radix Glycyrrhizae albumen, its N-end primary structure sequence is SEQ ID NO:1:NPDGLIACYCGQYCW.
Described Radix Glycyrrhizae molecular weight of albumen is that 31.0 kDa, iso-electric point are 4.5.
An extracting method for Radix Glycyrrhizae albumen, through the extracting of phosphate buffered saline buffer water, ion-exchange chromatography Macro-Prep High Q and hydrophobic chromatography POROS R1, from traditional Chinese medicine of licorice root medicine materical crude slice, separation obtains an electrophoretically pure Radix Glycyrrhizae albumen.It specifically comprises the following steps:
1) preparation of Radix Glycyrrhizae protein crude extract:licorice piece is pulverized through miniature high-speed pulverizer, with mass ratio 1:5, by Radix Glycyrrhizae powder with containing the phosphoric acid buffer (0.02 mol/L, pH7.2) of 0.1 mol/L NaCl, mixes, and is placed in 3-5 ℃, stirring and leaching 10-14 h; The licorice extract obtaining by 1-3 layer filtered through gauze, discards filter residue, by filtrate at 3-5 ℃ through the centrifugal 10-20 min of 10000-12000g, collect supernatant liquor; Supernatant liquor is placed in to 3-5 ℃, with ammoniacal liquor, is adjusted to pH9.5-10.5, the dehydrated alcohol of slow precooling, makes ethanol final concentration reach 40% while stirring; Standing 3-5 h, with the centrifugal 10-20 min of 10000-12000 g, collects supernatant liquor; Continuation slowly adds dehydrated alcohol to the ethanol massfraction of precooling to supernatant liquor be 50%, standing 10-12 h, the centrifugal 10-20 min of 11000-13000 rpm, collecting precipitation; After precipitation uses Tris-hydrochloride buffer (0.02 mol/L, pH8.0) fully to dissolve, with same concentrations Tris-hydrochloride buffer dialysed overnight, the solution obtaining is Radix Glycyrrhizae protein crude extract;
2) ion-exchange chromatography of Radix Glycyrrhizae albumen is separated:the first extract stream of 70-80 mL Radix Glycyrrhizae albumen is added to Tris-hydrochloride buffer (0.02 mol/L, pH8.0) balance Macro-Prep High Q chromatographic column (Bio-RAD company, column volume: 10*250 mm), with same concentration Tris-hydrochloride buffer, continue after 3 times of column volumes of balance, again with Tris-hydrochloride buffer (0.02 mol/L containing 0～0.5 mol/L NaCl, pH8.0) linear gradient elution 200 mL, finally with Tris-hydrochloride buffer (0.02 mol/L containing 0.5 mol/L NaCl, pH8.0) 1 column volume of wash-out, flow velocity 1 mL/min, it is 280 nm that ultraviolet spectrophotometer is measured wavelength, through the component of chromatographic separation, automatically to distribute, collecting instrument collects, after with SDS-PAGE, carry out protein ingredient evaluation,
3) hydrophobic chromatography of Radix Glycyrrhizae albumen is separated:g2 protein ingredient separated through High-Q and that dialysed is further carried out to purifying with hydrophobic chromatography POROS@R1, POROS R1 hydrophobic chromatography post (Applied Biosystems, 10 μ m, 2.1*100 mm) with deionized water balance, wash-out moving phase is: A deionized water, B acetonitrile, carries out gradient elution with 100%A-100% B linear gradient.Flow velocity 1.0 mL/min, applied sample amount: 5 mL, ultraviolet detection wavelength 280 nm, collect instrument through the component of chromatographic separation automatically to distribute and collect, after with SDS-PAGE, carry out protein ingredient evaluation;
4) essential property of Radix Glycyrrhizae albumen characterizes:with SDS-PAGE, measure the molecular size range of this purified Radix Glycyrrhizae albumen obtaining, with etc. point focusing measure the iso-electric point of this Radix Glycyrrhizae albumen, with Edman degradation, measure the N-end primary structure sequence of this Radix Glycyrrhizae albumen.
A Radix Glycyrrhizae protein nano particle, is comprised of described Radix Glycyrrhizae albumen, and 23 Radix Glycyrrhizae protein monomers form a spherical protein nano particle, and its median size of this nano particle is 74.09 ± 0.69 nm, and surface charge is negativity, and scope is at-23 ± 2mV.
The preparation method of described Radix Glycyrrhizae protein nano particle comprises: the pH 7.9-8.0 salts solution that this Radix Glycyrrhizae albumen is formulated as to 1 mg/mL, through 60-100 ℃ of heating 1 hour, application TSK gel G6000PW gel chromatography is removed unreacted Radix Glycyrrhizae albumen, obtains Radix Glycyrrhizae protein nano particle.Its concrete steps comprise: this Radix Glycyrrhizae albumen is formulated as to the pH 7.9-8.0 salts solution of 1 mg/mL, through 60-100 ℃ of heating 1 hour, application TSK gel G6000PW gel chromatography was removed unreacted Radix Glycyrrhizae albumen, can obtain Radix Glycyrrhizae protein nano particle.And apply dynamic light scattering technique dynamic light scattering, multi-angle laser light scattering and scanning electron microscope Radix Glycyrrhizae protein nano particle is carried out to property research, the Radix Glycyrrhizae protein nano particle median size that the present invention prepares is 74.09 ± 0.69 nm, surface charge is negativity, and zeta potential value is-23 ± 2 mV.
Take Cyclosiversioside F-Radix Glycyrrhizae protein nano particle that this Radix Glycyrrhizae albumen prepared as carrier, median size is 74.09 ± 0.69 nm, and charging ratio is 51.2%.
Radix Glycyrrhizae protein nano particle has all shown extremely low cytotoxicity in vitro, and to different cell strains, as Hep-G2, Caco-2, L02 and NR8383 have shown different cellular affinities.
Described salts solution is Tris-hydrochloric acid soln or phosphate solution.
The invention has the advantages that: the Radix Glycyrrhizae dietary protein origin the present invention relates to is in food, safe.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 Radix Glycyrrhizaes, for numerous crowds take for many years, safe.The Radix Glycyrrhizae protein nano particle In vitro cell experiment that the present invention obtains has shown utmost point hypotoxicity, and medicine stowage capacity is strong, rule of surface, and size distribution is even, good stability.The Radix Glycyrrhizae protein nano particle relating to is expected to be applied to send in the body of multi-medicament.
Accompanying drawing explanation
Fig. 1 Radix Glycyrrhizae protein crude extract ion-exchange chromatography and SDS-PAGE electrophorogram.
The SDS-PAGE electrophorogram of each chromatographic peak is collected in the ion-exchange of Fig. 2 Radix Glycyrrhizae protein crude extract, and wherein M is expressed as albumen Maker.
Fig. 3 Radix Glycyrrhizae albumen hydrophobic chromatography and SDS-PAGE electrophorogram.
Fig. 4 Radix Glycyrrhizae albumen hydrophobic chromatography is collected the SDS-PAGE electrophorogram that obtains each chromatographic peak, and wherein M is expressed as albumen Maker.
The electron microscopic observation figure of Fig. 5 Radix Glycyrrhizae protein nano particle.Wherein A represents 0.22 μ m cellulose acetate membrane blank; B represents Radix Glycyrrhizae protein nano particle.
Fig. 6 Radix Glycyrrhizae protein nano particle size distribution figure.
Fig. 7 Radix Glycyrrhizae protein nano granulosa cell toxicity, wherein L-02 is that normal liver cell, Hep-G2 human liver cancer cell, Caco-2 are that human colon carcinoma epithelial cell and MDCK are dog renal epithelial cell.
Fig. 8 Radix Glycyrrhizae protein nano granulosa cell binding specificity, wherein L-02 is that normal liver cell, Hep-G2 human liver cancer cell, Caco-2 are that human colon carcinoma epithelial cell and MDCK are dog renal epithelial cell.
Embodiment
embodiment 1: the extraction of Radix Glycyrrhizae albumen
Licorice piece is pulverized through miniature high-speed pulverizer.With mass ratio 1:5, by Radix Glycyrrhizae powder with containing the phosphoric acid buffer (0.02 mol/L, pH7.2) of 0.1 mol/L NaCl, mix, be placed in 4 ℃, stirring and leaching 12 h.The licorice extract obtaining by two layers of filtered through gauze, discards filter residue.By filtrate at 4 ℃ through centrifugal 15 min of 10,000 g, collect supernatant liquor.Supernatant liquor is placed at 4 ℃, with ammoniacal liquor, is adjusted to pH10.0, the dehydrated alcohol of slow precooling, makes ethanol final concentration reach 40% while stirring.Standing 4 h, with centrifugal 15 min of 10,000 g, collect supernatant liquor.Continuation slowly adds dehydrated alcohol to the alcohol concn of precooling to supernatant liquor be 50%, standing 12 h, centrifugal 15 min of 12000 rpm, collecting precipitation.After precipitation uses Tris-hydrochloride buffer (0.02 mol/L, pH8.0) fully to dissolve, with same concentrations Tris-hydrochloride buffer dialysed overnight, the solution obtaining is Radix Glycyrrhizae protein crude extract.
Radix Glycyrrhizae protein crude extract stream is added to Tris-hydrochloride buffer (0.02 mol/L, pH8.0) balance Macro-Prep High Q chromatographic column (Bio-RAD, column volume: 10*250 mm), with same concentration Tris-hydrochloride buffer, continue after 3 times of column volumes of balance, again with Tris-hydrochloride buffer (0.02 mol/L, pH8.0) linear gradient elution 200 mL containing 0～0.5 mol/L NaCl, finally with 1 column volume of Tris-hydrochloride buffer (0.02 mol/L, pH8.0) wash-out containing 0.5 mol/L NaCl.Radix Glycyrrhizae protein crude extract High Q color atlas and each component S DS-PAGE electrophorogram are shown in accompanying drawing 1,2.
G2 protein ingredient separated through High-Q and that dialysed is further carried out to purifying by hydrophobic chromatography.POROS@R1 hydrophobic chromatography post (Applied Biosystems, 10 μ m, 2.1*100 mm) is with deionized water balance.Wash-out moving phase is: A deionized water, B acetonitrile, carries out gradient elution with 100%A-100% B linear gradient.The protein component M5 that wash-out obtains is electrophoretically pure Radix Glycyrrhizae albumen, and Radix Glycyrrhizae protein crude extract POROS R1 color atlas and each component electrophorogram are shown in accompanying drawing 3,4.
The basic biochemical property of Radix Glycyrrhizae albumen, it is characterized by molecular weight 31.0 kDa, iso-electric point is that 4.5, N-end primary structure sequence is NPDGLIACYCGQYCW.
embodiment 2: the preparation of Radix Glycyrrhizae protein nano particle
This Radix Glycyrrhizae albumen is formulated as to the Tris-hydrochloride buffer (pH 7.9) of 1 mg/mL, through 60-100 ℃ of heating 1 hour, application TSK gel G6000PW gel chromatography was removed unreacted Radix Glycyrrhizae albumen, can obtain Radix Glycyrrhizae protein nano particle.Radix Glycyrrhizae protein nano particle electron microscopic observation figure is shown in accompanying drawing 5.
With laser particle analyzer, measure its granularity and surface potential, recording its particle diameter is 74.09 ± 0.69 nm, and surface potential is-23 ± 2 mV.Radix Glycyrrhizae protein nano particle size distribution figure is shown in accompanying drawing 6.
embodiment 3: the vitro cytotoxicity of Radix Glycyrrhizae protein nano particle is measured
Adopted normal liver cell (L-02), human liver cancer cell (Hep-G2), human colon carcinoma epithelial cell (Caco-2) and dog renal epithelial cell (MDCK) to measure the vitro cytotoxicity of Radix Glycyrrhizae protein nano particle.Cell is all used containing 20% calf serum RPMI1640 nutrient solution, in 37 ℃, in 5% CO2 culture environment, cultivates.During mensuration, cell is by 4 * 10
4individual/mL is connected to 96 orifice plates, and 6 parallel holes are established for every group in 200 μ L/holes.Cultivate 24 hours, discard substratum, the sample after doubling dilution is added with every hole 100 μ L amounts.Add not containing the substratum 100 μ L of serum simultaneously, be used as blank.After continuing to cultivate 24 h, with mtt assay, measure cell proliferation rate, calculation formula is as follows:
Survival rate=(the blank group of A590 application of sample group-A590) blank group * 100% of/A590
Radix Glycyrrhizae protein nano granulosa cell toxicity test shows, except Caco-2 extracellular, concentrations of nanoparticles lower than 224 ug/mL to suppressing to suppress the increment of cell.Caco-2 Growth of Cells increment is low, may be that the nano particle of high density has suppressed due to the Absorption And Metabolism of Caco.The vitro cytotoxicity of Radix Glycyrrhizae protein nano particle is shown in accompanying drawing 7.
embodiment 4: the cellular affinity of Radix Glycyrrhizae protein nano particle is measured
100 μ L fluorescein isothiocyanate (10 mg/mL) dye liquors are added in 4 mL licorice root particles samples, in 4 ℃ of standing 24 h in darkroom, carry out mark.Use Sephadex G25 to remove the Radix Glycyrrhizae protein nano particulate samples that obtains mark after free FITC.
Cell is by 4 * 10
5individual/hole access Tissue Culture Plate, 0.75 mL/ hole, adds the Radix Glycyrrhizae protein nano particle liquid of mark in hand-hole.Other holes add not containing blood serum medium 0.75 mL, as blank.Lucifuge, 37 ℃, 5% CO2, cultivation 24 h.Lucifuge supernatant discarded, PBS cleans 3 times, adds 0.75 mL not containing blood serum medium, is placed in (magnification 200*) under laser scanning co-focusing microscope and observes.
This experiment shows that Radix Glycyrrhizae protein nano particle can be incorporated into above-mentioned 4 kinds of cell surfaces, but combination degree notable difference is the strongest with the scavenger cell NR8383 Cell binding that derives from lung.The laser scanning co-focusing microscope of the cellular affinity of Radix Glycyrrhizae protein nano particle is observed and is seen accompanying drawing 8.
embodiment 5: Radix Glycyrrhizae protein nano particle loads Cyclosiversioside F ability and measures
Accurately the Cyclosiversioside F reference liquid (being dissolved in chromatographically pure methyl alcohol) of preparation 10 mg/mL, gets 50 μ L reference liquids and adds in 1.995 mL Radix Glycyrrhizae protein samples, and making Cyclosiversioside F final concentration is 0.25 mg/mL.Get and contain 0.25 mg/mL Cyclosiversioside F and not containing each 1.6 mL of Radix Glycyrrhizae protein liquid of Cyclosiversioside F, at 100 ℃, be incubated 60 min.Get Radix Glycyrrhizae albumen 1 mL containing Cyclosiversioside F (0.25 mg/mL), add 25 ℃ of 100 kDa super filter tubes, centrifugal 60 min of 5000 g.Add Tris-hydrochloric acid (0.02 mol/L, pH8.0) 1 mL, repeatedly rinse behind super filter tube upper sample pond, the centrifugal 60min of 5000 g, sees through after ultra-filtration membrane completely until liquid, measures Astragaloside content, is free Astragaloside content.Medicine charging ratio calculation formula is as follows:
Charging ratio=(total AST content-free AST content)/total AST content
After measured, under above-mentioned experiment condition, Radix Glycyrrhizae albumen is 51.2% to Cyclosiversioside F charging ratio.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing 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> Radix Glycyrrhizae protein nano particle and preparation method thereof
<130>1
<160>1
<170>PatentIn version 3.3
<210>1
<211>15
<212>PRT
<213> Radix Glycyrrhizae albumen
<400>1
Asn Pro Asp Gly Leu Ile Ala Cys Tyr Cys Gly Gln Tyr Cys Trp
1 5 10 15
Claims (6)
1. a Radix Glycyrrhizae albumen, is characterized in that: the N-end primary structure sequence of described Radix Glycyrrhizae albumen is SEQ ID NO:1:NPDGLIACYCGQYCW.
2. a kind of Radix Glycyrrhizae albumen according to claim 1, is characterized in that: described Radix Glycyrrhizae molecular weight of albumen is that 31.0 kDa, iso-electric point are 4.5.
3. the extracting method of a kind of Radix Glycyrrhizae albumen as claimed in claim 1, it is characterized in that: through the extracting of phosphate buffered saline buffer water, ion-exchange chromatography Macro-Prep High Q and hydrophobic chromatography POROS R1, from traditional Chinese medicine of licorice root medicine materical crude slice, separation obtains an electrophoretically pure Radix Glycyrrhizae albumen.
4. a Radix Glycyrrhizae protein nano particle, it is characterized in that: Radix Glycyrrhizae protein nano particle is comprised of the albumen of Radix Glycyrrhizae described in claim 1,23 Radix Glycyrrhizae protein monomers form a spherical protein nano particle, its median size of this nano particle is 74.09 ± 0.69 nm, surface charge is negativity, and scope is at-23 ± 2 mV.
5. the preparation method of an a kind of Radix Glycyrrhizae protein nano particle as claimed in claim 4, it is characterized in that: the preparation method of described Radix Glycyrrhizae protein nano particle comprises: the pH 7.9-8.0 salts solution that this Radix Glycyrrhizae albumen is formulated as to 1 mg/mL, through 60-100 ℃ of heating 1 hour, application TSK gel G6000PW gel chromatography is removed unreacted Radix Glycyrrhizae albumen, obtains Radix Glycyrrhizae protein nano particle.
6. the preparation method of a kind of Radix Glycyrrhizae protein nano particle according to claim 5, is characterized in that: described salts solution is Tris-hydrochloric acid soln or phosphate solution.
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| CN114796002A (en) * | 2022-04-22 | 2022-07-29 | 研译(杭州)生物科技有限公司 | Nano-carrying and transdermal delivery system of active ingredients and preparation method and application thereof |
| CN115040633A (en) * | 2022-07-25 | 2022-09-13 | 中国中医科学院中医基础理论研究所 | New use of protein extract of peony and licorice decoction |
| CN115089694A (en) * | 2022-07-25 | 2022-09-23 | 中国中医科学院中医基础理论研究所 | Liquorice protein extract and new application thereof |
| CN115350163A (en) * | 2022-08-26 | 2022-11-18 | 华中科技大学 | Application of liquiritin drug-loaded nanoparticles to preparation of medicine for treating psoriasis |
| CN116983386A (en) * | 2023-08-02 | 2023-11-03 | 中国中医科学院中医基础理论研究所 | New use of licorice protein extract |
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