CN114404307A

CN114404307A - Arginine/lysine polypeptide microcapsule inclusion and preparation method and application thereof

Info

Publication number: CN114404307A
Application number: CN202111554126.XA
Authority: CN
Inventors: 孙云起; 郭朝万; 魏瑞敬; 聂艳峰; 胡露; 王娟
Original assignee: Guangdong Marubi Biological Technology Co Ltd
Current assignee: Guangdong Marubi Biological Technology Co Ltd
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2022-04-29
Anticipated expiration: 2041-12-17
Also published as: CN114404307B

Abstract

The invention discloses an arginine/lysine polypeptide microcapsule inclusion and a preparation method and application thereof, wherein the microcapsule inclusion comprises the following raw materials: compounding arginine/lysine polypeptide, glycerol, polyglyceryl nonionic surfactant and glucan, and the balance being water; wherein the compound arginine/lysine polypeptide comprises red scorpion toxin and conotoxin peptide. The microcapsule inclusion adopts polyglyceryl nonionic surfactant as carrier material, so that the compound polypeptide can be spontaneously wrapped in the microcapsule bilayer to form a high-stability microcapsule inclusion structure, and the compound polypeptide can play the effects of resisting aging, wrinkle and oxidation. The preparation method of the microcapsule inclusion avoids the addition of excipient and plasticizer required by the traditional liposome preparation, and the preparation method is simple. The microcapsule inclusion can be conveniently and stably dispersed in cosmetic matrixes such as skin care emulsions, creams, cleaning faces, essences, astringent and the like of different types, and can be prepared into cosmetics or skin care products for application.

Description

Arginine/lysine polypeptide microcapsule inclusion and preparation method and application thereof

Technical Field

The invention relates to the technical field of microcapsule inclusion, in particular to an arginine/lysine polypeptide microcapsule inclusion and a preparation method and application thereof.

Background

The microcapsule technology is a technology in which dispersed fine solid particles, liquid or gaseous substances are wrapped with a natural or synthetic polymer material to form fine particles having a semipermeable or sealed capsule membrane. The process of encapsulation is called microencapsulation, and the formed micro-particles are called microcapsules. Microcapsules have two basic components, the substance enclosed within the microcapsule being referred to as the core material, and the outer "shell" being referred to as the wall material. In the preparation process of the microcapsule, the wall material can isolate the core material from the external environment, and then the core material is released under proper conditions.

The traditional preparation technology of the microcapsule comprises a spray drying method, a supercritical fluid method, a solvent diffusion method and the like. However, the spray drying method has certain requirements on temperature for preparing the microcapsules, which can cause structural changes of some heat-sensitive medicines, so that the application range of the spray drying method is limited. Although the microcapsule prepared by the supercritical fluid method is environment-friendly and can avoid the use of organic solvents, the equipment is expensive, the parameter control and structure modification technology is not mature, and the industrial mass production is difficult to realize. Although the process for preparing the microcapsule by the solvent diffusion method is simple and easy to empty, the problem of organic solvent residue exists.

Disclosure of Invention

In order to overcome the defects of the prior art, one of the purposes of the invention is to provide an arginine/lysine polypeptide microcapsule inclusion body, which adopts a double-layer molecular carrier structure which is formed by polyglyceryl nonionic surfactant and is similar to the traditional liposome as an inclusion material to coat the compounded arginine/lysine polypeptide, so that the arginine/lysine polypeptide can be better absorbed by the skin, and the permeability is obviously improved; the second purpose of the invention is to provide an arginine/lysine polypeptide microcapsule inclusion, which avoids the addition of an excipient and a plasticizer required by the traditional liposome preparation, and the preparation method is simple; the invention also aims to provide an application of the arginine/lysine polypeptide microcapsule inclusion, and the arginine/lysine polypeptide microcapsule inclusion has good compatibility of a cosmetic formula and can not damage the system stability of cosmetics or skin care products when being used in the cosmetics or the skin care products.

One of the purposes of the invention is realized by adopting the following technical scheme:

an arginine/lysine polypeptide microcapsule inclusion comprises the following raw materials in parts by weight:

0.002-0.01% of compound arginine/lysine polypeptide, 10-30% of glycerol, 0.2-1.5% of polyglyceryl nonionic surfactant, 0.0001-0.1% of glucan and the balance of water; the compound arginine/lysine polypeptide comprises polypeptide A and polypeptide B, wherein the polypeptide A is red scorpion toxin, and the polypeptide B is conotoxin peptide.

Further, the polyglyceryl nonionic surfactant is polyglyceryl-10 diisostearate and/or polyglyceryl-10 laurate.

Still further, the polyglyceryl nonionic surfactant is a composition of 0.1 to 1% of polyglyceryl-10 diisostearate and 0.1 to 0.5% of polyglyceryl-10 laurate.

Further, the mass ratio of the polypeptide A to the polypeptide B is (3-9): 1.

further, the polypeptide A is purchased from Zhejiang surge peptide biology, Inc., and has the model of ZPC-019S; the polypeptide B is purchased from Zhejiang surge peptide biology GmbH and has the model of ZPC-018S.

The second purpose of the invention is realized by adopting the following technical scheme:

the preparation method of the arginine/lysine polypeptide microcapsule inclusion comprises the following steps:

1) fully stirring and dissolving the compound arginine/lysine polypeptide, polyglycerol-10 diisostearate, polyglycerol-10 laurate, glycerol and glucan to obtain a uniform mixed solution.

2) And (3) performing wrapping auxiliary treatment on the mixed solution to obtain a microcapsule inclusion aqueous suspension, and removing impurities to obtain the arginine/lysine polypeptide microcapsule inclusion.

Further, in the step 2), the wrapping auxiliary treatment is one of high-voltage pulse electric field assistance, ultrasonic wave assistance and free diffusion. Preferably high voltage pulsed electric field assisted.

Further, in step 2), the wrapping auxiliary treatment is high-voltage pulse electric field auxiliary treatment, and the technical parameters of the high-voltage pulse electric field are as follows: the field intensity is 0.5 kV/cm-2 kV/cm, the number of pulse treatments is 10-60, and the material conductivity is 2000 mu S/cm-6000 mu S/cm.

The third purpose of the invention is realized by adopting the following technical scheme:

in the application of the arginine/lysine polypeptide microcapsule inclusion, the arginine/lysine polypeptide microcapsule inclusion is dispersed in cosmetic matrixes such as different types of skin care emulsions, creams, face cleaning agents, essences, lotions and the like to prepare cosmetics or skin care products.

Compared with the prior art, the invention has the beneficial effects that:

(1) the microcapsule inclusion body adopts polyglyceryl nonionic surfactant as a carrier material, the surfactant has a hydrophilic end and a hydrophobic end, the hydrophilic end automatically points to an organic solvent outwards, and the hydrophobic end inwards gathers and solubilizes part of water to form the microcapsule body, so that the microcapsule body has the capability of solubilizing polypeptide and automatically forms a bilayer, compound arginine/lysine polypeptide can be spontaneously wrapped in the microcapsule bilayer, a high-stability microcapsule inclusion body structure is formed, and glucan is added to contribute to the stability of the inclusion body. The compound arginine/lysine polypeptide contained in the microcapsule inclusion can respectively act on the presynaptic terminal and the postsynaptic terminal in the nerve-muscle conduction pathway in the aspect of molecular mechanism. The polypeptide A (erythrosin) is a bionic peptide at the C end of SNAP-25 consisting of 26 peptides, competes with SNAP-25 protein for a binding site of SNARE compound species at the presynaptic end, so that the formation of SNARE compound is efficiently competitively inhibited, the release of acetylcholine (Ach) is reduced, and facial muscle relaxation is realized to achieve the wrinkle reduction effect. The polypeptide B is a mu-type conotoxin bionic peptide for biologically simulating conotoxin, mainly acts on the postsynaptic end of a nerve, naturally relaxes facial muscles by limiting the conduction of current of the nerve muscles, and thus achieves the effect of reducing wrinkles.

(2) The preparation method of the microcapsule inclusion comprises the following steps: the double-layer molecular carrier structure which is formed by the nonionic surfactant and is similar to the traditional liposome is adopted, the arginine/lysine polypeptide in the composition can be spontaneously wrapped in the microcapsule bilayer formed by the nonionic surfactant after being uniformly mixed, the addition of an excipient and a plasticizer required by the traditional liposome preparation is avoided, and the preparation method is simple.

(3) The auxiliary coating treatment adopts a high-voltage pulse electric field technology, effectively improves the coating efficiency of arginine/lysine polypeptide in the microcapsule coating body, and the vesicle coating body has a structure similar to a double-layer phospholipid structure of skin cells, so that the compounded arginine/lysine polypeptide can be better absorbed by the skin, and the permeability is obviously improved.

(4) The average particle size of the prepared arginine/lysine polypeptide microcapsule inclusion is 100-200 nm, the average encapsulation rate of the compounded arginine/lysine polypeptide is 10-40%, and the prepared arginine/lysine polypeptide microcapsule inclusion can be conveniently and stably dispersed in cosmetic matrixes such as skin care emulsions, creams, cleaning faces, essences, toning lotion and the like of different types to prepare cosmetics or skin care products.

Drawings

FIG. 1 is a graph of the effect of a single polypeptide and an arginine/lysine combination polypeptide on nematode locomotion;

FIG. 2 shows the effect of a single polypeptide and an arginine/lysine combination polypeptide on nematode motility following excitation;

FIG. 3 is a flow chart of the ultrafiltration centrifugation method.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.

0.002-0.01% of compound arginine/lysine polypeptide, 10-30% of glycerol, 0.1-1% of polyglycerol-10 diisostearate, 0.1-0.5% of polyglycerol-10 laurate, 0.0001-0.1% of glucan and the balance of water; wherein the compound arginine/lysine polypeptide is prepared from the following components in a mass ratio of (3-9): 1, wherein the polypeptide A is purchased from Zhejiang surge peptide biology, Inc., and has the model of ZPC-019S; the polypeptide B is purchased from Zhejiang surge peptide biology GmbH and has the model of ZPC-018S.

The polypeptide A (erythrosin) is a bionic peptide at the C end of SNAP-25 consisting of 26 peptides, competes with SNAP-25 protein for a binding site of SNARE compound species at the presynaptic end, so that the formation of SNARE compound is efficiently competitively inhibited, the release of acetylcholine (Ach) is reduced, facial muscle relaxation is realized to achieve the wrinkle reduction effect, and the wrinkle improvement effect is remarkable in a real-time and long-acting manner. The polypeptide B is a mu-type conotoxin bionic peptide (conotoxin peptide) for biologically simulating conotoxin, mainly acts on the postsynaptic end of a nerve, naturally relaxes facial muscles by limiting the conduction of current of the nerve muscles, and thus achieves the effect of reducing wrinkles.

Example 1

0.002% of compound arginine/lysine polypeptide, 10% of glycerol, 0.1% of polyglycerol-10 diisostearate, 0.1% of polyglycerol-10 laurate, 0.0001% of glucan and the balance of water; wherein the mass ratio of the compound arginine/lysine polypeptide is 3: 1, wherein the polypeptide A is red scorpion toxin purchased from Zhejiang surge volume peptide biology, and has the model of ZPC-019S; the polypeptide B is conotoxin peptide purchased from Zhejiang surge volume peptide biology, and has the model of ZPC-018S.

2) And (3) carrying out high-voltage pulse electric field auxiliary treatment on the mixed solution to obtain a microcapsule inclusion aqueous suspension, and removing impurities to obtain the arginine/lysine polypeptide microcapsule inclusion. The technical parameters of the high-voltage pulse electric field are as follows: the field intensity is 0.5kV/cm, the number of pulse treatments is 10, and the material conductivity is 2000 mu S/cm.

Example 2

0.005% of compound arginine/lysine polypeptide, 30% of glycerol, 1% of polyglycerol-10 diisostearate, 0.5% of polyglycerol-10 laurate, 0.1% of glucan and the balance of water; wherein the mass ratio of the compound arginine/lysine polypeptide is 9: 1, wherein the polypeptide A is red scorpion toxin purchased from Zhejiang surge volume peptide biology, and has the model of ZPC-019S; the polypeptide B is conotoxin peptide purchased from Zhejiang surge volume peptide biology, and has the model of ZPC-018S.

2) And (3) carrying out high-voltage pulse electric field auxiliary treatment on the mixed solution to obtain a microcapsule inclusion aqueous suspension, and removing impurities to obtain the arginine/lysine polypeptide microcapsule inclusion. The technical parameters of the high-voltage pulse electric field are as follows: the field intensity is 2kV/cm, the number of pulse treatments is 60, and the material conductivity is 6000 muS/cm.

Example 3

0.01 percent of compound arginine/lysine polypeptide, 20 percent of glycerol, 0.5 percent of polyglycerol-10 diisostearate, 0.2 percent of polyglycerol-10 laurate, 0.005 percent of glucan and the balance of water; wherein the mass ratio of the compound arginine/lysine polypeptide is 5: 1, wherein the polypeptide A is red scorpion toxin purchased from Zhejiang surge volume peptide biology, and has the model of ZPC-019S; the polypeptide B is conotoxin peptide purchased from Zhejiang surge volume peptide biology, and has the model of ZPC-018S.

2) And (3) carrying out high-voltage pulse electric field auxiliary treatment on the mixed solution to obtain a microcapsule inclusion aqueous suspension, and removing impurities to obtain the arginine/lysine polypeptide microcapsule inclusion. The technical parameters of the high-voltage pulse electric field are as follows: the field intensity is 1kV/cm, the number of pulse treatments is 40, and the material conductivity is 5000 mu S/cm.

Example 4

Example 4 differs from example 3 in that: in the step 2) of example 4, the mixed solution is subjected to ultrasonic-assisted treatment to obtain an aqueous suspension of microcapsule inclusion, and the arginine/lysine polypeptide microcapsule inclusion is obtained after impurity removal. Wherein the temperature of the ultrasonic auxiliary treatment is 40 ℃, the ultrasonic power is 500W, and the time is 10 min. The formulation and preparation of the remaining arginine/lysine polypeptide microcapsule encapsulates are the same as in example 3, step 1) of the preparation process.

Example 5

Example 5 differs from example 3 in that: in the step 2) of example 5, the mixed solution is subjected to free diffusion auxiliary treatment to obtain an aqueous suspension of the microcapsule inclusion, and the arginine/lysine polypeptide microcapsule inclusion is obtained after impurity removal. Wherein the temperature of the free diffusion auxiliary treatment is 40 ℃ and the time is 30 min. The formulation and preparation of the remaining arginine/lysine polypeptide microcapsule encapsulates are the same as in example 3, step 1) of the preparation process.

Comparative example 1

Comparative example 1 differs from example 3 in that: the arginine/lysine polypeptide microcapsule inclusion only contains the polypeptide A, does not contain the polypeptide B, and the rest components and the preparation method are the same as the example 3.

Comparative example 2

Comparative example 2 differs from example 3 in that: the arginine/lysine polypeptide microcapsule inclusion of comparative example 2 contained only polypeptide B and no polypeptide a, and the remaining components and preparation method were the same as those of example 3.

Comparative example 3

Comparative example 3 differs from example 3 in that: comparative example 3 contained only the compounded arginine/lysine polypeptide without microencapsulation of glycerol, polyglycerol-10 diisostearate, polyglycerol-10 laurate, and dextran.

Comparative example 4

Comparative example 4 differs from example 3 in that: the arginine/lysine polypeptide microcapsule encapsulate of comparative example 4 contained no dextran, and the remaining components and preparation method were the same as in example 3.

Comparative example 5

Comparative example 5 arginine/lysine polypeptide microcapsule inclusion is prepared by mixing arginine/lysine polypeptide and carrier material, adding appropriate amount of anhydrous ethanol to dissolve completely, placing in a rotary evaporator, and recovering ethanol under reduced pressure to form film. Measuring a surfactant solution, adding the surfactant solution into a round-bottom flask for film formation, and ultrasonically washing the film for a certain time to obtain the arginine/lysine polypeptide microcapsule solution. Wherein the carrier material is rapeseed lecithin with the purity of 95 percent, and the mass ratio of the carrier to the composite polypeptide is 2.5: 1. the surfactant solution is tween-40.

Evaluation and test of Effect

Effect of one, different kinds of peptides on nematode movement

1. Sample peptides tested: polypeptide a (erythrosin), polypeptide B (conotoxin peptide), snake-like venom peptide and the compounded arginine/lysine polypeptide of example 3.

2. The setting method of the blank control group comprises the following steps:

(1) coli OP50 was plated onto nematode NGM medium.

(2) And (3) picking the synchronized nematodes to the culture medium prepared in the step (1) for 3 days, and collecting the nematodes in a 2mL centrifuge tube to obtain 1mL nematode aqueous suspension of 3000 nematodes/mL.

(3) 1mL of neostigmine mesylate with a concentration of 0.5mg/mL was added to the aqueous nematode suspension of step (2).

(4) And (3) after neostigmine mesylate is treated for 4 hours, taking nematode liquid in a blank culture medium, and observing the behavior characteristics of the nematodes. And counting the head swing frequency of the nematodes within 30s, counting 10 nematodes in each plate, and counting 3 nematodes in each group.

3. Test set setting method:

(1) the samples tested were mixed with E.coli OP50 and plated onto nematode NGM medium at a final polypeptide concentration of 5%.

As can be seen from FIG. 1, the four peptides can block the nerve impulse capability between nerves and muscles, and achieve the muscle relaxation effect. The polypeptide A can inhibit the head swing frequency of the nematode to 24.06 percent; the polypeptide B can inhibit the head swing frequency of the nematode to 17.30 percent; the snake venom-like peptide can inhibit the head swing frequency of the nematode to 9.02%; the compounded arginine/lysine polypeptide can inhibit the head swing frequency of the nematode to 33.08 percent.

As can be seen from FIG. 2, the four peptides have the ability to resist nerve-to-muscle nerve impulses under the action of excitatory drugs. The polypeptide A can inhibit the head swing frequency of the nematode to 9.72 percent; the polypeptide B can inhibit the head swing frequency of the nematode to 5.80 percent; the snake venom like peptide can inhibit the head swing frequency of the nematode to 8.02%; the compounded arginine/lysine polypeptide can inhibit the head swing frequency of the nematode to 17.59 percent.

Second, the influence of the preparation methods of different microcapsule inclusion bodies on indexes

The microcapsules of examples 3 to 5 and comparative examples 4 to 5 were taken and tested for encapsulation efficiency, particle size, loss rate and stability, as shown in table 1.

The specific detection method is as follows:

the method for measuring the wrapping rate comprises the following steps: ultrafiltration and centrifugation. As shown in FIG. 3, the ultrafiltration centrifugation method is to put the liposome into an ultrafiltration tube equipped with an ultrafiltration membrane, and centrifuge at a proper rotation speed, so that the free drug can pass through the ultrafiltration membrane under the action of centrifugal force, and the liposome is entrapped, thereby realizing the separation of the two. The wrapping rate + loss rate was 100%.

The particle size measurement method comprises the following steps: microscopy. The basic working principle of the method is that a particle image amplified by a microscope is transmitted to a computer through a CCD camera and a graphic acquisition card, the computer carries out edge recognition and other processing on the images, the projection area of each particle is calculated, the particle size of each particle is obtained according to the equivalent projection area principle, and then the number of the particles in a set particle size interval is counted, so that the particle size distribution can be obtained.

And (3) stability determination: the pH stability and the thermal stability were examined, and the stability was evaluated by examining the encapsulation efficiency at a water bath temperature of 0 to 60 ℃ at a pH of (0 to 10), respectively.

TABLE 1 index data for microcapsules prepared by different preparation methods

Group of	Percent of wrapping%	Particle size/nm	Rate of loss/%)	Stability of
					Example 3	93.15±5.64	26.54±2.06	6.8±2.45	++++
Example 4	62.18±1.95	39.84±1.64	37.01±1.94	+++
					Example 5	29.16±4.62	67.95±6.94	70.99±2.3	+++
Comparative example 4	34.76±8.94	69.78±2.66	65.24±1.49	+
					Comparative example 5	46.81±5.91	50.16±1.24	54.16±0.97	+++

As can be seen from table 1, the encapsulation efficiency of example 3 is the highest, and the encapsulation efficiency and particle size data of the microcapsule inclusion of example 3 are superior to those of examples 4 and 5, which shows that the encapsulation efficiency of the compound arginine/lysine polypeptide in the microcapsule inclusion is more effectively improved by using the high-voltage pulsed electric field technology to assist in processing the inclusion compared with ultrasonic processing and free diffusion, so that the arginine/lysine polypeptide can be better absorbed by the skin, and the permeability is significantly improved. In comparative example 4, since glucan was not present, the inclusion rate and inclusion stability were reduced. Comparative example 5 the conventional liposome solvent diffusion method was used to prepare the microcapsule solution, but the encapsulation efficiency and encapsulation stability were lower than those of example 3, which shows that the spontaneous encapsulation ability of the formulated arginine/lysine polypeptide of the polyglyceryl nonionic surfactant is better than that of the conventional lecithin liposome, because the nonionic surfactant has an "amphiphilic" structure, i.e. the hydrophilic group and the hydrophobic (oleophilic) group are independently oriented to the water solvent, the hydrophobic group is inwardly gathered to solubilize a part of water to form the vesicle, and the microcapsule has the ability to solubilize the polypeptide, thereby forming the vesicle encapsulation form with high stability structure.

Third, cell proliferation efficacy test of microcapsule encapsidation

Sample preparation: example 3, comparative examples 1 to 3

Cell proliferation assay procedure: the cell suspension was diluted to the desired concentration of 4 x 104cfu/ml with culture medium, 100 μ l of the cell suspension was pipetted into the cell plate using a 30-300 μ l row gun, and an equal amount of PBS was added to the outermost periphery of the cell plate, and the plate was plated and labeled on the upper cover of the cell plate with the following labels: cell name, operator name, test name, date. Putting into a carbon dioxide incubator for incubation for 12 h. After the cells adhere to the wall, when the area of the cell plating plate is about 30-40%, sucking out the culture medium, adding 100 mu L of serum-free culture medium into each hole, marking the upper cover of the cell plate, and putting the cell plate into a carbon dioxide incubator for incubation for 16 h. After incubation for 16h, the plating area of the cells is about 60-65%, if the test sample is a solution, a filter is needed to filter, the test sample is diluted by a serum-free culture medium, the highest concentration is 10 times of the dilution of the mother solution, then the dilution is performed in half and half in sequence, the concentration is 5, the dilution is added into a 96-well plate, 100 mu l of the dilution is added into each well, 3 multiple wells are arranged, and if the test sample is powder or thick paste, the operation is repeated after the sample is diluted to the proper concentration by a proper solvent. Meanwhile, a blank group and a positive control group (containing 10% FBS) are set. For each sample, a solvent control is required, and the dilution concentration of the solvent is the same as that of the sample. Marking on the upper cover of the cell plate, wherein the marking contents are as follows: the time when the sample was applied and the different sample areas are clearly identified. And putting the mixture into a carbon dioxide incubator for incubation for 48 hours, and observing the cell morphology and the cell quantity under a microscope periodically. And (3) after incubation, taking the ultra-clean workbench, removing the culture medium in the holes by using a liquid aspirator, turning off a lamp of the ultra-clean workbench to keep a light-tight state, diluting the CCK-8 solution by 10 times by using the culture medium, pouring the diluted solution into a sample adding groove, sucking 100 mu l of the diluted solution by using a row gun, adding the diluted solution into a cell plate, accurately recording the time, putting the cell plate into a carbon dioxide incubator for incubation for 0.5h, opening the cover, putting the cell plate into an enzyme-labeling instrument, and measuring the absorbance at the wavelength of 450 nm.

The cell antioxidation method comprises the following operation steps: diluting HepG2 cells with a culture medium until the cell density is 6 x 105 cells/mL, adding 100 mu L of PBS into each hole of a 96-hole plate (blackboard) after uniformly mixing, adding 100 mL of PBS into the marginal space, placing the cells in an incubator for incubation for 24 hours, removing the culture medium PBS, cleaning the cells once, adding 100 mu L of samples into each hole, incubating the cells in the incubator for 1 hour, washing the PBS, continuously adding 100 mu L of 600 mu M ABAP (diluted by an oxidation culture medium) into each hole, reading the cells in a microplate reader immediately after the cells are diluted by the microplate reader, and setting parameters of the microplate reader as follows: the excitation wavelength is 485nm and the emission wavelength is 535nm at 37 ℃, and data is recorded once every 5min for 12 times. The antioxidant effect of the sample on the cells can be obtained. The data are shown in Table 2.

TABLE 2 data of antioxidant effect of the final product of each group

As can be seen from Table 2, the comparative example 1 only contains the polypeptide A, the comparative example 2 only contains the polypeptide B, the cell proliferation rate and the cell antioxidant capacity of the polypeptide A and the polypeptide B are far lower than those of the example 3, the specific polypeptide A and the specific polypeptide B are adopted for compounding in the example 3, the anti-aging, tightening and antioxidant capacities can be effectively improved, and the two compounded arginine/lysine polypeptides have better activity respectively aiming at different action targets on the pre-synapse and the post-synaptic of the nerve conduction pathway responsible for facial muscle contraction.

The composite polypeptide of comparative example 3 was not microencapsulated, and the cell proliferation rate and cell antioxidant capacity were also lower than those of example 3. This is because example 3 is a microcapsule encapsulation prepared by using a polyglyceryl nonionic surfactant, and has better stability and higher transdermal absorption rate.

Fourth, testing human body efficacy

1. The method for testing the light-streak efficacy comprises the following steps: the finished products of the example 3 and the comparative examples 1 to 3 are used as raw materials and are respectively added into a basic formula for volunteer trial, and comparison before and after use and comparison of wrinkle proportion are carried out.

2. Test subject

(1) Informed consent was issued and all subjects voluntarily enrolled in the test received oral and paper informed consent in accordance with local regulations and regulations. Informed consent explains the nature, purpose and potential risk of participation in the study, and emphasizes the willingness to participate in the test, subjects can be withdrawn from the study at any time for any reason. All subjects can be asked about the study, giving sufficient time to consider before signing. All informed consent signatures must be made prior to study initiation.

(2) And (3) inclusion standard: the Chinese healthy population, the age of 25-45 years (test starting time), should be insensitive to common cosmetics, and the subject should not participate in other clinical studies for about three months.

(3) Subject information: the number of people: 20 persons/group, total 5 groups; minimum age: age 21 years old; maximum age: age 41 years old; average age: 27.17 years of age; standard deviation: 4.73 Weekly.

3. Data analysis method

And (3) performing statistical analysis by using SPSS software, if the data is in normal distribution, analyzing the difference of skin data changes before and after the test sample by using a t test method, and if the data is in abnormal distribution, performing corresponding statistical analysis by using a rank sum test. And (5) making a conclusion whether the test sample is valid according to the statistical analysis result, and performing double-side test to obtain a test level alpha of 0.05. The delta difference is the difference between the background value of the skin before application of the sample and the value of the skin immediately after application of the sample. The rate of change is relative to the rate of change before use.

4. Test flow

1) Products such as foundation make-up, sun-screening, isolation and color cosmetics cannot be used on the test day, the tested part is cleaned by clear water, and the test part is heated and dried under the constant temperature and humidity environment (21.0 +/-1.0 ℃; humidity: 50% +/-10%) for 30 min;

2) arranging subject data acquisition before using the sample and for 28 days, each time using the skin test instrument for skin parameter and image acquisition;

3) after the test is finished, the subject completes the questionnaire;

4) statistical analysis was performed using SPSS software to analyze the data before and after use of the test samples. And making a conclusion whether the calculation result is valid or not.

5. Wrinkle area fraction analysis

The smaller the wrinkle area ratio, the smaller the wrinkle area, the more the wrinkle area is reduced, and the better the wrinkle fading effect of the sample is. The data are shown in table 3.

TABLE 3 wrinkle area ratio mean data for each group of samples

As can be seen from Table 3, compared with comparative examples 1-2, the anti-wrinkle effect of example 3 is significantly better, and therefore it is verified that the compound polypeptide has better bioactivity and more significant skin care effects of light lines, compactness and lifting compared with single polypeptide A and single polypeptide B. Example 3 compared to comparative example 3, the microcapsules of example 3 were coated to allow the microcapsules to enter the stratum corneum more easily, penetrate the epidermis layer quickly and directly to the dermis layer, and release slowly in the dermis layer to prolong the skin care effect.

The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. An arginine/lysine polypeptide microcapsule inclusion is characterized by comprising the following raw materials in parts by weight:

2. The arginine/lysine polypeptide microcapsule inclusion according to claim 1, wherein the polyglyceryl nonionic surfactant is polyglyceryl-10 diisostearate and/or polyglyceryl-10 laurate.

3. The arginine/lysine polypeptide microcapsule inclusion according to claim 1, wherein the polyglyceryl nonionic surfactant is a combination of 0.1 to 1% polyglyceryl-10 diisostearate and 0.1 to 0.5% polyglyceryl-10 laurate.

4. The arginine/lysine polypeptide microcapsule inclusion body according to claim 1, wherein the mass ratio of the polypeptide A to the polypeptide B is (3-9): 1.

5. the arginine/lysine polypeptide microcapsule inclusion according to claim 1, wherein polypeptide a is purchased from Zhejiang surge peptide biology, Inc., type ZPC-019S; the polypeptide B is purchased from Zhejiang surge peptide biology GmbH and has the model of ZPC-018S.

6. The method of making an arginine/lysine polypeptide microcapsule encapsulate according to any one of claims 1 to 5, comprising the steps of:

1) fully stirring and dissolving the compound arginine/lysine polypeptide, polyglycerol-10 diisostearate, polyglycerol-10 laurate, glycerol and glucan to obtain a uniform mixed solution;

7. The method of claim 6, wherein in step 2), the encapsulation-assisted treatment is one of high-voltage pulsed electric field-assisted, ultrasonic-assisted and free diffusion.

8. The method for preparing arginine/lysine polypeptide microcapsule inclusion complex of claim 7, wherein in the step 2), the encapsulation auxiliary treatment is high-voltage pulse electric field auxiliary, and the technical parameters of the high-voltage pulse electric field are as follows: the field intensity is 0.5 kV/cm-2 kV/cm, the number of pulse treatments is 10-60, and the material conductivity is 2000 mu S/cm-6000 mu S/cm.

9. The use of the arginine/lysine polypeptide microcapsule inclusion complex according to any one of claims 1 to 5, wherein the arginine/lysine polypeptide microcapsule inclusion complex is dispersed in cosmetic bases of different types of skin care emulsions, creams, cleansing, essences and lotions to prepare cosmetics or skin care products.