CN103333222A - Pearl natural active peptide having whitening efficacy, and extraction method and application thereof - Google Patents
Pearl natural active peptide having whitening efficacy, and extraction method and application thereof Download PDFInfo
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- CN103333222A CN103333222A CN2013102898039A CN201310289803A CN103333222A CN 103333222 A CN103333222 A CN 103333222A CN 2013102898039 A CN2013102898039 A CN 2013102898039A CN 201310289803 A CN201310289803 A CN 201310289803A CN 103333222 A CN103333222 A CN 103333222A
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Abstract
The invention belongs to the technical field of biomedical materials, and discloses a pearl natural active peptide having a whitening efficacy, a method for extracting the pearl natural active peptide from nacre, and an application of the pearl natural active peptide. The pearl natural active peptide is obtained by extracting nanometer pearl powder as a raw material through heating water extraction and ultrasonic concussion conditions based on the full exposure of the effective active components of the nanometer pearl powder, and separating and purifying through technologies comprising ultrafiltration for removing macromolecular substances, membrane separation for removing salts, gel chromatographic purification and the like, and the relative molecular mass of the pearl natural active peptide is accurately detected through high performance liquid chromatography. The pearl natural active peptide has the characteristics of basically zero addition of any reagents in the whole extraction and purification processes, guarantee of the natural activities, and good safety, and can be widely used in the fields of cosmetics and medicines for inhibiting the synthesis of melanin in cells as a whitening agent.
Description
Technical field
The invention belongs to biological medicine material technology field, relate to a kind of pearl natural radioactivity peptide of Magnesium Ascorbylphosphate, also relate to the method for from nacre, extracting this kind pearl natural radioactivity peptide, and the application that relates to this kind pearl natural radioactivity peptide.
Background technology
In recent years, developing rapidly and grow in strength along with pearl sector, China's pearl production becomes ascendant trend year by year, yet the rate of obtaining of high-grade pearl is but very low, even reach ten thousand/ degree, therefore, the relatively poor outer inferior pearl production of level of profile constantly increases, accumulate for a long time, cause the pearl resource not to be fully used.According to statistics, in the freshwater cultured pearl, can not account for about 40% of pearl ultimate production as the inferior pearl that bullion is sold at present.How adopting suitable working method that a large amount of inferior pearls is converted into pearl medicine, healthcare products and the makeup with high added value, is the problem that vast pearl business men and scientific research personnel pay close attention to most.
Contain the various active composition in the pearl, as the anti-ageing factor-porphyrin, the light base Europe phosphorus amino acid that beautifies the skin effect, taurine, carotenoid, albumen, small molecule active peptide, VITAMIN etc.Modern study shows that pearl can promote the metabolism of body, suppresses the lipofuscin growth, increases the epidermic cell vigor, and delaying cell aging has beauty treatment and brightens removing beverage, removing heat to brighten vision, effects such as detoxify and promote granulation.Clinically prove that also it is evident in efficacy to cardiovascular and cerebrovascular diseases, hypertension, diabetes, insomnia, constipation, osteoporosis, hyperosteogeny, old pain in the waist and lower extremities etc., also be used to nurse one's health alcoholic liver etc.Pearl protein is the main ingredient of organic substrate in the nacreous layer, discovers, pearl active albumen can significantly improve bone density, has good function of resisting osteoporosis.And other effects still are in the preliminary study stage, and its research in fields such as medicinal, beauty treatment does not then appear in the newspapers.In recent years, along with the particularly fast development of life science, bioseparation engineering of scientific research, novel separation and purification means provide effective way for the exploitation of pearl active albumen.
Application and Development for the pearl functional component rests in the comparatively fixing technology pattern relatively at present, the crude extract of method preparation such as utilize traditional water to carry, enzymolysis is mentioned in acid is widely used in the additional product of pearl, the blindness and the inefficiencies that cause the pearl active components utilising, and be almost blank for the exploitation of pearl whitening functional component.For overcoming above deficiency, the present invention utilizes biological chemistry and Protocols in Molecular Biology that the pearl active composition is deeply developed, a kind of pearl natural radioactivity peptide of Magnesium Ascorbylphosphate is provided, and the method for from nacre, extracting this kind pearl natural radioactivity peptide, also have the application of this kind pearl natural radioactivity peptide.This is pearl as the optimal path of the bright skin prepared using of whitening, and has effectively improved the utility value of pearl simultaneously, realizes the comprehensive utilization of pearl, increases economic benefit.
Summary of the invention
The pearl natural radioactivity peptide that the purpose of this invention is to provide a kind of Pear Power activity.
Purpose of the present invention also provides the extracting method of the pearl natural radioactivity peptide of this kind Pear Power activity.
Another object of the present invention provides the application of the pearl natural radioactivity peptide of this kind Pear Power activity.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
A kind of pearl natural radioactivity peptide of Magnesium Ascorbylphosphate is raw material with the nanometer Margarita powder, utilizes external energy auxiliary water formulation to extract, obtain through separation and purification, for solid content is 0.2～1% the aqueous solution or is flaxen powder, Magnesium Ascorbylphosphate, its molecular weight≤3000 dalton.
Utilize the nano-pulverization technology to make after the effective constituent of pearl fully comes out, utilize the auxiliary extraction process by water of external energy to extract water-soluble base, the pearl peptide of the tool whitening function that obtains through separation and purification.Described nano-pulverization technology is to utilize comminution by gas stream to prepare the natural nano pearl powder, just use pure physical method, under the situation of not destroying the pearl natural component, make the effective active composition of pearl come out fully, the release rate of effective active composition and burst size also can significantly improve.
Described external energy auxiliary water formulation refers to it be applied the pressure ultrasonic oscillation, to realize the nanometer degraded of nanometer Margarita powder in 80 ℃ of ultrapure water water-baths extraction pearl dissolvable matrix processes.Described separation and purification refers to utilize ultrafiltration removal macromolecular substance, electrodialysis removal inorganic salt, column chromatography (also claiming gel chromatography) that small-molecular peptides is further purified.This kind pearl natural radioactivity peptide is through biochemical reaction and melanoma cells model discrimination, and tool suppresses that melanocyte forms and to cell toxicological harmless effect.
The extracting method of the pearl natural radioactivity peptide of this kind Magnesium Ascorbylphosphate may further comprise the steps successively:
A. raw material is prepared: prepare particle diameter less than the nanometer Margarita powder of 100nm; The original active substance of pearl is fully exposed, be not destroyed simultaneously.Use pure physical method, under the situation of not destroying the pearl natural component, make the effective active composition of pearl come out fully, the release rate of effective active composition and burst size also can significantly improve.
B. extract: be the mixed of 1:5～4:5 by weight with nanometer Margarita powder with deionized water, put into ultrasonic water bath and shake 2～8h that bath temperature is 60～100 ℃; To realize the abundant extraction of pearl water-soluble active ingredient.
C. separate: mixed solution is put into the centrifugal 15～30min of whizzer, and centrifugal speed is 2500～3500r/min, collects supernatant liquor, gets filtrate through the filter membrane suction filtration, is pearl water extract;
D. ultrafiltration: pearl water extract takes off metafiltration liquid after the ultra-filtration centrifuge tube ultrafiltration, reconcentration gets concentrated solution;
E. electrodialysis: concentrated solution is removed the impurity mineral ion through electroosmose process, obtain the organic active liquid of small molecules;
F. column chromatography: the organic active liquid of small molecules is carried out the dextrane gel column chromatography, collect the component that there is absorption peak at the 280nm place, after lyophilize, obtain pearl natural radioactivity peptide.Press the peak and collect component under the 280nm, obtain 2 main elution peaks, collect second elution peak in a large number.Pearl natural radioactivity peptide is that solid content is 0.2～1% the aqueous solution, or the lyophilize powdered, can be in lyophilize under the vacuum condition, activeconstituents is the pale yellow powder shape, its molecular weight≤3000 dalton, can be directly as the functional component of skin-lightening cosmetic.
Preferably, the nanometer Margarita powder in the described steps A is the natural nano pearl powder that pearl is prepared from through pure physical method.Use pure physical method, under the situation of not destroying the pearl natural component, make the effective active composition of pearl come out fully, the release rate of effective active composition and burst size also can significantly improve.
Described pure physical preparation method comprises successively that the raw material pearl selects, cleans, step such as oven dry, coarse reduction, fine powder are broken, nanometer pulverizing, drying, the pulverizing of coacervate secondary.Wherein, the coarse reduction step is processed into particle diameter at 20～300 purpose pearl powders on Roughpulverizer pearl is sheared by force with pearl; The fine powder quick short steps are rapid for the pearl meal after the coarse reduction is put into ultrafine crusher, with airflow pulverization method the pearl meal are ground into particle diameter at the pearl fine powder of 2～10 μ m; The nanometer pulverising step is for to put into nano grinder with the pearl fine powder, soak the back and form pearl fine powder slurry, the concentration of pearl fine powder slurry is 30～50%, in nano grinder by strong shearing, impact and friction pulverized fully, dispersion and emulsification, make particle diameter at the nano-glass pearl powder slurry of 20～200nm; Drying step is with the rapid drying on drying plant of nano-glass pearl powder slurry, forms the loose body of reuniting; Coacervate secondary pulverising step is pulverized for loose body that dried nano-glass pearl powder is reunited carries out secondary with airflow pulverization method on ultrafine crusher, obtains particle diameter at the natural nano pearl powder of 20～200nm.Particle diameter is 70～75% in the ratio of 40～100nm in this natural nano pearl powder.
Preferably, the nanometer Margarita powder among the described step B and deionized water are put into ultrasonic water bath and are shaken 4h by weight being 2:5, and bath temperature is 80 ℃.
Preferably, the mixed solution among the described step C is put into the centrifugal 20min of whizzer, and centrifugal speed is 3000r/min, and the employed filter membrane of suction filtration is 0.45 μ m filter membrane.
Preferably, the ultra-filtration centrifuge tube among the described step D is the 3kD ultra-filtration centrifuge tube.Need select suitable ultrafiltration pipe during ultrafiltration, usually answer the molecular weight cut-off should be greater than 1/3 of target protein molecular weight, be 35kD such as the target protein molecular weight, just can select the ultrafiltration pipe of 10kD molecular weight cut-off, if the target protein molecular weight is about 10kD, then can use the ultrafiltration pipe of molecular weight cut-off 3kD.
Preferably, the impurity mineral ion in the described step e comprises Ca
2+, Mg
2+, K
+, Na
+, CO
3 2+, OH
-With SO
4 2-
Preferably, the dextrane gel in the described step F is the SephadexG-25 dextrane gel.The commodity of SephadexG sephadex are called Sephndex, and the dextran of different size model represents that with English alphabet G the Arabic number of G back is 10 times that gel gets water number.For example, G-25 is every gram gel suction 2.5 grams when expanding, and same G-200 restrains dried glue suction 20 grams of every gram.The kind of sephadex has G-10, G-15, G-25, G-50, G-75, G-100, G-150 and G-200.Therefore, " G " reflection, the crosslinking degree of gel, degrees of expansion and distribution range.Sephadex G-25 sephadex G-25 separating ranges 1000-5000 is applicable to desalination, peptide and other micromolecular separation.
Preferably, further comprising the steps of:
G. detect: with the pearl natural radioactivity peptide that obtains with deionized water dissolving after, with high performance liquid chromatography (High Performance Liquid Chromatography, abbreviation HPLC) relative molecular mass of polypeptide product distributes before and after the detection purifying, testing conditions is: chromatographic column is TSKgel gel column G2000SWXL300mm * 7.8mm, mobile phase volume is than being Yi Jing ︰ Shui ︰ trifluoroacetic acid=30 ︰, 70 ︰ 0.1, the detection wavelength is UV280nm, and flow velocity is 1ml/min, and column temperature is 30 ℃; Be that 67000 bovine serum albumin, molecular weight are that 1335 vitamin B12 and molecular weight are that 614 Sleep-promoting factor B is made into and mixes mark with molecular weight, every kind of substances content is 5mg/ml, as standard model; Recording the accurate scope of its molecular weight is 100～2100 dalton.This composition carries out the effect checking through external tyrosine oxidase inhibition test, mouse black-in tumor cell test and all shows the good activity of whitening, and its effect all is better than arbutin.
The application of the pearl natural radioactivity peptide of this kind Magnesium Ascorbylphosphate in makeup, healthcare products or the medicine of preparation Magnesium Ascorbylphosphate.Pearl natural radioactivity peptide of the present invention, can be used as whitening agent and is used for makeup the significant inhibitory effect that is formed with of melanocyte in the cell through B16 melanoma cells model discrimination; In addition pearl powder as healthcare products, traditional Chinese medicine ingredients by people's life-time service, confirmed nontoxicly, therefrom the natural radioactivity peptide of Ti Quing is fool proof to human body.
Beneficial effect of the present invention: do not introduce any organic reagent fully in the pearl natural radioactivity peptide leaching process that the present invention obtains, guarantee that pure physical method extracts in the whole leaching process, guarantee the natural character of little peptide; Utilize the pure natural method to extract, through separation and purification, through biochemical reaction and melanoma cells model discrimination, tool suppresses that melanocyte forms and to the natural radioactivity peptide of cell toxicological harmless effect.The extraction and separation method technology that the present invention adopts is simple, cost is low, be easy to suitability for industrialized production; Adopt chromatographic technique that the natural radioactivity peptide has been carried out purifying and carried out molecular weight determination, not only can improve the state of the art of research and development of pearl and the economic benefit of pearl processing enterprise greatly, also establish certain basis for pearl active Products Development and industry conversion; Pearl natural radioactivity peptide of the present invention, can be used as whitening agent and is used for makeup the significant inhibitory effect that is formed with of melanocyte in the cell through B16 melanoma cells model discrimination; Simultaneously pearl powder as healthcare products, traditional Chinese medicine ingredients by people's life-time service, confirmed nontoxicly, therefrom the natural radioactivity peptide of Ti Quing is fool proof to human body.
Description of drawings
Below in conjunction with the drawings and specific embodiments the present invention is further described:
Fig. 1 is the Sephadex G25 gel-filtration curve of pearl active peptide sample;
Fig. 2 is the molecular weight distribution synoptic diagram of pearl active peptide sample;
Fig. 3 is that pearl active peptide is to the mouse black-in tumor cell inhibiting rate;
Fig. 4 is that pearl active peptide influences the mouse black-in tumor cell melanin content.
Embodiment
The pearl natural radioactivity peptide embodiment 1 of a kind of Magnesium Ascorbylphosphate of the present invention, be raw material with the nanometer Margarita powder, utilize external energy auxiliary water formulation to extract, obtain through separation and purification, for solid content is 0.2～1% the aqueous solution or is flaxen powder, Magnesium Ascorbylphosphate, its molecular weight≤3000 dalton.
The extracting method of the pearl natural radioactivity peptide of this kind Magnesium Ascorbylphosphate may further comprise the steps successively:
A. raw material is prepared: prepare particle diameter less than the nanometer Margarita powder of 100nm; The original active substance of pearl is fully exposed, be not destroyed simultaneously.Use pure physical method, under the situation of not destroying the pearl natural component, make the effective active composition of pearl come out fully, the release rate of effective active composition and burst size also can significantly improve.
B. extract: be the mixed of 1:5～4:5 by weight with nanometer Margarita powder with deionized water, put into ultrasonic water bath and shake 2～8h that bath temperature is 60～100 ℃; To realize the abundant extraction of pearl water-soluble active ingredient.
C. separate: mixed solution is put into the centrifugal 15～30min of whizzer, and centrifugal speed is 2500～3500r/min, collects supernatant liquor, gets filtrate through the filter membrane suction filtration, is pearl water extract;
D. ultrafiltration: pearl water extract takes off metafiltration liquid after the ultra-filtration centrifuge tube ultrafiltration, reconcentration gets concentrated solution;
E. electrodialysis: concentrated solution is removed the impurity mineral ion through electroosmose process, obtain the organic active liquid of small molecules;
F. column chromatography: the organic active liquid of small molecules is carried out the dextrane gel column chromatography, collect the component that there is absorption peak at the 280nm place, after lyophilize, obtain pearl natural radioactivity peptide.Press the peak and collect component under the 280nm, obtain 2 main elution peaks, collect second elution peak in a large number.As shown in Figure 1, be the Sephadex G25 gel-filtration curve of pearl active peptide sample.
Nanometer Margarita powder in the described steps A is the natural nano pearl powder that pearl is prepared from through pure physical method.
Nanometer Margarita powder among the described step B and deionized water are put into ultrasonic water bath and are shaken 4h by weight being 2:5, and bath temperature is 80 ℃.
Mixed solution among the described step C is put into the centrifugal 20min of whizzer, and centrifugal speed is 3000r/min, and the employed filter membrane of suction filtration is 0.45 μ m filter membrane.
Ultra-filtration centrifuge tube among the described step D is the 3kD ultra-filtration centrifuge tube.
Impurity mineral ion in the described step e comprises Ca
2+, Mg
2+, K
+, Na
+, CO
3 2+, OH
-With SO
4 2-
Dextrane gel in the described step F is the SephadexG-25 dextrane gel.
Further comprising the steps of:
G. detect: with the pearl natural radioactivity peptide that obtains with deionized water dissolving after, with high performance liquid chromatography (High Performance Liquid Chromatography, abbreviation HPLC) relative molecular mass of polypeptide product distributes before and after the detection purifying, testing conditions is: chromatographic column is TSKgel gel column G2000SWXL300mm * 7.8mm, mobile phase volume is than being Yi Jing ︰ Shui ︰ trifluoroacetic acid=30 ︰, 70 ︰ 0.1, the detection wavelength is UV280nm, and flow velocity is 1ml/min, and column temperature is 30 ℃; Be that 67000 bovine serum albumin, molecular weight are that 1335 vitamin B12 and molecular weight are that 614 Sleep-promoting factor B is made into and mixes mark with molecular weight, every kind of substances content is 5mg/ml, as standard model.
As shown in Figure 2, obtain as the data in the table 1, be the molecular weight distribution of pearl active peptide behind the purifying:
Table 1
| Sequence number | Retention time (min) | Area (%) | Molecular weight (Da) |
| 1 | 9.147 | 15.53 | 2101.49 |
| 2 | 9.816 | 36.59 | 1110.438 |
| 3 | 10.248 | 13.39 | 735.5348 |
| 4 | 11.122 | 25.96 | 319.6537 |
| 5 | 12.018 | 8.53 | 136.0336 |
By among Fig. 2 and the table 1 as can be known, recording the accurate scope of its molecular weight is 100～2100 dalton.This composition carries out the effect checking through external tyrosine oxidase inhibition test, mouse black-in tumor cell test and all shows the good activity of whitening, and its effect all is better than arbutin.
The application of the pearl natural radioactivity peptide of this kind Magnesium Ascorbylphosphate in preparation skin-lightening cosmetic or medical science whitening and speckle dispelling product.
Natural peral bioactive peptide of the present invention is through the good whitening effect of effect checking tool.Adopt tyrosine oxidase inhibition test, mouse black-in tumor cell experiment that the melanocyte growing amount is measured below.
Tyrosinase inhibition rate is measured:
According to table 2 application of sample, 1% arbutin is compared as one group of sample.
Table 2
| ? | Blank (ml) | Blank (ml) | Sample contrast (ml) | Sample (ml) |
| Sample | — | — | 1.0 | 1.0 |
| PBS | 2.5 | 2.0 | 1.5 | 1.0 |
| L-tyrosine | 1.0 | 1.0 | 1.0 | 1.0 |
| Tyrosine oxidase | — | 0.5 | — | 0.5 |
| Cumulative volume | 3.5 | 3.5 | 3.5 | 3.5 |
Wherein, PBS is PBS damping fluid, i.e. phosphoric acid buffer.
Earlier by after having added sample, PBS, L-tyrosine in the table 2, do not add tyrosine oxidase, solution is placed 37 ℃ of water-bath constant temperature 10min, make mixed solution reach 37 ℃ of the temperature of enzymatic reaction; In needs add the group of tyrosine oxidase, add tyrosine oxidase then, again solution is placed 37 ℃ of water-bath constant temperature 10min after, make enzymatic reaction fully reaction under optimum temperuture.Under 475nm, measure its absorbance, and with massfraction be that 1% arbutin compares.The tyrosinase activity inhibiting rate=(T1-C1)/T1*100%; C1: the absorbancy of expression sample, T1: the absorbancy that expression is blank.
As shown in Figure 3, obtain as the data in the table 3, be the inhibiting rate of different concns pearl active peptide to tyrosine oxidase:
Table 3
| Sample concentration (μ g/ml) | C1（A475nm） | T1（A475nm） | Tyrosinase activity inhibiting rate (%) |
| 25 | 0.275 | 0.419 | 34.37 |
| 50 | 0.097 | 0.419 | 76.84 |
| 100 | 0.012 | 0.419 | 97.13 |
| 150 | 0.006 | 0.419 | 98.56 |
| 200 | 0.007 | 0.419 | 98.32 |
| The 1wt% arbutin | 0.010 | 0.419 | 97.37 |
The mouse black-in tumor cell test:
1. melanoma cells proliferation rate detection method: the melanoma cells that will be cultured to for 3 generations is inoculated in 96 orifice plates, every hole 1 * 10
4Individual cell is at 37 ℃ and to contain massfraction be 5%C0
2Under hatch 24h, abandon supernatant liquor, add pearl water extract.If 6 concentration (10 μ g/mL, 50 μ g/mL, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL, 250 μ g/mL), each concentration is established 3 multiple holes, and set up control group directly to add substratum 200 μ L (contain cell but do not contain medicine), blank group does not only add cell for substratum, and arbutin is as medicine group positive control.After continuing to cultivate 72h, abandon supernatant liquor, the MTT solution 20 μ L that every hole adds 5 μ g/mL put into 37 ℃, 5%C0
2Incubator 4h abandons supernatant, and every hole adds DMSO150 μ L, and wherein, DMSO is dimethyl sulfoxide (DMSO) (Dimethyl sulfoxide is called for short DMSO), behind the room temperature vibration 10min, puts and measures each hole absorbance under the microplate reader 490nm wavelength.
Cell appreciation rate=[(the average light absorption value of each concentration of the testing sample-average light absorption value of blank group)/(the average light absorption value of the control group-average light absorption value of blank group)] * 100%.
2. melanoma cells melanocyte output detection method: melanoma cells is inoculated in 6 orifice plates.Every hole 3ml, putting 37 ℃, to contain massfraction be 5%CO
2After hatching 24h in the incubator, abandon supernatant liquor, every hole adds and contains the substratum that concentration is 20,50,80 μ g/ml pearl peptides respectively, and control group only is substratum, and arbutin is as positive control, and wherein, substratum is the RPMI-1640 cell culture medium.Cultivating behind the 48h with massfraction is 0.25% trysinization collecting cell, washes 2 times with PBS, counts under the mirror.Add the 0.2ml distilled water respectively and made cell suspension 1 minute, add then under the mixture room temperature of 500 μ l ethanol and 500 μ l ether and placed 15 minutes, 3000r/min is centrifugal, and 5min removes supernatant liquor, add 1ml1mol/L and contain the NaOH solution that massfraction is 10%DMSO, wherein, DMSO is dimethyl sulfoxide (DMSO) (Dimethyl sulfoxide, be called for short DMSO), at 80 ℃ of water bath heat preservation 2h, add the 4ml distilled water NaOH solution dilution is become 0.2mol/L, after under spectrophotometer, measuring the light absorption value of 475nm wavelength, survey absorbance in the 475nm microplate reader.
Melanin content=(mean value of medicine group absorbancy/cell count)/(mean value of control group absorbancy/cell count) * 100%.Concrete outcome is please referring to accompanying drawing 4.
The result of comprehensive tyrosine oxidase inhibition test, mouse black-in tumor cell test shows, the pearl natural radioactivity peptide concentration of a kind of Magnesium Ascorbylphosphate of the present invention is that concentration is that the pearl natural radioactivity peptide of 100 μ g/ml reaches 97.13% to tyrosinase inhibitory activity, massfraction is that 1% arbutin is 97.37% to tyrosinase inhibitory activity, therefore, concentration is that the pearl natural radioactivity peptide of 100 μ g/ml and massfraction are that the whitening effect of 1% arbutin is suitable.Concentration is that the arbutin of 50 μ g/ml has the obvious suppression effect, hypopigmentation about 47.2% to the chromogenesis of B16 cell.When being respectively 20,50,80 μ g/ml, pearl natural radioactivity peptide concentration to the equal showed cell toxicity not of B16 cell, and it melanicly is formed with obvious restraining effect.When pearl natural radioactivity peptide concentration was 80 μ g/ml, the melanocyte of B16 cell produced and has reduced 53.6%, and effect is better than the arbutin that concentration is 50 μ g/ml.
The above only is specific embodiments of the invention, but technical characterictic of the present invention is not limited thereto, and any those skilled in the art is in the field of the invention, and the variation of doing or modify all is encompassed among the claim of the present invention.
Claims (10)
1. the pearl natural radioactivity peptide of a Magnesium Ascorbylphosphate, it is characterized in that: be raw material with the nanometer Margarita powder, utilize external energy auxiliary water formulation to extract, obtain through separation and purification, for solid content is 0.2～1% the aqueous solution or is flaxen powder, Magnesium Ascorbylphosphate, its molecular weight≤3000 dalton.
2. the extracting method of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 1 is characterized in that: may further comprise the steps successively,
A. raw material is prepared: prepare particle diameter less than the nanometer Margarita powder of 100nm;
B. extract: be the mixed of 1:5～4:5 by weight with nanometer Margarita powder with deionized water, put into ultrasonic water bath and shake 2～8h that bath temperature is 60～100 ℃;
C. separate: mixed solution is put into the centrifugal 15～30min of whizzer, and centrifugal speed is 2500～3500r/min, collects supernatant liquor, gets filtrate through the filter membrane suction filtration, is pearl water extract;
D. ultrafiltration: pearl water extract takes off metafiltration liquid after the ultra-filtration centrifuge tube ultrafiltration, reconcentration gets concentrated solution;
E. electrodialysis: concentrated solution is removed the impurity mineral ion through electroosmose process, obtain the organic active liquid of small molecules;
F. column chromatography: the organic active liquid of small molecules is carried out the dextrane gel column chromatography, collect the component that there is absorption peak at the 280nm place, after lyophilize, obtain pearl natural radioactivity peptide.
3. the extracting method of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 2 is characterized in that: the nanometer Margarita powder in the described steps A is the natural nano pearl powder that pearl is prepared from through pure physical method.
4. the extracting method of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 2 is characterized in that: the nanometer Margarita powder among the described step B and deionized water are by weight being 2:5, and putting into ultrasonic water bath shakes 4h, and bath temperature is 80 ℃.
5. the extracting method of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 2, it is characterized in that: the mixed solution among the described step C is put into the centrifugal 20min of whizzer, centrifugal speed is 3000r/min, and the employed filter membrane of suction filtration is 0.45 μ m filter membrane.
6. the extracting method of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 2, it is characterized in that: the ultra-filtration centrifuge tube among the described step D is the 3kD ultra-filtration centrifuge tube.
7. the extracting method of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 2, its spy
Levy and be: the impurity mineral ion in the described step e comprises Ca
2+, Mg
2+, K
+, Na
+, CO
3 2+, OH
-With SO
4 2-
8. the extracting method of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 2, it is characterized in that: the dextrane gel in the described step F is Sephadex G-25 dextrane gel.
9. the extracting method of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 2 is characterized in that: further comprising the steps of,
G. detect: with the pearl natural radioactivity peptide that obtains with deionized water dissolving after, the relative molecular mass that detects purifying front and back polypeptide product with high performance liquid chromatography distributes, testing conditions is: chromatographic column is TSKgel gel column G2000SWXL300mm * 7.8mm, mobile phase volume is than being Yi Jing ︰ Shui ︰ trifluoroacetic acid=30 ︰, 70 ︰ 0.1, the detection wavelength is UV280nm, flow velocity is 1ml/min, and column temperature is 30 ℃; Be that 67000 bovine serum albumin, molecular weight are that 1335 vitamin B12 and molecular weight are that 614 Sleep-promoting factor B is made into and mixes mark with molecular weight, every kind of substances content is 5mg/ml, as standard model; The molecular weight that records pearl natural radioactivity peptide is 100～2100 dalton.
10. the application of the pearl natural radioactivity peptide of a kind of Magnesium Ascorbylphosphate as claimed in claim 1 in preparation skin-lightening cosmetic or medical science whitening and speckle dispelling product.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN107440969A (en) * | 2017-09-05 | 2017-12-08 | 广东科玮生物技术股份有限公司 | A kind of sun-proof renovation agent and its application |
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Address after: 311804 Zhejiang city of Shaoxing province Zhuji City Lake Phoenix Technology Park Zhejiang changshengniao pearl biotechnology Co Ltd Patentee after: Zhejiang changshengniao health Polytron Technologies Inc Address before: 311804 Zhejiang city of Shaoxing province Zhuji City Lake Phoenix Technology Park Zhejiang changshengniao pearl biotechnology Co Ltd Patentee before: Zhejiang Changshengniao Pearl Biotechnology Co., Ltd. |