CN111012689A

CN111012689A - Preparation method of astragalus polypeptide and application of astragalus polypeptide in preparation of cosmetics

Info

Publication number: CN111012689A
Application number: CN201911316152.1A
Authority: CN
Inventors: 戴跃锋; 何广文; 颜少慰; 钱景茹
Original assignee: Hunan Yujia Cosmetics Manufacturing Co ltd
Current assignee: Hunan Yujia Cosmetics Manufacturing Co ltd
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2020-04-17
Anticipated expiration: 2039-12-19
Also published as: CN111012689B

Abstract

The invention relates to the technical field of polypeptide extraction, in particular to a preparation method of astragalus polypeptide and application of the astragalus polypeptide in preparation of cosmetics. The method provided by the invention comprises the steps of crushing astragalus, treating by supercritical carbon dioxide fluid, cracking by using cracking liquid, and filtering to obtain filtrate; and sequentially salting out, cleaning, drying, grinding, carrying out enzymolysis, decoloring and ultrafiltering on the filtrate to obtain the astragalus polypeptide. The method reasonably controls each parameter in the extraction step, thereby keeping the results of multiple extractions consistent. By controlling the molecular weight, the astragalus polypeptide with good physiological function and permeation effect is obtained. Experiments show that the astragalus polypeptide provided by the invention can inhibit the expression of MMPs and promote the expression of Tolloid-like protein 1precursor, and has good permeation effect.

Description

Preparation method of astragalus polypeptide and application of astragalus polypeptide in preparation of cosmetics

Technical Field

The invention relates to the technical field of polypeptide extraction, in particular to a preparation method of astragalus polypeptide and application of the astragalus polypeptide in preparation of cosmetics.

Background

The polypeptide is a protein fragment, has a structure between amino acid and protein, is widely present in animals and plants, and participates in regulating various physiological activities of organisms. Many active substances of the human body exist in the form of peptides. Peptides are involved in the fields of hormones, nerves, cell growth and reproduction of the human body, and are important in regulating physiological functions of various systems and cells in the body, activating enzyme systems in the body, promoting permeability of intermediate metabolic membranes, or finally producing specific physiological effects by controlling DNA transcription or affecting specific protein synthesis. Peptides are important substances involved in various cellular functions in the human body. Peptides can synthesize cells and modulate the functional activities of cells. Peptides act as neurotransmitters in the human body, transmitting information. The peptide can be used as a transportation tool in human body, and can transport various nutrient substances eaten by human body, various vitamins, biotin, calcium and trace elements beneficial to human body to various cells, organs and tissues of human body. The peptide is an important physiological regulator of human body, can comprehensively regulate the physiological function of human body, enhance and exert the physiological activity of human body, and has important biological function. In recent years, medical researches on biological polypeptides show that the polypeptides have the characteristics of high activity and easy absorption, and based on the special physiological activity of small-molecule peptides, the peptides are more and more commonly applied to cosmetics in recent years, and various research results show that the polypeptides can essentially improve a series of problems of skin.

Most of the mainstream polypeptides in the market are amino acid synthesis sources, and although the synthesis cost of the polypeptides is continuously reduced and the synthesis efficiency is improved along with the continuous development of synthesis technology, the current situation that the existing sources of the polypeptides are chemical synthesis and the price of the synthesized polypeptides is still high cannot be changed. The chemical synthesis means does not meet the trend of the current consumers for natural cosmetics, and the price is relatively expensive, so that the common consumers cannot enjoy the effective solution of the skin problem caused by the high-concentration polypeptide. The polypeptide with higher safety and lower cost compared with the synthesized polypeptide can be obtained by hydrolyzing the protein with natural sources, but the problems that the conventional protein hydrolysis polypeptide product cannot reasonably control the protein source, does not control the hydrolysis process of hydrolase, does not select polypeptide fragments with more prominent effects and the like cause that the quality and the effect of the polypeptide obtained by hydrolysis cannot be ensured, and whether the polypeptide can be absorbed by skin cannot be ensured.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing astragalus polypeptide, wherein the astragalus polypeptide prepared by the method has good physiological activity and good transdermal absorption effect, and the present invention also provides an application of the polypeptide in preparing cosmetics.

The preparation method of the astragalus polypeptide comprises the following steps:

pulverizing radix astragali, treating with supercritical carbon dioxide fluid, cracking with cracking solution, and filtering to obtain filtrate;

salting out, cleaning, drying, grinding, performing enzymolysis, decoloring and ultrafiltering the filtrate in sequence to obtain astragalus polypeptide;

the cracking solution comprises trihydroxymethyl aminomethane, sodium dodecyl sulfate, glycerol, 2-mercaptoethanol and L-cysteine;

the enzymolysis adopts trypsin and rice trypsin.

In the invention, the supercritical carbon dioxide fluid treatment has the extraction temperature of 60-80 ℃ and the extraction pressure of 150-350 bar. For example, the extraction temperature is 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃. The extraction pressure is 150bar, 200bar, 250bar, 300bar, 350 bar. In some embodiments, the supercritical carbon dioxide fluid treatment is carried out at an extraction temperature of 70 ℃ and an extraction pressure of 350 bar.

The astragalus membranaceus is crushed to the particle size of 2-4 mm and then treated by supercritical carbon dioxide fluid.

The invention adopts supercritical carbon dioxide fluid to extract the astragalus powder, and removes grease in the astragalus powder; in addition, under the condition that a small part of water exists, part of sugar is removed, so that the influence of the sugar on protein extraction is reduced; meanwhile, the supercritical carbon dioxide fluid has a destructive effect on the cell structure of the astragalus membranaceus tissue, and the cell structure is destroyed, so that the extraction of protein is facilitated.

After the supercritical carbon dioxide fluid is treated, the cracking solution is ground before cracking until the particle size is 10 +/-5 microns.

The grinding is carried out by adopting a grinder for 10-20 min. In a specific embodiment, the milling time is 15 min.

In the invention, the lysis solution consists of the following components in volume fraction:

the lysis solution is more beneficial to the dissolution of the protein from the astragalus tissue, and can also protect the stability of the amino acid sequence of the protein so that the protein is not degraded in the lysis process. Wherein the volume fraction of the tris is 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%. The volume fraction of the sodium dodecyl sulfate is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. The volume fraction of glycerol is 5%, 10%, 15% or 20%. The volume fraction of 2-mercaptoethanol is 0.01%, 0.03%, 0.05%, 0.07% or 0.1%. The volume fraction of L-cysteine is 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

In some embodiments, the lysate consists of the following volume fractions of components:

in the present invention, in the cleavage step: the mass ratio of the astragalus to the lysis solution is 1: 1-5; in some embodiments, the mass ratio of the astragalus to the lysate is 1:1, 1:2, 1:3, 1:4, or 1: 5. The stirring speed is 500-700 rpm, the pH value is 8.0-9.0, in some embodiments, the stirring speed is 600rpm, the pH value is 8.4, and the pyrolysis is performed for 1-5 hours at normal temperature. In some embodiments, the time for lysis is 1h, 2h, 3h, 4h, or 5 h.

In some embodiments, in the lysing step: the mass ratio of the astragalus to the lysate is 1: 4; the stirring speed was 600rpm, the pH was 8.4, and the cleavage was carried out at room temperature for 2 hours. The normal temperature is 18-30 ℃.

After the cracking, the filtration is carried out, and the aperture of the filtration is 0.8 μm and 0.45 μm in sequence.

And after the filtration, removing residues and reserving filtrate. In the invention, ammonium sulfate is adopted for salting out; the mass ratio of the ammonium sulfate to the filtrate is 1 (0.1-1.0); the salting-out condition is that the mixture is stirred to dissolve and then stands for 6 hours at room temperature. In some embodiments, the mass ratio of ammonium sulfate to filtrate is 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, or 1: 1.0. In some embodiments, the mass ratio of ammonium sulfate to filtrate is 1: 0.528. The room temperature is 18-30 ℃.

The salting out is followed by filtration. The filtration is performed by a vacuum filtration device. Wherein the pore size of the filter disc is 0.45 μm. Filtering, removing the filtrate, and collecting protein precipitate.

In the invention, deionized water is adopted for cleaning. The temperature of the deionized water is 0 to-4 ℃, and in a specific embodiment, the temperature of the deionized water is-4 ℃. The volume ratio of the washed deionized water to the salting solution was 2: 1.

In the invention, the drying temperature is 35-45 ℃, and in the specific embodiment, the drying temperature is 40 ℃.

After drying, a grinding machine is adopted for grinding for 30 minutes until the particle size is 0.2 mu m +/-0.05 mu m.

In the enzymolysis step, the ground extract powder is dissolved by ammonium bicarbonate solution, and enzymolysis is carried out by trypsin and chymotrypsin at 37 ℃ and 600rpm for 12-24 hours by stirring and enzymolysis; wherein the mass ratio of the trypsin to the extract powder is (0.01-1.0): 1, and the mass ratio of the chymotrypsin to the extract powder is (0.01-1.0): 1. In some embodiments, the mass ratio of trypsin to extract powder is 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, or 1.0: 1. The mass ratio of the chymotrypsin to the extract powder is 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1 or 1.0: 1. In some embodiments, the mass ratio of trypsin to chymotrypsin is (1-10): (1:10). For example, the mass ratio of trypsin to chymotrypsin may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. The enzymolysis time is 12, 14, 16, 18, 20, 22 and 24 hours.

In some specific embodiments, the enzymolysis condition is 37 ℃, and the enzymolysis is carried out for 18 hours under stirring at 600 rpm; wherein the mass ratio of the trypsin to the extract powder is 0.08:1, and the mass ratio of the chymotrypsin to the extract powder is 0.08: 1.

In the invention, parameters including temperature, pH value, enzyme types, dosage and the like are optimized, so that the consistency of each hydrolysate is ensured.

The concentration of the ammonium bicarbonate solution is 50 mmol/L.

In the invention, the intercepted molecular weight of the ultrafiltration is less than 1000Da, 1000-3500 Da and 3500 Da-5000 Da respectively.

Preferably, the ultrafiltration has a molecular weight cut-off of < 1000 Da.

The astragalus polypeptide prepared by the method is provided.

The astragalus polypeptide prepared by the method is applied to preparing cosmetics.

The invention also provides a cosmetic which comprises the astragalus polypeptide prepared by the method and a cosmetic matrix.

A method of skin care which is administering the cosmetic provided by the present invention.

The cosmetic provided by the invention can improve the collagen production capacity of epidermal cells, so that the cosmetic has the effects of whitening, resisting wrinkles, recovering skin elasticity and/or resisting aging.

The method provided by the invention comprises the steps of crushing astragalus, treating by supercritical carbon dioxide fluid, cracking by using cracking liquid, and filtering to obtain filtrate; and sequentially salting out, cleaning, drying, grinding, carrying out enzymolysis, decoloring and ultrafiltering on the filtrate to obtain the astragalus polypeptide. The method reasonably controls each parameter in the extraction step, thereby keeping the results of multiple extractions consistent. By controlling the molecular weight, the astragalus polypeptide with good physiological function and permeation effect is obtained. Experiments show that the astragalus polypeptide provided by the invention can inhibit the expression of MMPs and promote the expression of Tolloid-like protein 1precursor, and has good permeation effect.

Drawings

FIG. 1 shows mass spectra of polypeptides after hydrolysis according to examples 4-6;

FIG. 2 shows the polypeptide distribution after hydrolysis in examples 4-6;

FIG. 3 shows the results of electrophoresis of the polypeptide-treated fibroblast protein obtained in example 7, wherein 3-a shows electrophoresis of blank composition fibroblast protein without polypeptide treatment; 3-b shows the electrophoresis of fibroblast proteins after polypeptide treatment with a molecular weight of less than 1000 Da; 3-c shows the electrophoresis of the fibroblast protein after the polypeptide with the molecular weight of 1000-3500 Da is processed; 3-d shows the electrophoresis of the fibroblast protein after the polypeptide treatment with the molecular weight of 3500-5000 Da; 3-e shows the electrophoresis of fibroblast proteins after treatment with a polypeptide having a molecular weight greater than 5000 Da;

FIG. 4 shows the effect of polypeptides of different molecular weights on the expression level of Tolloid-like protein 1 precursor;

FIG. 5 shows the effect of different molecular weight polypeptides on the expression level of MMPs-1;

FIG. 6 shows an apparatus for testing transdermal absorption efficiency;

fig. 7 shows the permeation effect after 2 h.

Detailed Description

The invention provides a preparation method of astragalus polypeptide and application thereof in preparing cosmetics, and a person skilled in the art can refer to the content and appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The instruments and reagents adopted by the invention are all common commercial products and can be purchased in the market. The radix astragali is from inner Mongolia. The invention is further illustrated by the following examples:

example 1

1. Supercritical carbon dioxide fluid treatment of astragalus membranaceus powder

The experimental conditions were: the size of the astragalus powder is 2-4 mm, the extraction temperature is 70 ℃, and the extraction pressure is 350 bar.

2. The astragalus powder treated by the supercritical carbon dioxide fluid is broken as a result of the structure, the whole astragalus powder becomes relatively fluffy, and then the astragalus powder is ground by a grinding machine, wherein the grinding time is preferably 15 minutes, and the particle size of the astragalus powder obtained after grinding is about 10 microns.

3. And (3) carrying out protein extraction on the astragalus powder treated by the supercritical carbon dioxide fluid by adopting an optimized cracking solution:

the specific composition of the lysate is as follows:

No.	name of raw materials	Volume fraction (%)
			1	Tris (hydroxymethyl) aminomethane	0.78
2	Sodium dodecyl sulfate	0.5
			3	Glycerol	10
4	2-mercaptoethanol	0.05
			5	L-cysteine	0.1
6	Water (W)	88.57

-an extraction process: astragalus powder: lysate (w/w) ═ 1: 4; the pH of the lysate was 8.4; stirring speed is 600rpm, temperature is normal temperature, and extraction time is 2 hours

4. After extraction is finished, stirring is continued, a valve connected with a delivery pump at the bottom of the protein extraction tank is opened, the mixture of the astragalus powder and the lysate is delivered to a plate and frame filter for filtration, the aperture of a filter sheet of the plate and frame filter is preferably the combination of 0.8 and 0.45 micrometer, filtrate after filtration is delivered to a protein settling tank for protein settling, and the astragalus powder is retained in the plate and frame filter in the form of residues.

5. In the protein settling tank, the astragalus protein is obtained by salting out method. Ammonium sulfate is preferably selected as a raw material by a salting-out method, and the astragalus protein solution is optimized: ammonium sulfate 1:0.528 at normal temperature. Slowly adding ammonium sulfate to completely dissolve while stirring the radix astragali protein solution in the settling tank, stopping stirring after completely dissolving, and standing for 6 hours to completely separate out radix astragali protein.

6. Standing for 6 hours, opening a valve at the bottom of a protein settling tank, transferring the precipitated protein solution into a vacuum filtration device for filtration, retaining the astragalus protein on a filter sheet, and treating the astragalus protein with deionized water at the temperature of-4 ℃ according to the weight ratio of deionized water: protein washing was performed at a ratio of 2:1 for the protein solution, and the salt remaining in the protein was washed off.

7. The obtained protein is placed in an oven at 40 ℃ for drying, and then a grinding machine is adopted for grinding, the grinding time is preferably 30 minutes, and the particle size of the astragalus protein powder obtained after grinding is about 0.2 micron.

8. Repeating the steps 2-7, and extracting for 3 times.

By adopting the extraction of the embodiment, the weight of the total astragalus protein finally obtained is 2.96g, and the mass fraction of the obtained astragalus protein in the astragalus medicinal material is 14.8%.

Example 2

1. And (3) extracting protein from the astragalus powder with the size of 2-4 mm:

the specific composition of the lysate is as follows:

2. After extraction is finished, stirring is continued, a valve connected with a delivery pump at the bottom of the protein extraction tank is opened, the mixture of the astragalus powder and the lysate is delivered to a plate and frame filter for filtration, the aperture of a filter sheet of the plate and frame filter is preferably the combination of 0.8 and 0.45 micrometer, filtrate after filtration is delivered to a protein settling tank for protein settling, and the astragalus powder is retained in the plate and frame filter in the form of residues.

3. In the protein settling tank, the astragalus protein is obtained by salting out method. Ammonium sulfate is preferably selected as a raw material by a salting-out method, and the astragalus protein solution is optimized: ammonium sulfate 1:0.528 at normal temperature. Slowly adding ammonium sulfate to completely dissolve while stirring the radix astragali protein solution in the settling tank, stopping stirring after completely dissolving, and standing for 6 hours to completely separate out radix astragali protein.

4. Standing for 6 hours, opening a valve at the bottom of a protein settling tank, transferring the precipitated protein solution into a vacuum filtration device for filtration, retaining the astragalus protein on a filter sheet, and treating the astragalus protein with deionized water at the temperature of-4 ℃ according to the weight ratio of deionized water: protein washing was performed at a ratio of 2:1 for the protein solution, and the salt remaining in the protein was washed off.

5. The obtained protein is placed in an oven at 40 ℃ for drying, and then a grinding machine is adopted for grinding, the grinding time is preferably 30 minutes, and the particle size of the astragalus protein powder obtained after grinding is about 0.2 micron.

By adopting the extraction of the embodiment, the weight of the obtained astragalus protein is 1.78g, and the extraction efficiency of the astragalus protein is 60.14%.

Example 3

the specific composition of the lysate is as follows:

The steps are not repeated, the extraction is only carried out once, the weight of the total astragalus protein finally obtained is 2.56g, the mass fraction of the obtained astragalus protein in the astragalus medicine is 12.8%, and the extraction efficiency of the protein is 86.49% (the extraction efficiency is the weight of the protein extracted once/the total protein content).

Example 4

Taking the astragalus protein powder prepared in the embodiment 3, stirring and dissolving the astragalus protein powder and 50mM ammonium bicarbonate according to the proportion of 1:100, and controlling the temperature at 37 ℃; after the astragalus protein powder is completely dissolved, trypsin is added to hydrolyze the astragalus protein. The hydrolysis conditions are preferably: the mass ratio of the astragalus protein to the trypsin is 1: 0.08; the temperature is 37 ℃; the reaction time is 18 hours; the stirring speed was 600 rpm. The progress of hydrolysis was monitored by MS analysis (FIGS. 1-2).

The results show that when trypsin is used alone, the distribution of astragalus peptides with the molecular weight of more than 500 is more, and particularly, the distribution of the astragalus peptides with the molecular weight of more than 1000 is obviously more than that of other two treatment modes.

Example 5

Taking the astragalus protein powder prepared in the embodiment 3, stirring and dissolving the astragalus protein powder and 50mM ammonium bicarbonate according to the proportion of 1:100, and controlling the temperature at 37 ℃; after the astragalus protein powder is completely dissolved, chymotrypsin is added to hydrolyze the astragalus protein. The hydrolysis conditions are preferably: the mass ratio of the astragalus protein to the chymotrypsin is 1: 0.08; the temperature is 37 ℃; the reaction time is 18 hours; the stirring speed was 600 rpm. The progress of hydrolysis was monitored by MS analysis (FIGS. 1-2).

The results show that part of astragalus polypeptides still remain above 500 molecular weight, especially above 1000 molecular weight, when treated with chymotrypsin alone.

Example 6

Taking the astragalus protein powder prepared in the embodiment 3, stirring and dissolving the astragalus protein powder and 50mM ammonium bicarbonate according to the proportion of 1:100, and controlling the temperature at 37 ℃; after the astragalus protein powder is completely dissolved, adding trypsin and chymotrypsin to hydrolyze the astragalus protein. The hydrolysis conditions are preferably: the mass ratio of the chymotrypsin to the astragalus protein is 1: 0.08; the temperature is 37 ℃; the reaction time is 18 hours; the stirring speed was 600 rpm. The progress of hydrolysis was monitored by MS analysis (FIGS. 1-2).

The results show that after the treatment of matching trypsin and chymotrypsin, the distribution of the astragalus polypeptides with the molecular weight of 300-500 is obviously improved, and the distribution of the astragalus polypeptides with the molecular weight of more than 1000 is obviously reduced. In conclusion, the astragalus protein can be effectively hydrolyzed by the matching treatment of trypsin and chymotrypsin to obtain the astragalus polypeptide with lower molecular weight.

Example 7

After the hydrolysis reaction described in example 6 was carried out for 18 hours, the temperature of the hydrolyzed solution was heated to 80 ℃ and the stirring speed was raised to 800rpm, while adding 0.5% activated carbon and stirring for 2 hours. The hydrolysis reaction is terminated by inactivating trypsin and chymotrypsin through a warmer temperature, and simultaneously, residual pigment in the protein is adsorbed by using activated carbon at a high temperature, so that the color of the final product is reduced.

After hydrolysis and decoloration are finished, a valve between a protein hydrolysis tank and a plate and frame filter is opened, a pressure pump is started, the mixed liquid of the hydrolyzed astragalus polypeptide solution and the active carbon is conveyed to the plate and frame filter for filtration, the filtration size of a filter sheet is preferably 0.45 micron, and the filtrate is conveyed to a protein hydrolysate tank for storage.

The hydrolysate tank is connected with the high-pressure pump and then connected with the ultrafiltration device. In the ultrafiltration device, firstly, a filtration membrane with MWCO of 5000 is adopted to carry out membrane separation on the astragalus protein hydrolysate, and the astragalus protein hydrolysate polypeptide with the molecular weight of below 5000 is obtained after separation.

Replacing a membrane separation element in the ultrafiltration device with a filtration membrane with MWCO of 3500, separating the obtained astragalus protein hydrolysis polypeptide with the molecular weight of below 5000, obtaining the astragalus protein hydrolysis polypeptide with the molecular weight of between 3500 and 3500-5000 after separation, and marking the astragalus protein hydrolysis polypeptide with the molecular weight of between 3500-5000 as peptide1

The membrane separation element in the ultrafiltration device was replaced with a filtration membrane having an MWCO of 1000, and the astragalus proteolysis polypeptide having a molecular weight of 3500 or less obtained in example-7 was separated to obtain an astragalus proteolysis polypeptide having a molecular weight of 1000 or less (denoted as peptide2) and a molecular weight of 1000 to 3500 (denoted as peptide 3).

Effect verification

In example 7, the solubility of the astragalus polypeptide solutions of different molecular weights obtained after passing through the ultrafiltration device was measured, and finally the astragalus polypeptide solution of 1000ppm concentration was prepared by adding 30% of 1, 3-butanediol, 2.5% of 1, 2-hexanediol and 67.5% of water and directly applied to the cosmetics.

And evaluating the efficacy of the astragalus polypeptide of each molecular fragment through a cell model, and preferably selecting the fragment with excellent anti-aging capability. The evaluation method adopts SDS-PAGE protein analysis, and evaluates the expression quantity of the protein related to the synthesis of the collagen in the fibroblasts to investigate the anti-aging capability of each astragalus protein hydrolyzed polypeptide.

-experimental materials:

astragalus proteolytic polypeptide (less than 1000Da, 1000-3500 Da, 3500-5000 Da, 5000Da or more), DMEM, FBS, DTT (dithioreitol), Iodoacetoamide, TEMED, isoproyl alcohol, Glycine, Glycerol, Sodium thiosulphate, silver nitrate, 37% formaldehide, Sodium carbonate, urea, Thiourea, CHAPS, SDS (Sodium do sulfite), APS (ammonium persulfate), Agarose, Acrylamide, 2D SDS-PAGE-stages, Top TM-TT 3CAR, fibroblast P9, and mobiline Dry strip pH 3-10

Experimental methods

Two-dimensional electrophoresis

The astragalus proteolytic polypeptides are solubilized using a cell culture medium (DMEM + 10% FBS + 1% AA) containing 100ppm (1mg/10 ml) of astragalus proteolytic polypeptides, and the culture medium containing astragalus proteolytic polypeptides is added to fibroblasts for culturing at 37 ℃ with 5% CO₂All cells were collected after 48 hours of culture. After the collected cells were washed with DPBS, lysis buffer (7M urea,2M thiourea, 4% CHAPS, 1% DTT, 2% carrier amphoyte, 40. mu.l/ml protease inhibitor cocktail) was added and lysed in a water bath at 30 ℃ for 5 hours. After 5 hours protein lysates were obtained by centrifugation at 13500rpm for 10 minutes. The supernatant obtained after centrifugation was quantified using the Bradford Assay Method.

Isoeletric focusing (IEF) Rehydation solution (7M urea,2M thiourea, 4% CHAPS, 1% DTT, 2% carrier ampholyte, 10% glycerol, 0.002% bromoketone blue) was placed 12 for rehydated. Then, 100. mu.g of the previously extracted fibroblast protein and 2% DNAse, 5% protien marker were introduced per immobiline DryStreps (pH 3-10line, 24-cm-long). Isoeletriccfousing was performed on the extracted fibroblast proteins using the EttanIPGphor 3(GE Healthcare, USA) system.

-soaking DryStrip with 1% DTT and an equilbration buffer (7M urea,2M thiourea, 2% SDS,50mM Tris-HCl, 30% glycerol, 0.002% bromophenol blue) before performing the Second dimension separation, allowing the disulfide bonds of the protein to open, followed by basification of the SH groups with 2.5% iodoacetamid.

The fibrillin was then isolated by transferring the Strip over a 12.5% polyacrylamide gels (25X 20cm) for second experimental electrophoresis (Step:10mA/gel 1h, Step2:40mV/gel5 h). After electrophoresis, the gel is dyed by a nitate silver staining method. The stained gel was scanned with a Umax Powerlook 2100XL Scanner and analyzed with an Image Master 2-D platinum program, the results of which are shown in FIG. 3.

1. Expression level of collagen synthesis-related enzyme Tolloid-like protein 1precursor

Test sample	Spot volume	Amount of increase (%)
			Blank sample	372.0313	-
Less than 1000Da	855.933	130.07
			1000～3500Da	754.678	102.85
3500～5000Da	543.566	46.11
			Above 5000Da	397.378	6.81

Tolloid-like protein 1precursor is one of important enzymes in protein synthesis, and experiments show that the astragalus membranaceus protein hydrolysis polypeptide prepared by the invention has a promotion effect on the growth of collagen, wherein the promotion effect of the astragalus membranaceus protein hydrolysis polypeptide with the length of less than 1000Da on the growth of collagen is the largest and reaches 130.07% (figure 4).

MMPs-1 (degradation of collagen I, II, III, IV) with reduced expression of matrix metalloproteinases

TABLE 2 reduction of MMPs expression

Test sample	Spot volume	Reduction amount (%)
			Blank sample	1652.68	-
Less than 1000Da	472.545	71.41
			1000～3500Da	770.757	53.36
3500～5000Da	927.367	43.89
			Above 5000Da	1095.73	33.70

MMPs-1 have degradation effect on collagen I, II, III and IV, and after the astragalus protein polypeptide prepared by the invention is treated, the expression level of MMps-1 in fibroblasts is reduced by a certain amount, wherein the effect of the astragalus protein hydrolytic polypeptide below 1000Da on inhibiting the expression of MMPs-1 is the best, and the expression level of MMPs-1 is reduced by 71.41% (figure 5).

Transdermal Performance testing

The polypeptides of different molecular weights prepared in example 7 were tested for transdermal absorption efficiency using Franz dispersion cell.

Experimental equipment and materials: franz diffusion cell device, stratum corneum skin, phosphoric acid buffer solution, acetic acid, deionized water, MS, polypeptide sample each 100ppm

Experimental method:

1. experimental setup (fig. 6) and test parameters:

receptor moiety: the Franz diffusion cell instrument was opened, the temperature of the circulating water was set at 32 ℃, the degassed phosphate buffer solution was filled into the diffusion cell and allowed to stand for 30 minutes to ensure that the dimension of the phosphate buffer solution reached 32 ℃.

Membrane section: soaking the stratum corneum skin stored at a low temperature of-20 ℃ in a phosphate buffer solution for 30 minutes, transferring the stratum corneum skin to the upper surface of a diffusion pool to cover a Receptor part of the diffusion pool, exhausting air below the skin, placing a cover part of a Donor above the stratum corneum skin, and locking and fixing the cover and the diffusion pool by using a lock catch;

donor part: 1ml of each of the hydrolyzate sample of example-6 (100ppm), the sample of example-9 (100ppm)

2. Sampling and analyzing: the time points for sampling analysis were 2,4,6,8, hours, 2ml per sample

3. Desalting the sample, and performing MS analysis under the following conditions: the method comprises the steps of adding 0.1% acetic acid into a 50% acetonitrile solution, adopting a scanning mode of Q1 MS (Q1), taking an electrode as a positive electrode, taking an ion source as turbo Spray, and scanning within the range of 300-1000 amu.

4. The permeability of the polypeptide having a molecular weight in the interval can be estimated by counting the total intensity of the ions in each interval of 300 to 1000amu with 50amu as the interval, and compared with the permeability of the hydrolysate and Peptide3 in example-6.

Results fig. 3 and fig. 7:

1) summary of the results

TABLE 3 Total Ionic Strength

From the data in the above table, it can be seen that the astragalus polypeptide having a molecular weight of 600 or more of example-6 hardly permeates after 2 hours of permeation, and the polypeptide molecules of the astragalus polypeptide Peptide3 permeate into the Receptor through the stratum corneum skin in each range of molecular weight of 300 to 1000. From the permeation results, it was also found that the permeation rate and permeation efficiency of Peptide3 were higher than those of the hydrolysate of example-6.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. The preparation method of the astragalus polypeptide is characterized by comprising the following steps:

the enzymolysis adopts trypsin and rice trypsin.

2. The method according to claim 1, wherein the supercritical carbon dioxide fluid treatment is carried out at an extraction temperature of 60 to 80 ℃ and an extraction pressure of 150 to 350 bar.

3. The method according to any one of claims 1 to 2, wherein the lysis solution consists of the following volume fractions:

4. a process according to any one of claims 1 to 3, wherein in the splitting step: the mass ratio of the astragalus to the lysis solution is 1: 1-5; stirring speed is 600rpm, pH value is 8.4, and normal temperature cracking is carried out for 1-5 hours.

5. The method according to any one of claims 1 to 4, wherein the salting out is performed using ammonium sulfate; the mass ratio of the ammonium sulfate to the filtrate is 1 (0.1-1.0); the salting-out condition is that the mixture is stirred to dissolve and then stands for 6 hours at room temperature.

6. The method according to any one of claims 1 to 5, wherein in the enzymolysis step, the ground extract powder is dissolved by ammonium bicarbonate solution, and enzymolysis is carried out by trypsin and chymotrypsin at 37 ℃ and 600rpm for 12 to 24 hours under stirring; wherein the mass ratio of the trypsin to the extract powder is (0.01-1.0): 1, and the mass ratio of the chymotrypsin to the extract powder is (0.01-1.0): 1.

7. The process according to any one of claims 1 to 6, wherein the ultrafiltration has a molecular weight cut-off of < 1000Da, 1000 to 3500Da, 3500Da to 5000Da, respectively.

8. An astragalus polypeptide prepared by the method of any one of claims 1 to 7.

9. Use of the astragalus polypeptide prepared by the method of any one of claims 1 to 7 in the preparation of cosmetics.

10. A cosmetic comprising the Astragalus polypeptide produced by the method of any one of claims 1 to 7 and a cosmetic base.