CN116003577B - Recombinant collagen for skin soothing and repairing and application thereof - Google Patents
Recombinant collagen for skin soothing and repairing and application thereof Download PDFInfo
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- CN116003577B CN116003577B CN202310123476.3A CN202310123476A CN116003577B CN 116003577 B CN116003577 B CN 116003577B CN 202310123476 A CN202310123476 A CN 202310123476A CN 116003577 B CN116003577 B CN 116003577B
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Abstract
The invention provides a recombinant collagen for skin relief repair and application thereof, and a nucleotide sequence for encoding the recombinant collagen for skin relief repair comprises a sequence shown as SEQ ID NO. 1. The amino acid sequence encoded by SEQ ID NO. 1 includes amino acids 950-1075 of type I collagen, and amino acids 594-729 and 1193-1199 of type III collagen. The recombinant collagen has the effects of moisturizing, supplementing skin collagen, promoting cell proliferation, resisting oxidation, resisting aging and the like, and has good histocompatibility, skin permeability and safety.
Description
Technical Field
The invention belongs to the technical field of skin care, and particularly relates to recombinant collagen for skin relief repair and application thereof.
Background
When the intracellular and extracellular environments of the skin change, the skin defending and repairing mechanism cannot adapt to the change, so that the damage to the skin and the aging state after the damage is gradually accumulated are caused. Studies have shown that free radicals are one of the important factors responsible for aging in humans. Various free radicals exist in human body, such as superoxide anion free radicals, hydroxyl free radicals, DPPH free radicals and the like, and the free radicals can cause cell and tissue decomposition and damage of metabolic functions. In addition to normal metabolism, the concentration of free radicals in the human body is also affected by factors such as solar radiation, pollution, exercise level, mental state, etc. Therefore, the skin care product containing the antioxidant component can eliminate excessive free radicals, delay the degradation of the body and inhibit the aging of the skin.
Schizophyllan is a beta-D-glucan produced from natural sources of schizophyllan, consists of a (1-3) -linked beta-D-glucopyranose residue main chain and a (1-6) -linked glucopyranose residue side chain, and has the capacity of resisting oxidation and radiation. Schizophyllan can inhibit the generation of free radicals by inhibiting cell oxidase systems and complexing transition metal ions induced to oxidize; can play a role in resisting radiation by removing free radicals, regulating gene expression, protein synthesis and other ways, prolong the apoptosis time of radiation damaged cells and inhibit lipid peroxidation.
Collagen is a biopolymer, the main component in animal connective tissue, and is also the most abundant and most widely distributed functional protein in mammals, accounting for 25-30% of the total protein. Collagen has good biocompatibility, biodegradability and bioactivity, so that the collagen has wide application in the fields of foods, medicines, tissue engineering, cosmetics and the like. The collagen includes type I collagen molecule, type III collagen molecule, type IV collagen molecule, type XI collagen molecule, etc., and has effects of moistening skin, promoting cell proliferation and enhancing cell barrier.
CN113057932a discloses a collagen skin care product for improving skin wrinkles, which comprises the following raw materials in parts by weight: the skin care product is prepared from 5-15 parts of active plants, 10-15 parts of glycerol, 1-2 parts of butanediol, 7-13 parts of active additives, 5-10 parts of collagen and 30-40 parts of deionized water by crushing, mixing, foaming and stirring, secondary mixing, sterilizing, adding collagen and final mixing.
CN102670477a discloses a collagen skin care product for improving skin defect, which has good treatment effect and no side effect. The skin care product comprises the following components in parts by weight: 10-30 parts of seaweed (rich in plant collagen), 10-30 parts of honey and 1-5 parts of vitamin E.
Therefore, it is very interesting to explore skin care compositions with multiple effects of antioxidant, anti-aging, repairing and moisturizing activities, and to strengthen skin injury repair from multiple angles.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a recombinant collagen and application thereof, in particular to a recombinant collagen for skin soothing and repairing and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides a recombinant collagen for skin relief repair, and the nucleotide sequence for encoding the recombinant collagen for skin relief repair comprises a sequence shown as SEQ ID NO. 1.
The nucleotide sequence shown in SEQ ID NO. 1 is:
GGGCCTCAAGGTATTGCTGGACAGCGTGGTGTGGTCGGCCTGCCTGGTCAGAGAGGAGAGAGAGGCTTCCCTGGTCTTCCTGGCCCCTCTGGTGAACCTGGCAAACAAGGTCCCTCTGGAGCAAGTGGTGAACGTGGTCCCCCTGGTCCCATGGGCCCCCCTGGATTGGCTGGACCCCCTGGTGAATCTGGACGTGAGGGGGCTCCTGGTGCCGAAGGTTCCCCTGGACGAGACGGTTCTCCTGGCGCCAAGGGTGACCGTGGTGAGACCGGCCCCGCTGGACCCCCTGGTGCTCCTGGTGCTCCTGGTGCCCCTGGCCCCGTTGGCCCTGCTGGCAAGAGTGGTGATCGTGGTGAGACTGGTCCTGCTGGTCCCGCCGGAGAACGAGGTGGCCCTGGAGGACCTGGCCCTCAGGGTCCTCCTGGAAAGAATGGTGAAACTGGACCTCAGGGACCCCCAGGGCCTACTGGGCCTGGTGGTGACAAAGGAGACACAGGACCCCCTGGTCCACAAGGATTACAAGGCTTGCCTGGTACAGGTGGTCCTCCAGGAGAAAATGGAAAACCTGGGGAACCAGGTCCAAAGGGTGATGCCGGTGCACCTGGAGCTCCAGGAGGCAAGGGTGATGCTGGTGCCCCTGGTGAACGTGGACCTCCTGGATTGGCAGGGGCCCCAGGACTTAGAGGTGGAGCTGGTCCCCCTGGTCCCGAAGGAGGAAAGGGTGCTGCTGGTCCTCCTGGGCCACCTGGTGCTGCTGGTACTCCTGGTCTGCAAGGAATGCCTGGACCTGGTCCTTGCTGTGGTGGTTAA
the invention creatively discovers a novel recombinant collagen sequence, wherein the nucleotide sequence for encoding the recombinant collagen for skin soothing repair comprises a sequence shown as SEQ ID NO. 1, and the amino acid sequence encoded by the SEQ ID NO. 1 comprises 950-1075 amino acids of type I collagen, 594-729 amino acids and 1193-1199 amino acids of type III collagen. The recombinant collagen has the effects of moisturizing, supplementing skin collagen, promoting cell proliferation, resisting oxidation, resisting aging and the like, and has good histocompatibility, skin permeability and safety.
In a second aspect, the present invention provides a composition having a soothing repair effect, the composition having a soothing repair effect comprising:
recombinant collagen and schizophyllan for skin relief repair as described in the first aspect.
The composition with the soothing and repairing effects comprises the recombinant collagen protein and schizophyllan for soothing and repairing skin, which are used in the first aspect, and the two components are matched for use, supplement each other, and have synergistic effects in the aspects of moisturizing, resisting oxidation, promoting cell proliferation, further improving skin injury repair and the like.
Preferably, the composition with the effect of relieving and repairing comprises the following components in parts by mass:
0.01-0.5 parts of recombinant collagen for skin relief repair and 0.1-5 parts of schizophyllan.
The weight part of the recombinant collagen for skin relief repair can be selected from 0.01 part, 0.1 part, 0.2 part, 0.3 part, 0.4 part or 0.5 part and the like;
the schizophyllan can be selected from 0.1 part, 1 part, 2 parts, 3 parts, 4 parts or 5 parts by mass and the like;
other specific point values in the numerical ranges are selectable, and will not be described in detail herein.
When the composition with the effect of relieving and repairing comprises 0.01-0.5 part of recombinant collagen for relieving and repairing skin and 0.1-5 parts of schizophyllan in parts by weight, the composition and the schizophyllan are combined for application, and the effect of repairing skin injury is better.
Preferably, the composition with soothing and repairing effects further comprises a humectant.
Preferably, the humectant comprises any one or a combination of at least two of glycerin, 1, 3-butanediol, sodium hyaluronate hydrolysate or ceramide 3; the at least two combinations include a combination of glycerin and 1, 3-butanediol, a combination of 1, 3-butanediol and sodium hyaluronate, or a combination of sodium hyaluronate and sodium hyaluronate, etc., and any other combination mode can be selected, and will not be described in detail herein.
Preferably, the humectant is glycerin.
When the humectant is specifically selected from glycerin, the combination of the recombinant collagen protein and schizophyllan polysaccharide for skin relief and repair can be matched with glycerin for use, and the humectant plays a stronger synergistic effect in the aspect of moisture preservation.
Preferably, the composition with the effect of relieving and repairing comprises the following components in parts by mass:
0.01-0.5 parts of recombinant collagen for skin relief repair according to the first aspect, 0.1-5 parts of schizophyllan and 5-50 parts of glycerol;
the weight part of the recombinant collagen for skin relief repair can be selected from 0.01 part, 0.1 part, 0.2 part, 0.3 part, 0.4 part or 0.5 part and the like;
the schizophyllan can be selected from 0.1 part, 1 part, 2 parts, 3 parts, 4 parts or 5 parts by mass and the like;
the mass part of the glycerol can be 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts or 50 parts and the like;
other specific point values in the numerical ranges are selectable, and will not be described in detail herein.
Preferably, the preparation method of the schizophyllan comprises the following steps:
fermenting and culturing schizophyllum in a culture medium containing radix puerariae, removing proteins in the fermentation culture solution by using a Sevag method, and adding an ethanol solution to precipitate polysaccharide in the fermentation culture solution to obtain the schizophyllum polysaccharide.
The schizophyllan polysaccharide is prepared by fermenting schizophyllan in a culture medium containing kudzuvine root, and the prepared schizophyllan polysaccharide has more remarkable effects in the aspects of antioxidation, cell repair, moisture preservation, immunoregulation and the like. Therefore, when schizophyllan prepared by adding the kudzuvine root into the culture medium is used for preparing the composition with the effect of relieving and repairing, the antioxidation, repairing and moisturizing effects of the composition are further enhanced.
In a third aspect, the present invention provides an inclusion having a soothing repair effect, the inclusion having a soothing repair effect comprising:
a microcapsule encapsulating material and a composition with a soothing and repairing effect as described in the second aspect, which is encapsulated inside the microcapsule encapsulating material.
The inclusion with the effect of relieving and repairing provided by the invention is a microcapsule wrapping material wrapping the composition with the effect of relieving and repairing outside, and after wrapping, the composition with the effect of relieving and repairing has stronger permeation effect, and the effects of moisturizing, antioxidation, relieving and repairing and the like exerted by the inclusion with the effect of relieving and repairing are further enhanced when the inclusion with the effect of relieving and repairing are applied to skin tissues.
Preferably, the microcapsule wrapping material is prepared from a raw material comprising a polyglyceryl nonionic surfactant;
preferably, the polyglyceryl nonionic surfactant comprises any one or a combination of at least two of polyglyceryl-10 diisostearate, polyglyceryl-10 laurate, or polyglyceryl-10 oleate. The at least two combinations include a combination of polyglycerin-10 diisostearate and polyglycerin-10 laurate, a combination of polyglycerin-10 laurate or polyglycerin-10 oleate, or a combination of polyglycerin-10 diisostearate and polyglycerin-10 oleate, etc., and any other combination mode may be selected, and will not be described in detail herein.
In a fourth aspect, the present invention provides a method for preparing an inclusion with a soothing repair effect according to the third aspect, the method comprising the steps of:
(1) Mixing a polyglyceryl nonionic surfactant with a composition having soothing and repairing efficacy as described in the second aspect;
(2) Performing wrapping auxiliary treatment on the mixture prepared in the step (1) to prepare the inclusion with the effect of relieving and repairing; the wrapping auxiliary treatment comprises high-voltage pulse electric field auxiliary treatment and/or ultrasonic auxiliary treatment.
The preparation method of the inclusion with the effect of relieving and repairing only needs to mix the effective components with the microcapsule wrapping material, and the inclusion can be prepared under the effect of wrapping auxiliary treatment.
Preferably, the wrapping auxiliary treatment in the step (2) is a high-voltage pulse electric field auxiliary treatment.
Preferably, the field intensity of the high-voltage pulse electric field auxiliary treatment is 0.5-2kV/cm, the number of pulse treatments is 10-60, and the conductivity of the material is 2000-6000 mu S/cm.
Specific numerical values in the range of 0.5-2kV/cm can be selected from 0.5kV/cm, 0.7kV/cm, 0.9kV/cm, 1.1kV/cm, 1.3kV/cm, 1.5kV/cm, 1.7kV/cm, 1.9kV/cm, 2kV/cm and the like;
specific values of the 10-60 can be 10, 20, 30, 40, 50m or 60 and the like;
specific values of 2000-6000. Mu.S/cm may be selected from 2000. Mu.S/cm, 3000. Mu.S/cm, 4000. Mu.S/cm, 5000. Mu.S/cm, 6000. Mu.S/cm, etc.;
other specific point values in the numerical ranges are selectable, and will not be described in detail herein.
In a fifth aspect, the present invention provides the use of a recombinant collagen according to the first aspect, a composition having a soothing repair effect according to the second aspect or an inclusion having a soothing repair effect according to the third aspect in the preparation of a skin care product.
Compared with the prior art, the invention has the following beneficial effects:
1. the nucleotide sequence of the recombinant collagen for skin soothing and repairing comprises a sequence shown as SEQ ID NO. 1, wherein the amino acid sequence coded by the SEQ ID NO. 1 comprises 950-1075 amino acids of type I collagen, 594-729 amino acids of type III collagen and 1193-1199 amino acids. The recombinant collagen has the effects of moisturizing, supplementing skin collagen, promoting cell proliferation, resisting oxidation, resisting aging and the like, and has good tissue compatibility, skin permeability and safety.
2. The composition with the soothing and repairing effects comprises the recombinant collagen protein and schizophyllan for soothing and repairing skin, which are used in the first aspect, and the two components are matched for use, supplement each other, and have synergistic effects in the aspects of moisturizing, resisting oxidation, promoting cell proliferation, further improving skin injury repair and the like.
3. The inclusion with the effect of relieving and repairing provided by the invention is a microcapsule wrapping material wrapping the composition with the effect of relieving and repairing outside, and after wrapping, the composition with the effect of relieving and repairing has stronger permeation effect, and the effects of moisturizing, antioxidation, relieving and repairing and the like exerted by the inclusion with the effect of relieving and repairing are further enhanced when the inclusion with the effect of relieving and repairing are applied to skin tissues.
4. The preparation method of the inclusion with the effect of relieving and repairing only needs to mix the effective components with the microcapsule wrapping material, and the inclusion can be prepared under the effect of wrapping auxiliary treatment.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited. The experimental methods in each example, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions recommended by the manufacturer.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control.
pET-28a (+) vectors in the following experiments were purchased from mikrin; schizophyllum commune is a schizophyllum commune GIM 5.43 strain purchased from the microbiology institute of cantonese province; BL21 (DE 3) chemocompetent cells were commercially available from Merck under the designation CMC0014-4X40 UL.
Preparation example 1
The preparation example provides recombinant collagen freeze-dried powder for skin soothing repair, and the preparation method of the recombinant collagen freeze-dried powder for skin soothing repair comprises the following steps:
(1) Constructing a recombinant plasmid: according to the nucleotide sequence shown in SEQ ID NO. 1, selecting a pET-28a (+) vector suitable for an expression vector escherichia coli host, and sending the pET-28a (+) vector to a sequencing company for synthesis to prepare a recombinant collagen expression vector pET-28a-1.
(2) Recombinant fermentation strain construction: transferring the pET-28a-1 expression vector into competent cells of escherichia coli (BL 21 (DE 3)) by using a chemical conversion method, screening positive clones in plate colonies after conversion by agarose gel electrophoresis, transferring the screened positive clones to 2mL of LB culture medium, culturing for 12 hours at 37 ℃ and 200rpm, diluting the cultured bacterial liquid by 1 time with glycerol with the volume fraction of 50%, packaging into a freezing tube, and freezing at the temperature of minus 80 ℃ to obtain the recombinant collagen fermentation strain.
(3) Fermenting and culturing recombinant fermentation strains: 10 mu L of LB recombinant collagen fermentation broth is coated on a solid LB plate, cultured for 12 hours in a 37 ℃ incubator, selected and inoculated in 50mL of LB liquid medium containing 50 mu g/mL kanamycin content, placed in a shaking table for culture at 200rpm at 37 ℃ for 14 hours, then inoculated in 200mL of LB liquid medium containing 50 mu g/mL kanamycin content in a ratio of 1:100, cultured at 37 ℃ until the bacterial broth OD=0.6, added with IPTG to achieve the final concentration of 1mM, continuously cultured for 4 hours, then centrifuged at 4000rpm for 15 minutes, and the bacterial cells are collected.
(4) Purifying recombinant collagen: the bacterial cells are subjected to ultrasonic disruption and centrifugation, the precipitate is washed by using a TritonX-100 washing solution, the supernatant is removed by centrifugation again, 6M guanidine hydrochloride is added into the precipitate, the pH is regulated to 8, after the precipitate is fully dissolved, the solution is subjected to nickel affinity chromatography resin, 20mM, 50mM, 100mM, 200mM and 500mM concentration gradient imidazole solution is used for eluting in sequence, eluent is concentrated by using a protein concentration column, the concentrated solution is collected and dialyzed overnight (dialysis bag, RC film, 14KD and the flat width is 44mM (Producer F600112)), then the protein concentration is detected by using a BCA kit, and the solution is uniformly mixed with the mannitol aqueous solution according to the proportion of 0.5mL of 5% mannitol aqueous solution by volume concentration per 5mg protein, and then the solution is pre-frozen at-40 ℃ for 10min by using a freeze dryer, and finally freeze-dried at-35 ℃ to obtain the recombinant collagen freeze-dried powder.
Preparation example 2
The preparation example provides schizophyllan, and the preparation method of the schizophyllan is as follows:
(1) Preparing schizophyllum commune fermentation products: the schizophyllum commune is inoculated into a fermentation medium according to the inoculation amount of 5 percent, and fermentation is carried out at the temperature of 28 ℃ and the speed of 160 rpm. After 120h of fermentation, the fermentation product is subjected to centrifugal treatment, and thalli and kudzuvine root powder are removed, so that the fermentation product is obtained. The fermentation medium comprises the following components: 0.6% of kudzuvine root powder, 0.3% of yeast extract, 0.05% of magnesium sulfate, 0.1% of monopotassium phosphate, 3% of glucose and the balance of water.
(2) Preparation of schizophyllan polysaccharide: adding distilled water with the same volume to the fermentation product for dilution, adding activated carbon with the volume fraction of 0.5%, carrying out water bath treatment at 80 ℃ for 40min for decolorization, centrifuging, filtering the filtrate, filtering the activated carbon by a 400-mesh screen to obtain clear and transparent fermentation liquor, eluting protein 5 times by using a Sevag method, then precipitating polysaccharide in the fermentation liquor by using 3 times of 95% ethanol, adding distilled water for redissolution after washing, dialyzing for 24h in running water and distilled water, concentrating the dialyzate, and freeze-drying to obtain the schizophyllan polysaccharide.
Preparation example 3
The preparation example provides a schizophyllan, and the preparation method of the schizophyllan is different from that of preparation example 2 only in that the fermentation medium does not contain kudzuvine root powder, and the rest operations are the same as those of preparation example 2.
Example 1
The embodiment provides an inclusion with a relieving and repairing effect, and the preparation method of the inclusion is as follows:
(1) 1 part of polyglycerol-10 diisostearate, 0.5 part of polyglycerol-10 laurate, 0.2 part of recombinant collagen prepared in preparation example 1, 0.3 part of schizophyllan prepared in preparation example 2 and 15 parts of glycerol are mixed, and 100 parts of water is added for supplement.
(2) Carrying out high-voltage pulse electric field auxiliary treatment on the mixed solution, and carrying out parameter selection: the field intensity is 0.5kV/cm, the pulse treatment number is 10, the material conductivity is 2000 mu S/cm, and the inclusion with the relieving and repairing effects is obtained after treatment.
Example 2
The embodiment provides an inclusion with a relieving and repairing effect, and the preparation method of the inclusion is as follows:
(1) 1 part of polyglycerol-10 diisostearate, 0.5 part of polyglycerol-10 laurate, 0.01 part of recombinant collagen prepared in preparation example 1, 5 parts of schizophyllan prepared in preparation example 2 and 50 parts of glycerol are mixed, and 100 parts of water is added for supplement.
(2) Carrying out high-voltage pulse electric field auxiliary treatment on the mixed solution, and carrying out parameter selection: the field intensity is 0.5kV/cm, the number of pulse treatments is 20, the material conductivity is 6000 mu S/cm, and the inclusion with the relieving and repairing effects is obtained after treatment.
Example 3
The embodiment provides an inclusion with a relieving and repairing effect, and the preparation method of the inclusion is as follows:
(1) 1 part of polyglycerol-10 diisostearate, 0.5 part of polyglycerol-10 laurate, 0.5 part of recombinant collagen prepared in preparation example 1, 0.1 part of schizophyllan prepared in preparation example 2 and 5 parts of glycerol are mixed, and 100 parts of water is added for supplement.
(2) Carrying out high-voltage pulse electric field auxiliary treatment on the mixed solution, and carrying out parameter selection: the field intensity is 2kV/cm, the pulse treatment number is 60, the material conductivity is 2000 mu S/cm, and the inclusion with the relieving and repairing effects is obtained after treatment.
Example 4
The present example provides an inclusion with a soothing and repairing effect, and the preparation method of the inclusion is different from example 1 only in that the preparation raw material does not contain glycerol, and the mass portion of glycerol is distributed to the recombinant collagen and schizophyllan in proportion, and the rest of operations are the same as example 1.
Example 5
This example provides an inclusion having a soothing and repairing effect, which is different from example 1 only in that schizophyllan polysaccharide prepared in preparation example 2 is replaced with schizophyllan polysaccharide prepared in preparation example 3, and the rest of the operations are the same as example 1.
Example 6
This example provides an inclusion having a soothing and repairing effect, which is different from example 1 only in that 0.2 parts of the recombinant collagen prepared in preparation example 1 and 0.3 parts of the schizophyllan polysaccharide prepared in preparation example 2 are replaced with a mixture of 0.5 parts of the recombinant collagen and the schizophyllan polysaccharide (the ratio of the recombinant collagen to the schizophyllan polysaccharide in the mixture is 0.01:5.1), and the rest of the operations are the same as in example 1.
Example 7
This example provides an inclusion having a soothing and repairing effect, which is different from example 1 only in that 0.2 parts of the recombinant collagen prepared in preparation example 1 and 0.3 parts of the schizophyllan polysaccharide prepared in preparation example 2 are replaced with a mixture of 0.5 parts of the recombinant collagen and the schizophyllan polysaccharide (the ratio of the recombinant collagen to the schizophyllan polysaccharide in the mixture is 0.6:0.05), and the rest of the operations are the same as in example 1.
Comparative example 1
This comparative example provides an inclusion having a soothing and repairing effect, which is different from example 1 only in that the raw material for preparation does not contain recombinant collagen, and the mass fraction of the recombinant collagen is distributed to schizophyllan, and the rest of the operations are the same as example 1.
Comparative example 2
This comparative example provides an inclusion having a soothing and repairing effect, which is different from example 1 only in that schizophyllan polysaccharide is not contained in the preparation raw material, and the schizophyllan polysaccharide is distributed to the recombinant collagen in parts by mass, and the rest of the operations are the same as example 1,
comparative example 3
This comparative example provides an inclusion having a soothing and repairing effect, which is different from example 1 only in that schizophyllan and recombinant collagen are not contained in the preparation raw material, and the parts by mass of schizophyllan and recombinant collagen are distributed to glycerin, and the rest of the operations are the same as example 1.
Test example 1
The present test example evaluates the antioxidant effect of the inclusion with the soothing repair effect of each example or comparative example in the following manner:
the cleaning efficiency of DPPH was used to evaluate the oxidation resistance of the examples and comparative examples. The specific evaluation method comprises the following steps: 0.5g of the inclusion bodies were immersed in 4mL of DPPH (100. Mu.M in 99wt% ethanol) solution, respectively (DPPH-only solution was used as a control group), and incubated in the dark for 60min. The absorbance value of the mixed solution at 517nm was then measured using an ultraviolet-visible spectrophotometer. The following equation is used for calculation: DPPH clearance= (control absorbance-experimental absorbance)/control absorbance x 100%, the results are shown in table 1.
TABLE 1
| Group of | DPPH clearance (%) |
| Example 1 | 67.3 |
| Example 2 | 62.4 |
| Example 3 | 66.5 |
| Example 4 | 67.9 |
| Example 5 | 42.7 |
| Example 6 | 33.9 |
| Example 7 | 51.0 |
| Comparative example 1 | 29.1 |
| Comparative example 2 | 18.2 |
| Comparative example 3 | 3.9 |
As can be seen from the data in table 1, compared with example 1, comparative example 1 and comparative example 2 lack one of recombinant collagen and schizophyllan respectively as preparation raw materials, and the DPPH clearance is lower than that of example 1, which indicates that schizophyllan and recombinant collagen complement each other, and have unexpected synergy in DPPH clearance; example 5 compared to example 1, the fermentation medium contained kudzuvine root powder with a lower DPPH clearance than example 1, indicating that the addition of kudzuvine root powder enhanced the antioxidant properties of the schizophyllan polysaccharide product; compared with example 1 in example 7, the weight ratio of the recombinant collagen to the schizophyllan is not in the range of (0.01-0.5): 0.1-5, and the DPPH clearance is lower than that in example 1, so that when the weight ratio of the recombinant collagen to the schizophyllan is in the range of (0.01-0.5): 0.1-5, the two components are matched and applied, and the oxidation resistance is stronger.
Test example 2
The present test example evaluates the moisturizing effect of the inclusion having the soothing repair effect of each example or comparative example in the following manner:
100 skin-healthy 18-40 year old volunteers were selected, and the male and female proportion was half, and randomly divided into 10 groups, and inclusion bodies with the soothing and repairing effects provided in examples and comparative examples were used. After the volunteers clean the arms, the skin moisture content before and after the volunteers use is measured by a skin tester (MC 750, german CK) by uniformly smearing the skin moisture content into a test area with the inner side of the arms and the diameter of 2cm according to the dosage of 2mg/cm, using the skin moisture content once a day and continuously using the skin moisture content for 30 days;
each subject was tested for skin moisture content before use and after 30 days of continuous use, three times per data, each group average was calculated, and skin moisture content change rate was calculated according to the following formula:
skin moisture content change rate= (skin moisture content after 30 days of use-skin moisture content before use)/skin moisture content before use x 100%; the test results are shown in table 2:
TABLE 2
As can be seen from the data in table 2, compared with comparative example 1 and comparative example 2, the preparation raw materials lack one of recombinant collagen and schizophyllan respectively, and the change rate of skin moisture content is lower than that of example 1, which indicates that the two components of schizophyllan and recombinant collagen complement each other, and the moisturizing effect of the inclusion can be enhanced; compared with the comparative example 3 and the comparative example 1, the preparation raw materials of the inclusion with the soothing and repairing effects respectively lack glycerol or the combination of the recombinant collagen and the schizophyllan, and the change rate of the skin moisture content is lower than that of the example 1, which shows that the glycerol, the recombinant collagen and the schizophyllan are combined for application, supplement each other, and have unexpected synergistic effect in improving the skin moisturizing effect.
Test example 3
The present test example evaluates the cell proliferation effect of the recombinant collagen of preparation example 1, the inclusion having the soothing repair effect of each example or comparative example, in the following manner:
after the HaCaT cells (human immortalized keratinocytes) with good growth state are paved on a 96-well plate for 17 hours, the serum-free basic culture medium is replaced for 7 hours, the old culture medium is discarded, a culture medium containing 1% of recombinant collagen aqueous solution or inclusion solution (the concentration of inclusion in the culture medium is 2 mug/mL) prepared in each example or comparative example is added, and the culture medium of a blank group does not contain inclusion, and the rest is consistent with an experimental group. After 48h incubation, cell viability was measured by CCK8 (absorbance at wavelength 450nm with reference wavelength 630 nm), each data was tested three times, the average value of each group was calculated, and the relative cell proliferation rate was calculated according to the following formula: relative cell proliferation rate= (experimental group absorbance-blank group absorbance)/blank group absorbance x 100%.
TABLE 3 Table 3
| Group of | Relative cell proliferation Rate (%) |
| Example 1 | 41.9 |
| Example 2 | 38.1 |
| Example 3 | 36.7 |
| Example 4 | 42.2 |
| Example 5 | 18.3 |
| Example 6 | 26.1 |
| Example 7 | 25.4 |
| Comparative example 1 | 13.1 |
| Comparative example 2 | 9.2 |
| Comparative example 3 | 1.6 |
| Preparation example 1 | 29.8 |
As can be seen from the data in table 3, the recombinant collagen of preparation example 1 has an excellent cell proliferation promoting effect. In addition, comparative example 1 and comparative example 2, which lack one of recombinant collagen and schizophyllan, respectively, as the preparation raw materials, compared with example 1, have lower relative cell proliferation rate than example 1, indicating that schizophyllan and recombinant collagen complement each other, and have unexpected synergy in promoting cell proliferation; example 5 compared with example 1, the fermentation medium contained kudzuvine root powder, which had a lower relative cell proliferation rate than example 1, indicating that the addition of kudzuvine root powder enhanced the ability of schizophyllan polysaccharide to promote cell proliferation.
Test example 4
The present test example evaluates skin relief repair of inclusion bodies having relief repair efficacy of each example or comparative example in the following manner:
(1) 66 guinea pigs were taken, each half of the male and female were removed from the right hind paw before 1d of the experiment, and the hair removal area was about 2cm 2 Guinea pigs were randomly divided into 11 groups, each containing 6 guinea pigs, and recorded as examples 1-7, comparative examples 1-3, and blank groups, respectively.
(2) Guinea pigs of examples 1 to 7, comparative examples 1 to 3 and blank groups were continuously 2d coated with 0.05g of inclusion solution prepared in examples or comparative examples, respectively, at dehairing, and the blank groups were coated with an equal amount of deionized water 1 time per day.
(3) On day 3 of the experiment, histamine phosphate solutions of 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), 0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v), and 0.1% (w/v) were prepared with physiological saline, respectively. The haired area of the right hind paw of the guinea pig was scratched with coarse sand paper to an extent of about 1cm 2 After the corresponding inclusion solution is smeared for 1 time locally, the smearing amount is 0.05g, the equivalent deionized water is smeared on a blank group, 0.01% (w/v) histamine phosphate solution is dripped on a scratch part after 10min, and whether a guinea pig returns within 3min is observedIf the right hind paw is licked, 0.02% (w/v) histamine phosphate solution 0.05mL is continuously added dropwise to the scratch part, and whether the guinea pig licks the right hind paw in 3min is observed. And so on, increasing the concentration according to the gradient every 3min, and dripping 0.05mL of histamine phosphate solution each time until the guinea pigs lick the right hind feet within 3 min. The average value of the itch causing threshold and the itch causing threshold difference ratio of guinea pigs of the same group were calculated with the cumulative total amount of histamine phosphate added at this time as itch causing threshold (. Mu.g), and the itch causing threshold difference ratio= (test group itch causing threshold-blank group itch causing threshold)/blank group itch causing threshold X100%, and the results are shown in Table 4.
TABLE 4 Table 4
From the data in Table 4, it can be seen that the higher the itching threshold, the stronger the corresponding skin barrier effect, i.e., the stronger the soothing and repairing function of the inclusion solution; compared with the comparative example 3 and the comparative example 1, the preparation raw materials of the inclusion with the soothing and repairing effects respectively lack glycerol or the combination of the recombinant collagen and the schizophyllan, and the difference rate of the itching threshold is lower than that of the example 1, which indicates that the glycerol, the recombinant collagen and the schizophyllan are combined for application, supplement each other, and have unexpected synergistic effect in the aspect of soothing and repairing skin. Compared with the example 1, the comparative example 1 and the comparative example 2 lack one of recombinant collagen and schizophyllan respectively as the preparation raw materials, and the difference rate of the itching threshold is lower than that of the example 1, which shows that the two components of the schizophyllan and the recombinant collagen supplement each other, so that the itching relieving and repairing effects of the inclusion can be improved; example 5 compared with example 1, the fermentation medium contains kudzuvine root powder, and the itching threshold difference rate is lower than that of example 1, which shows that the addition of kudzuvine root powder enhances the oxidation resistance of the schizophyllan polysaccharide product, so that the barrier effect of the skin can be improved; compared with example 1 in example 7, the ratio of the parts by weight of the recombinant collagen and the schizophyllan is not in the range of (0.01-0.5): 0.1-5, and the ratio of the difference of the itching threshold is lower than that in example 1, which shows that when the ratio of the parts by weight of the recombinant collagen and the schizophyllan is in the range of (0.01-0.5): 0.1-5, the two components are matched for application, and the skin soothing and repairing functions are stronger.
Test example 5
The inclusion with the effect of relieving and repairing prepared in the example is subjected to safety evaluation. The specific evaluation method comprises the following steps: the test is carried out according to the human skin patch test method in the human body safety test method of the seventh chapter in the cosmetic safety technical Specification 2015 edition. (1) Female volunteers required for the 18-60 year old compound experiments were selected, excluding criteria: (a) Antihistamines used in the last week or immunosuppressants used in the last month; (b) Any anti-inflammatory agent is applied to the tested part in the last two months; (c) a subject suffering from a clinically unhealed inflammatory skin condition; (d) insulin dependent diabetes mellitus; (e) Asthma or other chronic respiratory disease patients undergoing treatment; (f) receiving an anti-cancer chemotherapeutic within approximately 6 months; (g) patients with immunodeficiency or autoimmune disease; (h) lactating or pregnant women; (i) Bilateral mastectomy and bilateral axillary lymphadenectomy; (n) affecting the determination of the test result at the skin site due to scars, pigments, atrophy, port-marks or other imperfections; (o) participate in other clinical trial researchers; (p) highly sensitive body constitution; (q) non-volunteer participants or those who were unable to complete the prescribed content as required by the trial. (2) each example corresponds to a group of 10 volunteers; the selected area is not more than 50mm 2 A plaque test apparatus of about 1mm depth. The inclusion solution prepared in each example was placed in a plaque laboratory at an amount of about 0.025g. The control wells were empty of any material. The patch test with the test object is applied to the back or forearm of the subject by hypoallergenic tape, and is applied to the skin with palm light pressure for 24 hr. (3) Skin reactions were observed according to the criteria of Table 5 for 30min (after disappearance of the indentations), 24h and 48h, respectively, after removal of the subject plaque tester. And evaluated according to the criteria of table 5.
TABLE 5
The result shows that the product prepared by the invention is safe and has no stimulation and higher use value.
The applicant states that the present invention is illustrated by the above examples of the preparation process of the present invention, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be practiced by relying on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (15)
1. The recombinant collagen for skin soothing repair is characterized in that the nucleotide sequence for encoding the recombinant collagen for skin soothing repair is shown as SEQ ID NO. 1.
2. A composition having a soothing repair effect, characterized in that the composition having a soothing repair effect comprises:
the recombinant collagen and schizophyllan for skin relief repair of claim 1.
3. The composition with soothing repair function according to claim 2, wherein the composition with soothing repair function comprises, in parts by mass:
0.01-0.5 parts of recombinant collagen for skin relief repair and 0.1-5 parts of schizophyllan.
4. The composition with soothing repair function according to claim 2, wherein the composition with soothing repair function further comprises a humectant.
5. The composition with soothing repair effect according to claim 2, wherein the humectant comprises any one or a combination of at least two of glycerin, 1, 3-butylene glycol, sodium hyaluronate, hydrolyzed sodium hyaluronate, or ceramide 3.
6. The composition having a soothing repair effect according to claim 5, wherein the humectant is glycerin.
7. The composition with soothing repair function according to claim 2, wherein the composition with soothing repair function comprises, in parts by mass:
0.01-0.5 parts of recombinant collagen for skin relief repair according to claim 1, 0.1-5 parts of schizophyllan and 5-50 parts of glycerol.
8. The composition having a soothing repair effect according to claim 2, wherein the schizophyllan is prepared by a process comprising:
fermenting and culturing schizophyllum in a culture medium containing radix puerariae, removing protein in the fermentation culture solution by using a Sevag method, adding an ethanol solution to precipitate polysaccharide in the fermentation culture solution, and centrifuging to obtain the schizophyllum polysaccharide.
9. An inclusion having a soothing repair effect, characterized in that the inclusion having a soothing repair effect comprises:
a microcapsule encapsulating material and a composition having a soothing repair effect as claimed in any one of claims 2 to 8 encapsulated inside the microcapsule encapsulating material.
10. The inclusion with soothing repair function of claim 9, wherein the microcapsule encapsulating material is prepared from a material comprising a polyglyceryl nonionic surfactant.
11. The inclusion with soothing repair effect according to claim 10, wherein the polyglyceryl nonionic surfactant comprises any one or a combination of at least two of polyglyceryl-10 diisostearate, polyglyceryl-10 laurate, or polyglyceryl-10 oleate.
12. The method for preparing an inclusion having a soothing repair effect according to claim 9, wherein the method comprises the steps of:
(1) Mixing a polyglyceryl nonionic surfactant with the composition having a soothing repair effect as claimed in any one of claims 2 to 8;
(2) Performing wrapping auxiliary treatment on the mixture prepared in the step (1) to prepare the inclusion with the effect of relieving and repairing; the wrapping auxiliary treatment comprises high-voltage pulse electric field auxiliary treatment and/or ultrasonic auxiliary treatment.
13. The method of claim 12, wherein the wrapping auxiliary treatment in step (2) is a high-voltage pulsed electric field auxiliary treatment.
14. The method for preparing inclusion with relieving and repairing effects according to claim 12, wherein the field intensity of the high-voltage pulse electric field auxiliary treatment is 0.5-2kV/cm, the number of pulse treatments is 10-60, and the material conductivity is 2000-6000 μs/cm.
15. Use of the recombinant collagen according to claim 1, the composition with soothing repair efficacy according to any one of claims 2 to 8 or the inclusion with soothing repair efficacy according to any one of claims 9 to 11 for the preparation of a skin care product.
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