CN111714395B

CN111714395B - Polypeptide composition compounded by ginkgo biloba extract and application thereof

Info

Publication number: CN111714395B
Application number: CN202010696663.7A
Authority: CN
Inventors: 查建生; 徐金荣; 童莉; 吴杨生; 郑云云
Original assignee: Nanjing Sbaike Biotechnology Co ltd
Current assignee: Nanjing Sbaike Biotechnology Co ltd
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2022-10-04
Anticipated expiration: 2040-07-20
Also published as: CN111714395A

Abstract

The invention discloses a polypeptide composition compounded by ginkgo biloba extracts and application thereof, wherein the compounded composition comprises at least one acetyl micromolecule polypeptide, at least one palmitoyl micromolecule polypeptide, at least one solubilizer, at least one solvent-type polyalcohol, a water-soluble ginkgo biloba extract and water; the use of specific polyols and solubilizers and further increases the solubility and stability. The formula has excellent effects of resisting photoaging, inhibiting free radicals, and improving skin elasticity.

Description

Polypeptide composition compounded by ginkgo biloba extract and application thereof

Technical Field

The invention relates to the field of polypeptide and compound compounding, in particular to a polypeptide composition compounded by ginkgo biloba extracts and application thereof.

Background

Skin aging is generally classified into intrinsic aging and extrinsic aging, the latter being caused mainly by environmental factors such as ultraviolet radiation, smoking, wind, sun, and exposure to harmful chemicals. Since ultraviolet radiation in sunlight is a major factor in environmental factors leading to skin aging, extrinsic aging of skin is also called photoaging of skin. The ultraviolet rays are divided into three bands, wherein short-wave ultraviolet rays UVC are absorbed by the atmosphere and cannot reach the ground, and part of medium-wave ultraviolet rays UVB and almost all of long-wave ultraviolet rays UVA can penetrate through the atmosphere and reach the ground. The strong and weak penetration of UVB energy mainly damages epidermal cells, destroys skin barrier, inhibits immune function, and causes erythema and pigmentation. UVA is weak in energy but strong in penetrating power and can reach the dermis to damage collagen, elastic fibers and proteoglycan of the dermis. Skin photoaging is the result of the simultaneous effects of UVA and UVB on the epidermis and dermis layers and is mainly manifested by laxity, roughness, increased wrinkle formation, irregular pigmentation, vasodilatation, epidermal keratosis, abnormal proliferation, etc. at the exposed areas.

In the research of skin photoaging, skin damage caused by visible blue light is gradually emphasized in recent years. The blue light is the visible light with the shortest wavelength and the highest energy, the wavelength is within 380nm-500nm, and the blue light can come from various liquid crystal display screens such as sunlight, computers, televisions, flat panels, mobile phones and the like, and even can come from street lamps, indoor energy-saving lamps and other equipment. Compared with UVA and UVB, blue light can invade deeper into the skin, can influence the growth, metabolism and DNA damage of epithelial cells, and can even cause cell dysfunction and induce apoptosis. Blue light irradiation has a certain relation with skin photoaging, age, tumor occurrence and the like.

For skin photoaging problems, vitamin C or its corresponding derivatives are often selected in the art as antioxidants, such as ascorbyl tetraisopalmitate and ascorbyl glucoside. Vitamin C or a derivative thereof with a certain concentration has excellent antioxidant performance and can repair photoaging and blue light damaged skin to a certain extent, but the vitamin C only acts on a hydrophilic area of a cell membrane, the acting area is not wide enough, and the high-concentration vitamin C can generate irritation to the skin.

In recent years, it has been reported that bioactive peptides also have certain effect of improving skin photoaging, and are popular because of strong skin permeability and high stability. These bioactive peptides have been developed with a variety of effects, such as collagen production promotion, anti-inflammatory, antioxidant, neurotransmitter release prevention, and the like. The solubility of bioactive peptide can be divided into water solubility and fat solubility, but most of the polypeptides have poor solubility, the addition amount of the bioactive peptide in the formula is generally 100-2000ppm, and for the polypeptides containing long-chain acyl modification such as palmitoyl or myristoyl, the addition amount in the formula is more than 50ppm, and precipitation is easy to generate.

Ginkgo extract (A)Ginkgo bilobaL.) is dark powder obtained by crushing ginkgo leaves, extracting with alcohol, concentrating and drying, and the CAS No. 90045-36-6. The Chinese pharmacopoeia 2015 year edition discloses a preparation method of folium ginkgo extract by using ethanol to reflux and extract, and combining the extract, andthe total ginkgolic acid content should not exceed 10mg/kg, the total flavonol glycoside content should not be less than 24.0%, and the terpene lactone content should not be less than 6.0%. Ginkgolic acid is a general name of a series of derivatives of 6-alkyl or 6-alkenyl salicylic acid, toxicological studies show that the substance has sensitization, embryotoxicity, immunotoxicity and cytotoxicity, and particularly, the content of the substance is required to be reduced as much as possible when preparing a skin external cosmetic formula. The ginkgetin glycoside is mainly composed of quercetin, kaempferide and isorhamnetin, and is an important active component of folium Ginkgo extract. The terpene lactones are unique active ingredients of the ginkgo biloba extract, mainly ginkgolides and bilobalide, and the lactone components greatly reduce the water solubility of the ginkgo biloba extract, so that in the process of compounding cosmetics, the problem of irritation caused by overhigh ginkgolic acid is solved, and the solubility is further improved to meet the effective concentration of the ginkgo biloba extract.

Although those skilled in the art can adjust the pH by adding an acidic or basic substance or improve the solubility of the polypeptide in the formulation by adding a solubilizing agent, for the polypeptide and the ginkgo biloba extract with poor solubility, too many inactive ingredients affect the efficacy of the product and simultaneously consider the problems of appearance, stability, toxicity, skin irritation, etc. for a mature cosmetic formulation, which is not easy to solve.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a composition compounded by micromolecular polypeptide and ginkgo biloba extract for resisting and improving skin photoaging; it is a further object of the present invention to provide the use of the aforementioned compositions for combating photoaging, inhibiting free radicals, and improving skin elasticity.

The technical scheme is as follows: in order to achieve the above object, the present invention provides a polypeptide composition compounded with ginkgo biloba extract, comprising at least one acetyl small molecule polypeptide, at least one palmitoyl small molecule polypeptide, at least one solubilizer, at least one solvent-based polyol, a water-soluble ginkgo biloba extract and water; the number of amino acids of any small molecule polypeptide is 3~8, and the solvent-type polyol is dihydric alcohol and/or trihydric alcohol containing 3~5 carbon atoms.

The active ingredients of the polypeptide composition are micromolecule polypeptide and ginkgo biloba extract, at least two bioactive peptides are contained in the formula, the content of the active ingredients is improved as much as possible by utilizing the matching of a solubilizer and polyhydric alcohol on the premise of ensuring the stability of an aqueous solution and no precipitate, so as to provide a cosmetic formula for effectively resisting the photoaging and the blue light damage of the skin, and the content of the micromolecule polypeptide in the formula can be increased to be 0.05-5 wt% of water. The polypeptide composition is a clear and transparent brown yellow aqueous solution, and can be placed at room temperature for at least two years without precipitate separation.

The water-soluble ginkgo leaf extract is obtained by drying and crushing ginkgo green leaves, reflux-extracting by methanol and combining filtrate. On the basis, other modern filtering, concentrating and crystallizing processes can be combined to further improve the purity of the extract and reduce impurities. It should be noted that, in order to significantly reduce the content of ginkgolic acid and ensure the contents of flavonol glycosides and terpene lactones higher than those required in the chinese pharmacopoeia 2015, the water-soluble ginkgo extract provided by the present invention is preferably extracted by ethanol under reflux for multiple times, the filtrates are combined and then eluted repeatedly by using low concentration alcohol (10-30V/V%), and finally concentrated, dried and pulverized to obtain the water-soluble ginkgo extract. Flavonol glycoside not less than 26 wt%, terpene lactone not less than 6 wt%, and total ginkgolic acid less than 0.1 μ g ^-1 . The IC50 value of the water-soluble ginkgo biloba extract treated by the method on a B16 cell line is 1032 mug/mL. No toxicity to cells was observed at concentrations of 975. Mu.g/mL, 780. Mu.g/mL and 390. Mu.g/mL, respectively. Because the water solubility of the ginkgo terpene lactones is not high, the solubility of the common water-soluble ginkgo leaf extract in the market at 25 ℃ is not more than 0.05g/ml generally, and the solubility of the water-soluble ginkgo leaf extract obtained based on the method can be improved by 20-30 percent at 25 ℃.

In consideration of the characteristics of solubility, small molecular weight, easy absorption and the like, the amino acid content of any small molecular polypeptide is 3~8, and the small molecular polypeptide can be roughly divided into three types: the first type of small molecule polypeptide is acetyl small molecule polypeptide, which is selected from any one or the combination of two of acetyl hexapeptide-8 and acetyl octapeptide-3; the second kind of small molecule polypeptide is palmitoyl small molecule polypeptide, which is selected from any one or more of palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, and palmitoyl tetrapeptide-7.

Palmitoyl tripeptide-1, the molecular weight of 578.8, the amino acid sequence Pal-Gly-His-Lys, the water solubility of 100g is less than 0.01g (difficult to dissolve), and the palmitoyl tripeptide-1 is a Matrikine signal peptide, acts on the dermis layer, can promote the synthesis of extracellular matrix such as collagen and glycosaminoglycan, can effectively relieve wrinkles, and has stronger capacity of resisting ultraviolet irradiation.

Palmitoyl tripeptide-5, the molecular weight of 611.9, the amino acid sequence Pal-Lys-Val-Lys, the water solubility of 100g is less than 0.01g (insoluble), and the palmitoyl tripeptide-5 promotes the generation of collagen, protects the collagen from being decomposed by matrix metalloproteinase, effectively removes wrinkles and improves skin.

Palmitoyl pentapeptide-4, the molecular weight of which is 802.1, the amino acid sequence Pal-Lys-Thr-Thr-Lys-Ser, the water solubility of 100g is less than 0.01g (insoluble), and the palmitoyl pentapeptide-4 can promote the generation of collagen and elastin, eliminate static lines, help the skin to thicken and achieve the purpose of compacting the skin.

Palmitoyl tetrapeptide-7, the molecular weight of 694.9, the amino acid sequence Pal-Gly-Gln-Pro-Arg, the water solubility of 100g is less than 0.01g (difficult to dissolve), and the palmitoyl tetrapeptide-7 can effectively reduce the external injury and the generation of interleukin IL-6 in keratinocytes and fibroblasts caused by ultraviolet, thereby reducing the inflammatory reaction and glycosylation injury.

The molecular weight of the acetyl hexapeptide-8 is 888.9, the amino acid sequence of Ac-Glu-Glu-Met-Gln-Arg-Arg is 100g of water solubility of 0.01 to 1g (slightly soluble), and the acetyl hexapeptide-8 can locally block neurotransmission muscle contraction information, influence the skin sac nerve conduction, relax the facial muscles and achieve the purpose of smoothing dynamic wrinkles, static wrinkles and fine wrinkles; effectively reorganizes the elasticity of tissue collagen, can increase the activity of elastin, relax the lines of the face, smooth wrinkles and improve relaxation.

Acetyl octapeptide-3, the molecular weight 1073.19, the amino acid sequence Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp, the solubility is 0.01 to 1g (slightly soluble), the acetyl octapeptide-3 is a botulinum toxin mechanism, and the synthesis of SNARE receptors is inhibited, so that the excessive release of catecholamine and acetylcholine of skin is inhibited, the nerve transmission muscle contraction information is locally blocked, the facial muscle is relaxed, and the purpose of smoothing fine lines is achieved.

The total weight of the at least one acetyl small molecular polypeptide and the at least one palmitoyl small molecular polypeptide is 0.05-5 wt% of the polypeptide composition. Preferably, the at least one acetyl small molecular polypeptide and the at least one palmitoyl small molecular polypeptide account for 0.1 to 0.5 wt% of the polypeptide composition, wherein the acetyl small molecular polypeptide accounts for 0.1 to 0.4 wt% and the palmitoyl small molecular polypeptide accounts for 0.05 to 0.2 wt%. We find that when the small molecular polypeptide with the proportion is prepared into an aqueous solution, and 0.01-5 wt% of water-soluble ginkgo leaf extract is added, the small molecular polypeptide still can be separated out after standing for a long time, the precipitate can be temporarily eliminated by reducing the content of the small molecular polypeptide, but the precipitate still can be separated out after standing for 48 hours.

In order to optimize the solubility of the formula, the composition of the invention also needs at least one solubilizer and at least one solvent-type polyol, so as to balance the influence of 0.05 to 5wt% of effective content of the small-molecule polypeptide on the solubility. The traditional cosmetic solubilizer has high hydrophilicity, such as polyoxyethylene castor oil, fatty alcohol polyoxyethylene ether, polyglycerol fatty acid ester, tween-20 and the like, the addition amount of the traditional cosmetic solubilizer is at least more than 5wt%, and the traditional cosmetic solubilizer has high skin irritation.

The solubilising agent selected for use in the present invention is in particular selected from steareth-20 or behenyl behenate. The inventor finds that the stearyl alcohol polyether-20 or the behenyl alcohol can effectively eliminate the precipitate and ensure that the precipitate is not separated out for a long time and does not cause skin irritation after the stearyl alcohol polyether-20 or the behenyl alcohol is added with a solvent type polyol according to the adding amount of not more than 1.5 wt percent of the polypeptide composition. The polyalcohol is selected from any one or more of propylene glycol, butanediol, pentanediol and glycerol, and is added in an amount of 5-40 wt% of the polypeptide composition.

More preferably, the solubilizer is selected from steareth-20, which is not more than 1.5 wt% of the polypeptide composition; meanwhile, the solvent-type polyhydric alcohol is glycerol, and the addition amount of the glycerol is 30% of that of the polypeptide composition. The combination of steareth-20 with glycerin unexpectedly eliminates the solubility problem of the insoluble and insoluble components, while a higher proportion of glycerin helps to make the composition sticky in texture and provide moisture to the skin.

As a further optimization of the present invention, the polypeptide composition further comprises at least one preservative type polyol having a total number of carbon atoms of no more than 12, including but not limited to caprylyl glycol or ethylhexyl glycerin or a combination of both; the preservative type polyol has low molecular weight and good solubility, and the addition amount of the preservative type polyol is not more than 1 wt percent of the polypeptide composition.

The polypeptide composition has at least more than two biological activities from small molecular polypeptides, has excellent photo-aging resistance and blue light resistance after being added with the water-soluble ginkgo leaf extract, can effectively inhibit free radicals, and improves skin elasticity. The problem that each component is difficult to dissolve is solved by optimizing the solvent, and the bioactive material can be added into other various cosmetic products as a bioactive raw material, including but not limited to products such as emulsion, cream, essence, facial mask, eye cream and the like. The addition amount of the polypeptide composition is preferably 0.5-5 wt%, and can be added to 10 wt% to carry out strong repair if necessary.

The polypeptide composition disclosed by the invention integrates the effects of biological small-molecule polypeptides and a ginkgo leaf extract on the basis of solving the problem that palmitoyl small-molecule polypeptides are difficult to dissolve, and meanwhile, the polypeptide composition has the effects of resisting oxidation, inhibiting free radicals, promoting collagen synthesis, resisting blue light and preventing and solving the skin aging problem in multiple directions. The polypeptide composition of the present invention can be dissolved in a short time without precipitation, and the preferable range can ensure no precipitation for a long time. In addition, the ginkgo biloba extract in the polypeptide composition has good water solubility, the ginkgolic acid content is obviously lower than the standard of Chinese pharmacopoeia, the hidden danger of irritation is solved, and the excellent effect on blue light resistance is found.

The ginkgo extract has a plurality of purposes developed by people, such as the effects of improving blood circulation, resisting allergy, resisting broad-spectrum bacteria and the like, and can inhibit common skin pathogenic bacteria such as staphylococcus aureus, epidermophyton floccosum and the like. Besides the above effects, the water-soluble ginkgo leaf extract disclosed by the invention has an unexpected blue light resistant effect, and is compounded with functional small molecular polypeptides to complement each other. The water-soluble ginkgo leaf extract is subjected to a blue light damage test based on an EpiKutis model to detect SOD activity, has an obvious inhibiting effect on the increase of oxidative stress level caused by blue light irradiation under the condition of concentration of 0.5mg/mL, and has concentration dependence on the inhibiting effect.

Drawings

FIG. 1 is a graph showing the effect of each sample on collagen secretion in cells in Experimental example 3;

FIG. 2 is a skin elasticity test of test example 4;

FIG. 3 is a clinical anti-wrinkle test of test example 5;

FIG. 4 is a typical image of the subject in Experimental example 5;

FIG. 5 is the change of SOD activity in the epidermal model under different treatment conditions in Experimental example 6.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings.

Example 1

This example provides that the ginkgolic acid content is less than 0.1. Mu.g.g ^-1 The preparation method of the water-soluble ginkgo leaf extract. Oven drying green leaves of semen Ginkgo, and pulverizing; refluxing with 3-10 times of 40-60% ethanol for 2-3 times, filtering, mixing filtrates, and concentrating under reduced pressure until there is no ethanol. Then adding water again, stirring and refluxing, loading into macroporous adsorbent resin, repeatedly eluting with low concentration alcohol (10-30V/V%), concentrating, drying, and pulverizing to obtain water soluble folium Ginkgo extract. The water soluble folium Ginkgo extract has ginkgolic acid content of 0.06 μ g.g ^-1 28 percent of flavonol glycoside and 7.4 percent of terpene lactone, and the solubility is 0.06g/ml.

Example 2

The polypeptide composition 1 comprises the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.0%

30 percent of glycerin

The balance of water.

Example 3

The polypeptide composition 2 consists of the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.1 percent,

acetyl hexapeptide-8.0 percent,

0.5 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.5%,

30 percent of glycerin,

the balance of water.

Example 4

The polypeptide composition 3 consists of the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

0.2 percent of acetyl hexapeptide,

5.0 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.5%,

30 percent of glycerin,

the balance of water.

Example 5

The polypeptide composition 4 comprises the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.2%,

30 percent of glycerin

The balance of water.

Example 6

The polypeptide composition 5 comprises the following components in percentage by mass and volume:

0.05 percent of palmitoyl pentapeptide,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.5%,

the balance of water.

Example 7

The polypeptide composition 6 comprises the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

30 percent of glycerin,

the balance of water.

Example 8

The polypeptide composition 7 consists of the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

palmitoyl tetrapeptide-7.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.5%

30 percent of glycerin

The balance of water.

Example 9

The polypeptide composition 8 consists of the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

palmitoyl tetrapeptide-7.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.5%,

30 percent of glycerin,

0.4 percent of octyl glycol,

0.4 percent of ethyl hexyl glycerol,

the balance of water.

Example 10

The polypeptide composition 9 consists of the following components in percentage by mass and volume:

palmitoyl tripeptide-1.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

1.2 percent of Sanshan wasabi essence,

30 percent of glycerin,

0.8 percent of ethyl hexyl glycerol,

the balance of water.

Example 11

The polypeptide composition 10 comprises the following components in percentage by mass and volume:

palmitoyl tripeptide-5.05%,

palmitoyl tetrapeptide-7.05%,

0.2 percent of acetyl octapeptide,

1.5 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.5%,

35 percent of glycerin,

0.4 percent of octyl glycol,

0.4 percent of ethyl hexyl glycerol,

the balance of water.

Example 12

The polypeptide composition 11 consists of the following components in percentage by mass and volume:

palmitoyl tripeptide-1.05%,

palmitoyl tetrapeptide-7.05%,

0.2 percent of acetyl octapeptide,

0.05 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.5%,

30 percent of glycerin,

1.0 percent of the octyl glycol,

the balance of water.

Test example 1 solubility test

Dividing the obtained polypeptide composition into a plurality of groups, standing for different time, analyzing the dissolution characteristics of the polypeptide composition through light transmittance, selecting the wavelength corresponding to the maximum light transmittance position of the solution for determination in all solubility tests, and selecting an L5S ultraviolet spectrophotometer as an instrument.

This test first verified the water solubility characteristics of the water soluble ginkgo extract obtained in example 1, referenced as a 0.5 wt% solution, measured at 25 ℃, and the results are shown in table 1:

then, the water solubility of palmitoyl polypeptide and acetyl polypeptide are respectively determined, and as shown in tables 2 and 3, which are respectively measured at 25 ℃ by taking water as reference, the water solubility of the water-soluble ginkgo extract, palmitoyl pentapeptide-4 and acetyl hexapeptide-8 is not high, and the solubility is obviously reduced after the mass percentage of the prepared mixed solution is increased.

Table 4 shows the water solubility effect of polypeptide compositions 2-11, measured at 25 ℃ with reference to the solution prepared with polypeptide composition 1 of example 2. Compared with example 2, examples 3 and 4 show that under the condition of containing higher-concentration alcoholic solution and solubilizer, the palmitoyl small-molecule polypeptide is slightly increased, the content of acetyl small-molecule polypeptide is slightly increased, and the phenomenon that the complete solvent is not separated out within 3 months can be basically ensured. After the addition amount of the water-soluble ginkgo extract is increased to 5 percent, the solubility of the ginkgo extract is greatly influenced.

Examples 5, 6, 7 in comparison to example 2, it can be seen that the lowering of both solubilizer (steareth-20) and polyol (glycerol) resulted in slight, and increasing, precipitation over time; if the steareth-20 is lacked, the solubility of the instant compound meets the requirement, but the solubility performance is greatly reduced after two or three months, so the stability of the product dissolution can be improved by adding the steareth-20; and the combination of the polyhydric alcohol and the solubilizer can further remarkably improve the dissolving effect.

Examples 2 and 8 both provide preferred examples, and the addition of trace amounts of palmitoyl tetrapeptide-7 has little effect on solubility properties. The formulation does not affect dissolution performance by adding preservative in an amount of more than 1 wt%.

Test example 2 storage stability test

Test subjects: example 9 solution of formulated polypeptide composition 8.

The test method comprises the following steps:

1. sample was weighed in 1 portion (accurate to 0.1 g) plus CO free ₂ Heating to 40 deg.C, stirring, cooling to room temperature, and measuring pH with pH meter (METTLER TOLEPO).

2. Total number of colonies (CFU/g or CFU/mL)

A sample 5g was aseptically placed in 45ml of sterile saline, homogenized and shaken thoroughly to prepare a 1. And (3) taking a 1ml sterilization pipette to suck 1ml of the diluent, adding into 9ml of sterilized normal saline, uniformly mixing to prepare a diluent of 1.

Sucking 1ml of each dilution into a sterilized culture dish, injecting 20ml of nutrient agar cooled to 46 ℃, and mixing uniformly. After the nutrient agar solidified, the plate was inverted and cultured at 36 ℃ for 48 hours. Blank control was also performed.

3. Number of molds and yeasts (CFU/g or CFU/mL)

A sample (5 g) was aseptically dissolved in 45ml of sterilized physiological saline, homogenized, and shaken well to prepare a 1. And (3) taking a 1ml sterilization pipette to suck 1ml of the diluent, adding into 9ml of sterilized normal saline, uniformly mixing to prepare a diluent of 1.

Sucking 1ml of each dilution into a sterilized culture dish, injecting 20ml of Bengal red culture medium cooled to 46 ℃, and mixing uniformly. After the medium solidified, the plate was inverted and incubated at 28 ℃ for 72h. Blank control was also performed.

Test example 3 cellular collagen secretion test

Test subjects: the solution of the polypeptide composition 8 prepared in example 9, the solution of the polypeptide composition 9 prepared in example 10, and the solution of the polypeptide composition 10 prepared in example 11; control group: palmitoyl pentapeptide-4 in water (0.1 wt%).

In the experiment, a hydroxyproline kit (Solarbio) method is adopted to detect the influence of palmitoyl pentapeptide-4 and the polypeptide composition 8-10 on the collagen expression of NIH/3T3 cells (mouse fibroblasts).

Hydroxyproline (Hyp) accounts for 13.4% of collagen and is one of characteristic amino acids of collagen, so that the amount of collagen can be indirectly reflected by detecting the content of hydroxyproline.

Experimental methods

The fusion degree of NIH/3T3 cells reaches about 80 percent, the cells are digested by pancreatin, cell suspension is removed after cell density is adjusted, and the cells are evenly inoculated in a 6-hole plate. After the cells are completely attached to the wall, the old culture medium is completely sucked, a maintenance culture medium containing 0.4% FBS is added, the growth state of the cells is synchronized, after 24 h is cultured in a cell culture box, the old culture medium is completely sucked, and the maintenance culture medium containing TGF-beta 1 (10 ng/mL) and samples with different concentrations are respectively added. 48 And h, respectively collecting culture media of each hole, and detecting the influence of the sample on the collagen secretion of the NIH/3T3 cells by adopting a hydroxyproline determination kit method.

Results of the experiment

As can be seen from fig. 1 and table 6, both TGF- β 1, which is a positive drug, and the sample group were able to increase the hydroxyproline content of NIH/3T3 cells, and the effect was significant, and the sample group 3 (0.5% of example 9) had a significant difference (indicated by x) from the blank groupp< 0.001, vs. BC group).

Test example 4 clinical elasticity test

Test subjects: polypeptide composition 7 prepared in example 8 (1.0 wt% aqueous solution), polypeptide composition 11 prepared in example 12 (1.0 wt% aqueous solution); control group: acetyl hexapeptide-8 (0.1 wt% aqueous solution).

Subject: female 20 people aged 25-55 years, trial-face; the using method comprises the following steps: the face cream is uniformly smeared on the face once in the morning and at night every day, and the trial period is 56 days.

Evaluation parameters: ISE, instantaneous skin elasticity (N/m);

collecting an environment: the temperature is 20-25 ℃, and the humidity is 40-60%;

the instrument model is as follows: elastiMeter (ELM 1128).

The experimental results are as follows: as shown in fig. 2 and table 7, the skin elasticity of the subjects is significantly improved in all three groups of examples, wherein the effect of example 8 on the skin elasticity is more significant and is significantly better than that of acetyl hexapeptide-8 and example 12, and the combined effect of palmitoyl pentapeptide-4 and acetyl hexapeptide-8 is more significant.

Test example 5 clinical anti-wrinkle test

Experimental groups: example 8, example 12;

comparison: a single component hexapeptide-8.

1. Test procedure

Testing parts: a face;

the using method comprises the following steps: the medicine is uniformly applied to the face once a day in the morning and at night;

and (3) testing period: 56 days;

testing parameters:

volume (px) ³ ) Used for representing the three-dimensional size and depth of the wrinkle;

area (px) ² ) Used for representing the width and length of the wrinkle plane;

the area ratio (%) is used to indicate the ratio of wrinkle area to the selected skin;

and (3) testing environment: the temperature is 20-25 ℃, and the humidity is 40-60%;

the instrument model is as follows: visioFace 1000D.

2. Test results

Referring to FIG. 3, FIG. 4, and tables 8-10, the skin wrinkles of the subjects were significantly improved by the three groups of examples, wherein the reduction effect of example 8 on the volume, area and area ratio of wrinkles was significantly better than that of acetyl hexapeptide-8 and example 8. Therefore, the formula combining palmitoyl pentapeptide-4, palmitoyl tetrapeptide-7, acetyl hexapeptide-8 and the water-soluble ginkgo leaf extract has better wrinkle removing effect than other examples.

Test example 6 blue light-resistant Effect

The test is based on an EpiKutis blue light damage model, and the in-vitro anti-blue light efficacy of the sample to be tested is evaluated by detecting the activity of superoxide dismutase (SOD). The skin model is a 3D skin model (Epikutis) produced by Guangdong Boxi Biotechnology Limited. Blue light tube (PL-L18W/52, philips). The experimental samples were water-soluble ginkgo extract provided in example 1 (0.5 wt%) and polypeptide composition 7 provided in example 8 (1.0 wt%).

And (3) processing each group of skin models according to the method in the table, collecting the models after incubation and cleaning, adding protein extracting solution, and collecting upper-layer protein solution after uniform blowing. Concentration determination was performed using BCA protein kit (Solarbio). The SOD activity of each group of samples was measured according to the SOD (Solarbio) assay kit instructions.

The changes in the activity of the epidermal model SOD under different treatment conditions were as follows:

as shown in fig. 5, the SOD activity in the blue light injury group NC was significantly lower than that in the blank control BC group; the SOD activity of the positive control PC group is obviously higher than that of the NC group, and the experiment is proved to be effective. Example 8 SOD activity under the treatment conditions was significantly higher than that of the NC group and example 1 (## - #indicatesp< 0.01, by comparison with BC-groupp< 0.001, compared to NC panels).

Claims

1. A polypeptide composition compounded by ginkgo biloba extracts is characterized in that: comprises at least one acetyl small molecular polypeptide, at least one palmitoyl small molecular polypeptide, at least one solubilizer, at least one solvent-based polyol, a water-soluble ginkgo leaf extract and water; the number of amino acids of any small molecular polypeptide is 3~8, and the solvent-type polyol is dihydric alcohol and/or trihydric alcohol containing 3~5 carbon atoms; the polypeptide composition is a clear and bright solution;

the acetyl small molecular polypeptide is any one or the combination of acetyl hexapeptide-8 and acetyl octapeptide-3; the palmitoyl small molecule polypeptide is any one or combination of palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl pentapeptide-4 and palmitoyl tetrapeptide-7; the at least one acetyl small molecular polypeptide and the at least one palmitoyl small molecular polypeptide account for 0.05 to 5wt% of the polypeptide composition;

the solubilizer is stearyl polyether-20 or tribehenate behenyl alcohol; the addition amount of the solubilizer is not more than 1.5 wt percent of the polypeptide composition;

the solvent-type polyhydric alcohol is selected from any one or more of propylene glycol, butanediol, pentanediol and glycerol; the addition amount of the solvent-type polyhydric alcohol is 5-40 wt percent of the polypeptide composition;

the water-soluble folium Ginkgo extract flavonol glycoside is not less than 26 wt, terpene lactone is not less than 6 wt, and total ginkgoic acid is less than 0.1 μ g.g ^-1 (ii) a The addition amount of the water-soluble ginkgo leaf extract is 0.01-5 wt% of the polypeptide composition.

2. The ginkgo extract complex polypeptide composition of claim 1, wherein: the polypeptide composition also includes at least one preservative-type polyol having a total number of carbon atoms of no more than 12.

3. The ginkgo extract compounded polypeptide composition of claim 2, wherein: the preservative type polyhydric alcohol is octyl glycol or ethylhexyl glycerol; the preservative polyol is added in an amount not exceeding 1 wt% of the polypeptide composition.

4. The ginkgo extract compounded polypeptide composition according to claim 1, which comprises the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

stearyl polyether-20.5%,

30 percent of glycerin,

the balance of water.

5. The ginkgo extract compounded polypeptide composition according to claim 1, which comprises the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

palmitoyl tetrapeptide-7.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

1.5 percent of stearyl alcohol polyether,

30 percent of glycerin,

the balance of water.

6. The ginkgo extract compounded polypeptide composition according to claim 3, which comprises the following components in percentage by mass and volume:

palmitoyl pentapeptide-4.05%,

palmitoyl tetrapeptide-7.05%,

0.2 percent of acetyl hexapeptide,

0.5 percent of water-soluble ginkgo leaf extract,

1.5 percent of stearyl alcohol polyether,

30 percent of glycerin,

0.4 percent of octyl glycol,

0.4 percent of ethyl hexyl glycerol,

the balance of water.

7. The use of the polypeptide composition formulated with ginkgo biloba extract of claim 1 in the preparation of a skin care product for combating photoaging.

8. The use of the polypeptide composition formulated with ginkgo biloba extracts of claim 1 in the preparation of a skin care product for inhibiting free radicals.

9. The use of the polypeptide composition formulated with ginkgo biloba extract of claim 1 in the preparation of a skin care product for enhancing skin elasticity.

10. Use according to any one of claims 7 to 9, wherein the composition is added in an amount of 0.5 to 10 wt%.