CN105919864B - Bamboo-leaves flavones composite nanoparticle with white-skinned face function and preparation method thereof - Google Patents
Bamboo-leaves flavones composite nanoparticle with white-skinned face function and preparation method thereof Download PDFInfo
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- CN105919864B CN105919864B CN201610380410.2A CN201610380410A CN105919864B CN 105919864 B CN105919864 B CN 105919864B CN 201610380410 A CN201610380410 A CN 201610380410A CN 105919864 B CN105919864 B CN 105919864B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/0241—Containing particulates characterized by their shape and/or structure
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
- A61K8/20—Halogens; Compounds thereof
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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Abstract
The invention discloses a kind of bamboo-leaves flavones composite nanoparticle, which is made of the component of following three kinds of weight contents: 6~13.5% bamboo-leaves flavones, 70~86% acidic polysaccharose and 0.5~24% cationic cross linking agent.Bamboo-leaves flavones are bamboo extractive, and general flavone content is 20~80% in bamboo-leaves flavones；Acidic polysaccharose is plant origin, polysaccharide containing acidic groups；Cationic cross linking agent is the acceptable cationic cross linking agent of cosmetic field.The present invention further simultaneously discloses the preparation method of above-mentioned bamboo-leaves flavones composite nanoparticle.The bamboo-leaves flavones composite nanoparticle has white-skinned face function.
Description
Technical field
The invention belongs to daily use chemicals technical fields, are related to a kind of nanoparticle more particularly to a kind of bamboo with white-skinned face function
Leaf flavones composite nanoparticle and preparation method thereof is applied to daily use chemicals industry.
Background technique
In Cosmetic Market, whitening product is constantly subjected to greatly pay close attention to and pursue.By the use of Skin whitening care cosmetics,
Asians wants to obtain pale, bright and clean skin, and American-European consumer wishes that mitigating and eliminate the pigments such as senile plaque, chloasma sinks
Product.Normal human skin color depends primarily on the melanin content in skin, and the generation of melanin is occurred in melanocyte
In, under the catalysis of tyrosinase etc., it is gradually oxidized to DOPA, DOPA quinone by tyrosine, is eventually converted into the enzymatic of melanin
Reaction.Melanocyte by melanin transfer to Epidermal basal layer cells, then on move to cuticula, finally fall off and arrange with cuticula
It lets out.When dermal melanin, which is overrun, to be increased or be unevenly distributed, pigmentation will be caused, lead to skin darkening or form color spot.
Mainly there are arbutin, hydroquinone, kojic acid and its derivative using more extensive whitening additive currently on the market
Object etc., they are usually to pass through related speed limit enzymatic activity and the inhibition black such as inhibit melanocyte proliferation, inhibit tyrosinase
The transfer of element, to reach whitening effect.
Since Traditional skin whitening agent often has stronger sensitization and potential carcinogenicity, as consumer safety is anticipated
Know and be continuously improved, pure natural, nuisanceless and obvious effect plant whitening ingredient is increasingly pursued and favored.Therefore, it opens
Send out highly-safe, white-skinned face function is strong and the natural plant whitening agent that is easily added in formula becomes the research of cosmetic field
Hot spot.Bamboo-leaves flavones (Bamboo Leaf Flavonoids, BLF) are the leaves from Phyllostachys (Phyllostachys) kind
Obtained in, the plant flavone preparation with China's native resource characteristic and independent intellectual property right, raw material sources are abundant, preparation product
Matter is stable, flavor faint scent is gentle, is a kind of safe and non-toxic native compound, have remove free radical, anti-oxidant, anti-good fortune is penetrated,
The various biologicals effect such as anti-inflammatory, anti-aging, antibacterial, antiviral, raising immunity.
The excellent biological effectiveness of bamboo-leaves flavones especially outstanding anti-oxidant, antiradical activities make it make up in whitening
Product field has huge potential use.But, bamboo-leaves flavones there are dissolubility lower, color the same with other flavone compounds
Relatively deeply, it is not easy the problems such as being absorbed by the skin, limits its application in whitening preparation.Using pharmaceutical carrier technology (such as micro- glue
Capsule, liposome etc.) to bamboo-leaves flavones carry out package be effective method.Such as the patent that publication No. is 105342889 A of CN
Text discloses a kind of skin whitening, moisturizing composition comprising bamboo-leaves flavones, carries out microencapsulation to bamboo-leaves flavones at normal temperatures and pressures
It handles, has used the organic solvents such as butanediol, propylene glycol in preparation process.
Nanoparticle (Nanoparticles) refers to the absorption such as bioactive substance, drug, is attached to particle surface,
Or dissolve, be wrapped in inside particles, become the solid fraction colloidal particle of ultra micro microspheric form carrier, including nanosphere
(Nanospheres) and nanocapsule (Nanocapsules), diameter is between 1~1000nm.When particle enters nanoscale, by
In small-size effect, skin effect, macro quanta tunnel effect etc., it is swollen that nanoparticle can show low melting point, high reaction activity, height
A series of new features different from macroscopic material such as swollen coefficient, high intensity, high tenacity, high diffusivity rate, strong absorptivity.Just because of
These characteristics, nanoparticle have become the application study hot spot in the fields such as cosmetics, medicine, functional food.
Material currently used for preparing nanoparticle mainly has synthesis high molecular material and natural macromolecular material, wherein naturally
High molecular material has that good biocompatibility, biological degradability is good, toxic side effect is small, and has special biological effectiveness etc. excellent
Point, thus be more favored.The preparation method of natural polymer nanoparticle mainly have ionic cross-linking, coacervation, emulsion-crosslinking method,
Solvent evaporated method etc., wherein ionic cross-linking is that physical crosslinking reaction occurs by the interaction between opposite charges, in turn
The glomerate gel nanoparticle of shape has many advantages, such as that reaction condition is mild, convenient and efficient, small to natural component destruction, and
Obtained nanoparticle uniform particle diameter can design, while be avoided that the use of organic solvent, be to prepare natural polymer nanometer at present
The method of grain most prospect.
Summary of the invention
The bamboo-leaves flavones composite nanoparticle that the technical problem to be solved in the present invention is to provide a kind of with white-skinned face function and
Preparation method and use, the present invention use ionic cross-linking, normal temperature and pressure, without using organic solvent under conditions of prepare bamboo
Leaf flavones composite nanoparticle, simple process is mild, obtained nano particle diameter is uniform, quality is stable, white-skinned face function extremely
Significantly；Its white-skinned face function is significantly better than arbutin.
In order to solve the above technical problem, the present invention provides a kind of bamboo-leaves flavones composite nanoparticle, the bamboo-leaves flavones are multiple
It closes nanoparticle to be made of the component of following three kinds of weight contents: 6~13.5% bamboo-leaves flavones, 70~86% (preferably 75
~86%) acidic polysaccharose and 0.5~24% (preferably 4.5~16%) cationic cross linking agent.
Improvement as bamboo-leaves flavones composite nanoparticle of the invention: bamboo-leaves flavones are bamboo extractive, the leaf of bamboo
General flavone content (in terms of rutin) is 20~80% (w/w) in flavones.It is preferred that 40~60% (w/w).
Further improvement as bamboo-leaves flavones composite nanoparticle of the invention: the acidic polysaccharose is plant origin
, polysaccharide containing acidic groups；The cationic cross linking agent is the acceptable cationic cross linking agent of cosmetic field.
Further improvement as bamboo-leaves flavones composite nanoparticle of the invention:
The acidic polysaccharose is at least one of Yuan root polysaccharide, caulis bambusae in taenian polysaccharide, algal polysaccharides；
The cationic cross linking agent is at least one of ε-poly-D-lysine, calcium chloride.
The present invention goes back while providing the preparation method of above-mentioned bamboo-leaves flavones composite nanoparticle, includes the following steps:
1) bamboo-leaves flavones, are added in distilled water and acidic polysaccharose, heating stirring to bamboo-leaves flavones and acidic polysaccharose are complete
Dissolution, obtains bamboo-leaves flavones-acid polysaccharide solution；
The mass ratio of the bamboo-leaves flavones and distilled water is 1:500~4000；
2) cationic cross linking agent and distilled water are mixed with the mass ratio of 1:400~1000, and stirred to cationic crosslinked
Agent is completely dissolved；
3) the resulting bamboo-leaves flavones-acid of step 1), is added in step 2) acquired solution under conditions of low speed magnetic agitation
In property polysaccharide solution；Continue 10~30min of low speed magnetic agitation, it is (mixed to stablize to obtain bamboo-leaves flavones composite nanoparticle solution
Suspension).
Remarks illustrate: above-mentioned addition refers to be instilled with the speed of 40~200 drop per minute.
The resulting bamboo-leaves flavones composite nanoparticle solution of the present invention, partial size can save between 300~550nm at 4 DEG C
It is spare, it is saved after may also be lyophilized.
The improvement of preparation method as bamboo-leaves flavones composite nanoparticle of the invention:
The bamboo-leaves flavones composite nanoparticle solution obtains bamboo-leaves flavones composite nanoparticle powder through drying.
The further improvement of preparation method as bamboo-leaves flavones composite nanoparticle of the invention:
The revolving speed of low speed magnetic agitation in the step 3) is 100~300r/min.
The further improvement of preparation method as bamboo-leaves flavones composite nanoparticle of the invention:
The drying is freeze-drying or addition precipitating reagent separation drying after dialysis.
The present invention goes back while providing the purposes of above-mentioned bamboo-leaves flavones composite nanoparticle: having white-skinned face function.
The partial size of composite nanoparticle can be by changing the type of acidic polysaccharose and cationic cross linking agent and matching in the present invention
Than adjusting.
The structure and composition ratio of composite nanoparticle of the invention as shown in Figure 1, compound particle partial size 300~
Between 550nm (preferably 400~550nm).
The preparation method of bamboo-leaves flavones composite nanoparticle with white-skinned face function of the invention, is to pass through ionic cross-linking
It prepares.This method reaction condition is mild, uniform particle sizes are controllable, favorable reproducibility, does not use organic solvent, simple and easy to do, tool
There are environmental protection, efficient, low cost, while the active constituent of natural goods can be kept to the maximum extent.
Efficacy experiments according to the present invention prove that the bamboo-leaves flavones composite nanoparticle has brilliant white-skinned face function,
It is significantly better than arbutin to melanocyte tyrosinase activity, B16 cell amount and the active inhibiting rate of melanocyte.
The bamboo-leaves flavones composite nanoparticle with brilliant white-skinned face function of the invention, is suitably applied skin-lightening cosmetic neck
In domain, bamboo-leaves flavones composite nanoparticle of the present invention is added in cosmetic formulations (for example, with certain proportion
12.5-100 μ g/mL), the cosmetics of different dosage forms, such as lotion, frost, essence can be prepared into；Then according to conventional makeup
Product usage mode carries out use.The bamboo-leaves flavones composite nanoparticle solution that directly present invention can also be prepared applies
It smears in skin surface.
The present invention has the advantage that
(1) the bamboo-leaves flavones composite nanoparticle with brilliant white-skinned face function has been invented, white-skinned face function is better than positive right
According to arbutin.
(2) bamboo-leaves flavones composite nanoparticle is prepared using ionic cross-linking, simple process is easy, mild condition, repetition
Property is good, and preparation process is completed in aqueous solution completely, is not related to the use of any organic reagent and surfactant, avoids pair
The destruction of active skull cap components.
(3) nano particle diameter prepared by is between 300~550nm, and appearance is opalescence suspension, and quality and performance are steady
It is fixed.
(4) the bamboo-leaves flavones composite nanoparticle invented has excellent tyrosinase activity inhibiting effect, can be well
Inhibit the activity and the generation of melanin of melanocyte, significant effect is better than positive control arbutin.
(5) the bamboo-leaves flavones composite nanoparticle invented is made of natural goods (or treating as natural goods), and preparation process is not
It is non-stimulated to skin using organic crosslinking agent and surfactant, it can be used as the whitening function factor applied to daily chemical products, have
Vast market prospect.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is that the composed structure of bamboo-leaves flavones composite nanoparticle produced by the present invention is illustrated；
Fig. 2 is the granularmetric analysis result of bamboo-leaves flavones composite nanoparticle made from embodiment 1；
Fig. 3 is the granularmetric analysis result of bamboo-leaves flavones composite nanoparticle made from embodiment 2；
Fig. 4 is the granularmetric analysis result of bamboo-leaves flavones composite nanoparticle made from embodiment 3；
Fig. 5 is the granularmetric analysis result of bamboo-leaves flavones composite nanoparticle made from embodiment 4；
Fig. 6 is that the electron microscope of bamboo-leaves flavones composite nanoparticle produced by the present invention characterizes；
Fig. 7 is that the transmission electron microscope of bamboo-leaves flavones composite nanoparticle produced by the present invention characterizes.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.Embodiment is merely to illustrate the present invention, without
It should limit the scope of the invention.It is not specified in embodiment at particular technique or condition, is retouched according to pertinent literature in the art
The technical conditions stated carry out.Production firm person is not specified in agents useful for same, is the conventional products that can be obtained by purchase.
Caulis bambusae in taenian polysaccharide described in the present embodiment and Yuan root polysaccharide are the acid that inventor extracts from caulis bambusae in taenian and Yuan root respectively
Property plant polyose, the caulis bambusae in taenian polysaccharide can be published in " the caulis bambusae in taenian polysaccharide extracting process of " food industry science and technology " according to having disclosed
Optimization and its analysis of physical and chemical property " method be prepared；The Yuan root polysaccharide, which can refer to have disclosed, to be published in
" the Composition analysis of " International Journal of Biological Macromolecules "
And anti-hypoxia activity of polysaccharide from Brassica rapa L. ", patent CN
101524389A the method is prepared；Its purity is measured 90% or more through sulfuric acid-phynol method；Bamboo-leaves flavones and ε-are poly-
Lysine is commercially available；Bamboo-leaves flavones general flavone content is about 50%.
Embodiment 1, a kind of bamboo-leaves flavones composite nanoparticle preparation method with white-skinned face function:
(1) bamboo-leaves flavones of 4mg are dissolved in the distilled water of 2mL, heating stirring dissolution；
(2) step (1) acquired solution and stirring and dissolving is added in the caulis bambusae in taenian polysaccharide of 24mg, obtains bamboo-leaves flavones-caulis bambusae in taenian polysaccharide
Solution；
(3) ε-poly-D-lysine of 2mg is dissolved in the distilled water of 1mL；
(4) with revolving speed be 100~300r/min magnetic agitation under conditions of by step (3) acquired solution with 1 minute
(that is, about with the flow velocity of 200mL/h) instills in bamboo-leaves flavones-caulis bambusae in taenian polysaccharide solution；
(5) continue low speed (100~300r/min) and stir 30min, obtained opalescence suspension is the bamboo with white-skinned face function
Leaf flavones composite nanoparticle solution (I).
Embodiment 2, a kind of bamboo-leaves flavones composite nanoparticle preparation method with white-skinned face function:
(1) bamboo-leaves flavones of 4mg are dissolved in the distilled water of 2mL, heating stirring dissolution；
(2) step (1) acquired solution and stirring and dissolving is added in the caulis bambusae in taenian polysaccharide of 36mg, obtains bamboo-leaves flavones-caulis bambusae in taenian polysaccharide
Solution；
(3) ε-poly-D-lysine of 2mg is dissolved in the distilled water of 2mL；
(4) with revolving speed be 100~300r/min magnetic agitation under conditions of by step (3) acquired solution with 1 minute
(that is, about with the flow velocity of 300mL/h) instills in bamboo-leaves flavones-caulis bambusae in taenian polysaccharide solution；
(5) continue to stir at low speed 30min, obtained opalescence suspension is the bamboo-leaves flavones composite nano-granule with white-skinned face function
Sub- solution (II).
Embodiment 3, a kind of bamboo-leaves flavones composite nanoparticle preparation method with white-skinned face function:
(1) bamboo-leaves flavones of 4mg are dissolved in the distilled water of 16mL, heating stirring dissolution；
(2) step (1) acquired solution and stirring and dissolving is added in the Yuan root polysaccharide of 50mg, obtains bamboo-leaves flavones-Yuan root polysaccharide
Solution；
(3) ε-poly-D-lysine of 10mg is dissolved in the distilled water of 4mL；
(4) with revolving speed be 100~300r/min magnetic agitation under conditions of by step (3) acquired solution with 6 minutes
(that is, about with the flow velocity of 200mL/h) instills in bamboo-leaves flavones-Yuan root polysaccharide solution；
(5) continue to stir at low speed 30min, obtained opalescence suspension is the bamboo-leaves flavones composite nano-granule with white-skinned face function
Sub- solution (III).
Embodiment 4, a kind of bamboo-leaves flavones composite nanoparticle preparation method with white-skinned face function:
(1) bamboo-leaves flavones of 6mg are dissolved in the distilled water of 14mL, heating stirring dissolution；
(2) step (1) acquired solution and stirring and dissolving is added in the Yuan root polysaccharide of 50mg, obtains bamboo-leaves flavones-Yuan root polysaccharide
Solution；
(3) ε-poly-D-lysine of 10mg is dissolved in the distilled water of 4mL；
(4) with revolving speed be 100~300r/min magnetic agitation under conditions of by step (3) acquired solution with 5 minutes
(that is, about with the flow velocity of 200mL/h) instills in bamboo-leaves flavones-Yuan root polysaccharide solution；
(5) continue to stir at low speed 30min, obtained opalescence suspension is the bamboo-leaves flavones composite nano-granule with white-skinned face function
Sub- solution (IV).
The bamboo-leaves flavones composite nanoparticle solution that 1~embodiment of above-described embodiment 4 is prepared is dry by vacuum refrigeration
It is dry to constant weight, obtain bamboo-leaves flavones composite nanoparticle.
Experiment 1
Using Malvern Zetasizer Nano Zs90 nanometer particle size and potentiometric analyzer, to embodiment in the present invention
Gained bamboo-leaves flavones composite nanoparticle carries out granularmetric analysis in 1-4.As a result as shown in table 1 and Fig. 2-5, by embodiment 1-4 system
There must be the bamboo-leaves flavones composite nanoparticle of white-skinned face function, average grain diameter is between the section 400-550nm, particle diameter distribution index
PDI is 0.2 or so, illustrates its uniform particle diameter, dispersion degree is good.
Table 1, nanoparticle particle size determination analytical table
Experiment 2
Using transmission electron microscope (Transmission Electron Microscope, TEM) and electron microscope,
Morphological Characterization is carried out to 3 gained bamboo-leaves flavones composite nanoparticle of embodiment in the present invention.As shown in Figure 6 and Figure 7, embedding gained
Nanoparticle it is spherical in shape or spherical, partial size is more uniform, does not generate some huge Filamentous or random nanoparticles.
Experiment 3, the measurement of bamboo-leaves flavones composite nanoparticle flavones content
(1) experimental principle
Flavones content in bamboo-leaves flavones composite nanoparticle directly affects its white-skinned face function, using aluminum nitrate colorimetric method pair
General flavone content in embodiment 1-4 in bamboo-leaves flavones nanoparticle is measured.
(2) experimental method
1, the production of standard curve
Precision weighs rutin control 10mg in 100mL measuring bottle, adds methanol to dissolve and is diluted to scale, therefrom draw solution
0,0.5,1.0,2.0,3.0,4.0mL is diluted to 5mL respectively at 30% ethanol solution in 25mL colorimetric cylinder, is added, successively respectively plus
5% sodium nitrite solution 0.3mL, places 5min after shaking；10% aluminum nitrate solution 0.3mL is added, places 6min after shaking；Add
Enter 1.0mol/L sodium hydroxide solution 4.0mL, 30% ethanol solution is diluted to 10.0mL, places 10min after shaking up；It is managed with No. zero
It is by ordinate, concentration of trap with the cuvette of 1cm, the measurement absorbance at the wavelength of 510nm after shaking up for blank
Abscissa draws standard curve.
2, the measurement of bamboo-leaves flavones nanoparticle flavones content
The nanoparticle suspension high speed centrifugation (15000r/30min) that will have been prepared, measures the body of centrifuged supernatant
Product, takes supernatant 1ml, measures flavones content therein by the method in 1, and then obtain the flavones content in total supernatant, from
And calculate the flavones content in nanoparticle.
Nanoparticle flavones content (%)=(general flavone content in general flavone content-supernatant of addition)/addition is total
Flavones content × 100.
(3) experimental result
In table 2, bamboo-leaves flavones composite nanoparticle flavones content measurement (n=3,)
| Group | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| Flavones content (%) | 10.82±0.83 | 20.45±0.57 | 45.27±0.92 | 41.83±0.76 |
Since in the component of bamboo-leaves flavones composite nanoparticle, the ingredient with white-skinned face function is mainly bamboo-leaves flavones, because
The content of bamboo-leaves flavones and its white-skinned face function have substantial connection in this composite nanoparticle.It can be obtained by table 2, obtained by different embodiments
Bamboo-leaves flavones composite nanoparticle effective embedding has been carried out to bamboo-leaves flavones, wherein the flavones content of embodiment 3 reaches most
A height of 45.27%, that is, the embedding effect of embodiment 3 is best.
Test the evaluation of 4 white-skinned face functions
(1) experimental principle
Melanin content be determine skin color key factor, and the synthesis of melanin be occur melanocyte,
Under the catalysis of the correlation rate-limiting enzyme such as tyrosinase, it is gradually oxidized to DOPA, DOPA quinone by tyrosine, is eventually converted into melanin
Enzymatic reaction, therefore the approach that whitening agent plays effect usually has following three kinds: inhibit melanocyte proliferation, inhibit tyrosinase
Activity and the synthesis for inhibiting melanin.
The melanocyte in skin base of the basic structure of B-16 melanocyte, especially B16 cell function and human normal
This is consistent, and in the case where human primary's dermal melanin cell culture is extremely difficult, B-16 melanoma cells become medicine portion
Door carries out pigment studies of lesions and the widely used cell of skin-lightening cosmetic additive efficacy assessments.
The present invention is using B-16 melanocyte as test cell strain, and setting up arbutin is positive control, without pharmaceutically-active black
Plain cell is negative control, right in terms of inhibiting melanocyte proliferation, inhibiting tyrosinase activity and inhibit melanin genesis three
The white-skinned face function of gained embodiment 1-4 bamboo-leaves flavones composite nanoparticle of the invention is assessed.
(2) experimental method
1, tested material is handled
Setting up bamboo-leaves flavones composite nanoparticle obtained by embodiment 1-4 in the present invention is experimental group, and concentration is 12.5-100 μ
G/mL, it is spare after 0.22 μm of membrane filtration sterilizing using same concentration arbutin solution as positive controls.
2, the culture of cell and tested material prepare
Under aseptic condition, B-16 melanoma cells are inoculated in the training of the RPMI1640 culture medium containing 10% newborn bovine serum
It supports in bottle, 37 DEG C in carbon dioxide incubator, 5%CO2It is cultivated in saturated humidity environment, and changes liquid daily.When cell is grown
To convergence degree be 80%~90% when, through 0.25% trypsin digestion pass on, continue to cultivate.
3, tested material exposure
Nearly Fusion Strain is grown to through 0.25% trypsin digestion to cell, and collecting and adjusting cell density is 5 × 104
A/mL, every 100 μ L of hole is inoculated in 96 orifice plates, and supplements culture solution to 200 μ L.37 DEG C in carbon dioxide incubator, 5%CO2
It is cultivated in saturated humidity environment, overnight, more renews culture medium after adherent, the tested material solution of 200 μ L various concentrations is added in every hole,
For the volume fraction of DMSO less than 0.5%, 6 multiple holes are arranged in each concentration, and fresh medium is added instead of tested in blank control group
Object solution, changes liquid daily.
4, the measurement of cell proliferation rate
Cell fishplate bar and tested material exposure treatment are same as above.It is measured using mtt assay cell proliferation rate.When cultivating 44
It taking out, 20 μ L of 5mg/mL MTT solution is added in every hole, at 37 DEG C in carbon dioxide incubator, 5%CO2In saturated humidity environment
It is incubated for 4h, culture medium and MTT is then discarded, the DMSO of 150 μ L is added into each hole, to dissolve remaining MTT- formazan crystallization,
10min is shaken, measures light absorption value of each hole at 570nm with microplate reader after mixing.
Calculate cell proliferation rate:
Cell proliferation rate (%)=(OD570 (experimental ports)—OD570 (blank wells))/(OD570 (blank control wells)—OD570 (blank wells))×100
5, the measurement of intracellular tyrosine enzymatic activity relative inhibition
Cell fishplate bar and tested material exposure treatment are same as above.Intracellular tyrosine enzymatic activity is surveyed using dopa oxidase method
It is fixed.The cell that action time has been arrived abandons supernatant, and PBS buffer solution is washed 2 times, and 50 μ of 1%Triton-X100 aqueous solution is added in every hole
L is put into rapidly in -80 DEG C of refrigerators and freezes 30min, and 37 DEG C of thawings are completely severed cell (about 20min) after taking-up, is added 1%
10 hole μ L/ of L-DOPA solution, the cell of whitening agent culture is not added as control, 37 DEG C of reaction 2h set microplate reader and measure 475nm
Locate OD475Value.
Calculate tyrosinase relative inhibition:
Intracellular tyrosine enzymatic activity relative inhibition (%)=(1-ODExperimental group/ODBlank control group)/cell proliferation rate × 100
6, intracellular melanin content measurement
Cell fishplate bar and tested material exposure treatment are same as above.Culture solution is removed in culture 48h hypsokinesis, is washed 1 time, is added with PBS buffer solution
Enter the 0.25% pancreatin digestion of 100 μ L, then is blown and beaten sedimentation cell at counting after suspension, by density with PBS buffer solution (2-3mL)
It adjusts to 105A/mL or so is spare.Take spare cell suspension to add PBS buffer solution, it is each draw 1mL cell suspension be respectively placed in from
In heart pipe, liquid is discarded supernatant after 1500r/min centrifugation 10min, 200 μ L PBS buffer solution, which are first added, makes cell suspend again, so
1mL ethyl alcohol is added afterwards: ether solution (volume ratio 1: 1) places 20min to dissolve the opaque particle of non-melanocyte at room temperature,
3000r/min centrifugation 5min simultaneously discards supernatant liquid, and the 1mol/L NaOH solution that 1mL contains 10% dimethyl sulfoxide is added, is placed in 80
DEG C water-bath 45min, is completely dissolved cell mass.96 orifice plates are transferred to, each concentration 6 parallel, with microplate reader in 405nm wave
Strong point measures absorbance.
Calculate opposite melanin content:
Opposite melanin content (%)=(OD in individual cellsExperimental group/ODControl group)/cell proliferation rate × 100
(3) experimental result
1, influence of the bamboo-leaves flavones composite nanoparticle to B-16 cell proliferation rate
Dermal melanin cell tissue can be by melanin transfer into Epidermal basal layer cells, so as to cause local skin mistake
Black and pigmentation, therefore cell proliferation rate is the important indicator for judging whitening effect.
Table 3, different whitening agent to B-16 cell proliferation rate influence (n=3,)
Note:aCompared with negative control group, P < 0.05；bCompared with positive controls, P < 0.05.
As shown in table 3, B-16 melanoma cells are handled with the whitening agent of various concentration, with the increase of whitening agent concentration,
Compared with negative control group, the inhibiting rate of each group sample cell proliferation increases, and cell viability gradually decreases, survival rate decline.
Positive controls arbutin in all tested concentration cell survival rate reduce but it is unobvious, cell proliferation rate 90% a left side
It is right；When concentration reaches 50 μ g/mL, bamboo-leaves flavones composite nanoparticle obtained by each embodiment begins with suppression to B-16 cell Proliferation
Production is used, and wherein 3 inhibiting effect of embodiment is most obvious, and cell proliferation rate is (68.59 ± 0.53) %；In 100 μ g/mL concentration
Under, cell proliferation rate drops to (40.86 ± 0.83) %, and there were significant differences compared with positive control arbutin group (P < 0.05).
Therefore, bamboo-leaves flavones composite nanoparticle can influence the proliferation of melanoma cells to a certain extent；With positive controls black bearberry
Glycosides is compared, and dose-effect relationship is more obvious, i.e., with the increase of concentration, cell survival rate reduces amplitude and is noticeably greater than arbutin,
The proliferation of melanoma cells can be significantly inhibited in 50 μ g/mL and 100 μ g/mL, to become the possible approaches of white-skinned face function.
2, influence of the bamboo-leaves flavones composite nanoparticle to B-16 intracellular tyrosine enzymatic activity
Tyrosinase is a kind of conclusive rate-limiting enzyme during melanocyte synthesis of melanin, and catalytic action is mainly sent out
Life is converted to DOPA in tyrosine and DOPA is converted into the two reactions of DOPA quinone.Most of whitening agent is by inhibiting junket ammonia
Phytase activity or the oxidative pathway for blocking tyrosine generation melanin, so that the generation for reducing melanin reaches white-skinned face function, because
This whitening agent is the important evidence for measuring white-skinned face function to the inhibiting rate of tyrosinase activity.
Table 4, different whitening agent to B-16 intracellular tyrosine enzyme relative activity influence (n=3,)
Note:aCompared with negative control group, P < 0.05；bCompared with positive controls, P < 0.05.
As shown in table 4, positive control arbutin group and bamboo-leaves flavones composite nanoparticle are to intracellular tyrosine enzymatic activity
There is inhibiting effect, and there is dose-dependence, i.e., is improved with inhibiting rate of the increase of dosage to tyrosinase activity；Implement
In example 1-4, embodiment 3,4 gained bamboo-leaves flavones composite nanoparticles become apparent from the inhibitory effect of tyrosine enzyme activity, work as concentration
Tyrosinase activity is respectively (30.32 ± 2.30) % and (38.45 ± 1.96) % when for 100 μ g/mL, with positive control black bearberry
Glycosides group is compared to there were significant differences (P < 0.05).Illustrate that bamboo-leaves flavones composite nano-granule subgroup can significantly inhibit the intracellular junket ammonia of B-16
Sour enzyme relative activity.
3, influence of the bamboo-leaves flavones composite nanoparticle to B-16 cell melanin genesis
Melanin content is the principal element for determining skin color, and melanin is transferred to horn cell after being synthesized by melanocyte
Cuticula is moved on again, leads to skin darkening or forms color spot.Therefore, melanin content number be measure whitening agent white-skinned face function
Direct basis.The intracellular melanin content crossed by the sample treatment of variety classes and concentration is as shown in table 5.
Table 5, different whitening agent to B-16 cell with respect to melanin content influence (n=3,)
Note:aCompared with negative control group, P < 0.05；bCompared with positive controls, P < 0.05.
As shown in Table 5, with melanin content in the individual cells of blank control for 100%, then with the increase of concentration, each beauty
White dose of group melanin content decreases, and dose-dependence is presented；When concentration is 100 μ g/mL, obtained by embodiment 1-4
Bamboo-leaves flavones composite nanoparticle is obvious to the inhibitory effect of melanin genesis, and wherein embodiment 3,4 experimental groups are relatively black into the cell
Cellulose content is reduced to (37.65 ± 3.14) % and (45.35 ± 0.97) % respectively, has compared with positive control arbutin group significant
Difference (P < 0.05) illustrates that bamboo-leaves flavones composite nanoparticle can significantly inhibit the melanin genesis of B-16 melanocyte.
Conclusion: bamboo-leaves flavones composite nanoparticle has significant white-skinned face function, and whitening function is by black to B-16
Chromatophore proliferation, the synthesis of melanin, intracellular tyrosine enzyme relative activity etc. are many-sided to be inhibited to realize.In addition, in conjunction with
The result of comparison sheet 3-5 and table 2 can verify that as drawn a conclusion: the white-skinned face function of bamboo-leaves flavones nanoparticle contains with flavones contained therein
Amount is positively correlated, that is, embedding effect is directly proportional to white-skinned face function.
Comparative example 1-1, the dosage of Yuan root polysaccharide in embodiment 3 is changed to 70mg by 50mg, remaining is equal to embodiment 3.
Comparative example 1-2, the dosage of Yuan root polysaccharide in embodiment 3 is changed to 30mg by 50mg, remaining is equal to embodiment 3.
Comparative example 1-3, the Yuan root polysaccharide in embodiment 3 is changed to caulis bambusae in taenian polysaccharide, remaining is equal to embodiment 3.
Comparative example 2-1, ε-poly-D-lysine dosage in embodiment 3 is changed to 15mg by 10mg, remaining is equal to implementation
Example 3.
Comparative example 2-2, ε-poly-D-lysine dosage in embodiment 3 is changed to 5mg by 10mg, remaining is equal to embodiment
3。
Comparative example 2-3, ε-poly-D-lysine in embodiment 3 is changed to calcium chloride, remaining is equal to embodiment 3.
Comparative example 3-1, the dosage of bamboo-leaves flavones in embodiment 3 is changed to 8mg by 4mg, remaining is equal to embodiment 3.
Comparative example 3-2, the dosage of bamboo-leaves flavones in embodiment 3 is changed to 2mg by 4mg, remaining is equal to embodiment 3.
Comparative example 4-1, constant current flow rate pump in embodiment 3 is changed to 400mL/h by 200mL/h, that is, the time of instillation is by 6
Minute is changed to 3 minutes；Remaining is equal to embodiment 3.
Comparative example 4-2, constant current flow rate pump in embodiment 3 is changed to 100mL/h by 200mL/h, that is, the time of instillation is by 6
Minute is changed to 12 minutes；Remaining is equal to embodiment 3.
Bamboo-leaves flavones nanoparticle obtained by above-mentioned all comparative examples is subjected to flavones content measurement, acquired results are as follows:
Bamboo-leaves flavones nanoparticle obtained by table 6, different comparative example flavones content (n=3,)
| Comparative example | 1-1 | 1-2 | 1-3 | 2-1 | 2-2 |
| Flavones content (%) | 41.03±0.23 | 19.58±0.17 | 10.07±0.82 | 25.41±0.76 | 35.07±0.53 |
| Comparative example | 2-3 | 3-1 | 3-2 | 4-1 | 4-2 |
| Flavones content (%) | 23.28±0.55 | 39.58±0.26 | 15.04±0.43 | 30.22±0.72 | 36.24±0.83 |
As shown in table 6, the flavones content of bamboo-leaves flavones composite nanoparticle obtained by above-mentioned all comparative examples is measured,
Can be obtained by measurement result: nanoparticle obtained by all comparative examples has carried out effective embedding to bamboo-leaves flavones, wherein flavones content
Up to 41.03% (comparative example 1-1), the respectively less than flavones content of 3 gained bamboo-leaves flavones nanoparticle of embodiment
(45.27%).By 4 conclusions of experiment it is found that bamboo-leaves flavones composite nanoparticle obtained by above-mentioned all comparative examples all has whitening function
Effect, but it is not so good as the embodiment of the present invention 3 since it embeds effect, white-skinned face function is not so good as the embodiment of the present invention 3.
Above description describe the basic principles, principal features and advantages of the present invention.The present invention is not by above-described embodiment
Limitation, all any modification, equivalent replacement, combination, simplification, improvement made without departing from the spirit and scope of the present invention
Deng should be included within the scope of the present invention.
Claims (6)
1. bamboo-leaves flavones composite nanoparticle, it is characterized in that the bamboo-leaves flavones composite nanoparticle is by following three kinds of weight contents
Component is constituted: 6~13.5% bamboo-leaves flavones, 70~86% acidic polysaccharose and 0.5~24% cationic cross linking agent；
The acidic polysaccharose is at least one of Yuan root polysaccharide, caulis bambusae in taenian polysaccharide, and the cationic cross linking agent is that ε-poly relies ammonia
Acid；
The preparation method of the bamboo-leaves flavones composite nanoparticle is to include the following steps:
1) bamboo-leaves flavones, are added in distilled water and acidic polysaccharose, heating stirring to bamboo-leaves flavones and acidic polysaccharose are completely dissolved,
Obtain bamboo-leaves flavones-acid polysaccharide solution；
The mass ratio of the bamboo-leaves flavones and distilled water is 1:500~4000；
2) cationic cross linking agent and distilled water are mixed with the mass ratio of 1:400~1000, and stirred complete to cationic cross linking agent
Fully dissolved；
3), under conditions of low speed magnetic agitation that step 2) the acquired solution addition resulting bamboo-leaves flavones-acidity of step 1) is more
In sugar juice；Continue 10~30min of low speed magnetic agitation, obtains bamboo-leaves flavones composite nanoparticle solution.
2. bamboo-leaves flavones composite nanoparticle according to claim 1, it is characterized in that:
Bamboo-leaves flavones are bamboo extractive, and general flavone content is 20~80% in the bamboo-leaves flavones.
3. bamboo-leaves flavones composite nanoparticle according to claim 2, it is characterized in that:
The bamboo-leaves flavones composite nanoparticle solution obtains bamboo-leaves flavones composite nanoparticle powder through drying.
4. bamboo-leaves flavones composite nanoparticle according to claim 2 or 3, it is characterized in that:
The revolving speed of low speed magnetic agitation in the step 3) is 100~300r/min.
5. the preparation method of bamboo-leaves flavones composite nanoparticle according to claim 3, it is characterized in that:
The drying is freeze-drying or addition precipitating reagent separation drying after dialysis.
6. the purposes of the bamboo-leaves flavones composite nanoparticle as described in Claims 1 to 5 is any, it is characterized in that: having whitening function
Effect.
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| CN116139062A (en) * | 2021-05-26 | 2023-05-23 | 湖北爱莲生物科技有限公司 | A natural lotus cosmetic containing nanoparticles and its preparation method |
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