CN114085266B

CN114085266B - Peptide conjugate derivative based on palmitoyl tetrapeptide-7, preparation method and application

Info

Publication number: CN114085266B
Application number: CN202210012352.3A
Authority: CN
Inventors: 黄毅; 王辉平; 钱令页; 应佳伟
Original assignee: Zhejiang Pai Peptide Biology Co ltd Shenzhen Branch
Current assignee: Shenzhen Branch Of Zhejiang Peptide Biology Co ltd
Priority date: 2022-01-07
Filing date: 2022-01-07
Publication date: 2022-05-10
Anticipated expiration: 2042-01-07
Also published as: CN114085266A

Abstract

The invention discloses a palmitoyl tetrapeptide-7-based peptide conjugate derivative, a preparation method and application, and relates to the technical field of synthesis of biological peptide and derivatives thereof. The peptide conjugate derivative has an amino acid sequence as follows: Pal-Gly-Gln-Pro-Arg-R; the R is a compound containing amino in the structure and is conjugated with carboxyl in the structure of palmitoyl tetrapeptide-7 through amido bond; r above includes prosulfocarb. The palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention has more excellent anti-inflammatory and anti-radiation activities, further increases the generation of collagen, and improves the skin elasticity; and has more obvious anti-allergy effect, promotes self-rapid transdermal absorption, and generates better repairing effect on the skin.

Description

Peptide conjugate derivative based on palmitoyl tetrapeptide-7, preparation method and application

Technical Field

The invention belongs to the technical field of synthesis of biological peptides and derivatives thereof, and particularly relates to a palmitoyl tetrapeptide-7-based peptide conjugate derivative, a preparation method and application thereof.

Background

In further understanding of the microstructure and mechanism of skin, it was found that the processes of proliferation, renewal, metabolism and inflammation of skin cells are all regulated by specific sequence of oligopeptides, so that the introduction of specific bioactive peptides into skin care can fundamentally prevent and repair various skin problems. Because different types of peptides contain different amounts and types of amino acids, the activity of the peptides is diversified, so that oligopeptides with various effects are formed, and the oligopeptides have the cosmetic effects of promoting collagen generation, resisting free radical oxidation, diminishing inflammation, repairing, resisting edema, promoting hair regeneration, whitening and the like. Due to the remarkable cosmetic effect of the products, more and more effective bioactive peptides are applied to the cosmeceutical industry.

However, the direct application of oligopeptide compounds still has some defects, and the ideal effect cannot be achieved, so that modification treatment and the like are required. It is well known that the properties and characteristics of peptides can be altered by conjugating to the peptide a group that appropriately alters the properties of the peptide. Palmitoyl Tetrapeptide-7 (Palmitoyl Tetrapeptide-7) is also called as Palmitoyl Tetrapeptide-3, the amino acid sequence of which is Pal-Gly-Gln-Pro-Arg, abbreviated as Pal-GQPR, and belongs to one of Palmitoyl oligopeptide series polypeptides in signal peptide. The small molecular oligopeptide has the characteristics of no toxicity, high biological activity, specific biochemical physiological mechanism, high homology and easy absorption, and essentially solves various skin problems, thereby being concerned by the cosmetic industry. Currently, the synthetic scheme for palmitoyl tetrapeptide-7 is not much studied internationally and has been derived or conjugated to some extent.

Disclosure of Invention

The invention aims to provide a palmitoyl tetrapeptide-7-based peptide conjugate derivative, a preparation method and application, wherein the peptide conjugate derivative has more excellent anti-inflammatory and anti-radiation activities, further increases the generation of collagen, and improves the skin elasticity; and has more obvious anti-allergy effect, promotes self-rapid transdermal absorption, and generates better repairing effect on the skin.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a palmitoyl tetrapeptide-7-based peptide conjugate derivative having the amino acid sequence: Pal-Gly-Gln-Pro-Arg-R;

the R is a compound containing amino in the structure and is conjugated with carboxyl in the structure of palmitoyl tetrapeptide-7 through amido bond;

r includes prosulfocarb. The palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention is obtained by derivatizing palmitoyl tetrapeptide-7 by an amidation reaction with probethiamine, and provides more excellent efficacy. The palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention has higher anti-inflammatory activity, can more efficiently inhibit the generation of excessive interleukins, thereby inhibiting part of unnecessary inflammatory reaction, and particularly plays a role in inflammatory reaction possibly caused by other components added in a cosmetic product; and can also inhibit the production of interleukin when the skin is irradiated by ultraviolet rays, and has more excellent radiation-resistant effect. Meanwhile, the collagen can be further promoted to generate, the skin elasticity is obviously improved, and the self healing and regeneration capacity of the skin is enhanced. In addition, the palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention has higher hyaluronidase inhibition activity, and has more obvious anti-allergy effect when used alone. The palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention can be widely used as a raw material of a functional beauty skin care product, the functional activity is obviously improved, the transdermal absorption activity is better, and the addition usage amount of the palmitoyl tetrapeptide-7-based peptide conjugate derivative can be obviously reduced during application; can be applied to sensitive skin and has good anti-allergy repairing effect on the skin.

A process for the preparation of the palmitoyl tetrapeptide-7 based peptide conjugate derivative described above, comprising: the palmitoyl tetrapeptide-7 and the probenazole are subjected to condensation reaction under the action of a condensing agent.

In a preferred embodiment of the invention, the condensing agent comprises one of DCC, DPPA, DEPC; the agents used in conjunction with the condensing agent include organic bases or activators; the organic base is triethylamine; the activating agent is DMAP or HOSu; the solvent used in the condensation reaction comprises at least one of dichloromethane, chloroform, THF, DMF, methanol and ethyl acetate.

Further, the preparation method of the palmitoyl tetrapeptide-7-based peptide conjugate derivative specifically comprises the following steps:

dissolving palmitoyl tetrapeptide-7 in a methanol solution, adding a condensing agent, reacting for 2-3 h at room temperature, adding probethiamine and triethylamine, and reacting for 15-20 h at room temperature under stirring; and then distilling the solvent under reduced pressure, adding saturated sodium bicarbonate solution, washing the saturated sodium bicarbonate solution twice with diethyl ether, adjusting the pH of an aqueous phase to 2-3 by adding 1M sodium bisulfate aqueous solution, extracting the aqueous phase three times with ethyl acetate, washing an organic phase with distilled water and saturated sodium chloride aqueous solution in turn, drying the organic phase with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain the palmitoyl tetrapeptide-7-based peptide conjugate derivative.

In a preferred embodiment of the invention, the feed-to-liquid ratio of the palmitoyl tetrapeptide-7 to the methanol solution is 1 g: 4.5-7 mL; the mol ratio of the condensing agent to the palmitoyl tetrapeptide-7 is 1.1-1.3: 1; the molar ratio of the prosulfocarb to the palmitoyl tetrapeptide-7 is 1.1-1.5: 1; the molar ratio of the triethylamine to the palmitoyl tetrapeptide-7 is 2.05-2.4: 1.

A cosmetic product comprising a palmitoyl tetrapeptide-7-based peptide conjugate derivative as described above.

In a preferred embodiment of the present invention, the palmitoyl tetrapeptide-7-based peptide conjugate derivative may also be present in the form of a cosmetically acceptable salt or an alcogel. The palmitoyl tetrapeptide-7-based peptide conjugate derivative is compounded with alcohol to prepare the alcohol gel, so that the oil can be removed by virtue of an alcohol system, the transdermal resistance is reduced, the peptide and the derivative thereof which are beneficial to the efficacy penetrate through the stratum corneum, and the skin repairing efficacy of the peptide and the derivative thereof is better exerted.

In a preferred embodiment of the present invention, the peptide-conjugated derivative or the cosmetically acceptable salt or alcogel based on palmitoyl tetrapeptide-7 is added in the above-mentioned product in an amount of 0.001 to 1 wt% based on the total weight of the product.

In a preferred embodiment of the present invention, the cosmetically acceptable salt includes, but is not limited to, hydrochloride, sulfate, nitrate, phosphate, acetate, trifluoroacetate, benzenesulfonate, citrate.

In a preferred embodiment of the present invention, the above-mentioned products can be prepared as, but not limited to, preparations such as lotions, nourishing creams, skin lotions, facial masks, beauty lotions, muscle foundations, essential oils, and the like.

The palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel is a gel with alcohol as a dispersion system, and comprises the following components:

palmitoyl tetrapeptide-7-based peptide conjugate derivatives, and

at least one lower alcohol and/or polyol.

In a preferred embodiment of the invention, the lower alcohol comprises one or more of ethanol, propanol, hexanol or tert-butanol; the polyhydric alcohol comprises one or more of ethylene glycol, propylene glycol, 1, 2-butanediol, glycerol, maltitol, sorbitol, and panthenol.

In a preferred embodiment of the invention, isovalerylshikonin is further added into the polyhydric alcohol, and the addition amount is 0.5-3 mg/mL. Isovaleryl shikonin is added in the preparation process of the alcogel, and the effect of the isovaleryl shikonin on the skin can be effectively enhanced, so that bioactive substances can more quickly enter the skin to exert the effect of the bioactive substances, and the improvement and repair effect on the skin is enhanced; in addition, under the condition of existence of isovaleryl shikonin, the prepared alcogel has more excellent stability, and still has excellent activity after being stored for 28 days under a harsher condition. In addition, when isovalerylshikonin and the peptide conjugated derivative based on palmitoyl tetrapeptide-7 are compounded for use, the effect of the peptide conjugated derivative can be obviously improved, and the peptide conjugated derivative has good gain effects on anti-inflammatory reaction, radiation resistance, collagen generation promoting activity and anti-allergy effects.

The preparation method of the alcogel specifically comprises the following steps:

and (3) mixing the peptide conjugate derivative based on the palmitoyl tetrapeptide-7 with alcohol, stirring, heating to dissolve, placing at 40-55 ℃, standing and aging for 2-10 hours to obtain the alcohol gel.

In a preferred embodiment of the present invention, the concentration of the palmitoyl tetrapeptide-7-based peptide conjugate derivative in the alcogel is 1.6 to 12.5 mg/mL.

The invention also discloses the application of the peptide conjugate derivative or the alcogel in reducing skin inflammatory reaction, increasing skin elasticity and enhancing the radiation resistance of skin.

The invention also discloses the application of the peptide conjugated derivative in preparing a cosmetic product for increasing the skin elasticity and enhancing the radiation resistance of the skin.

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

the palmitoyl tetrapeptide-7-based peptide conjugate derivative has higher anti-inflammatory activity, can more efficiently inhibit the generation of excessive interleukin so as to inhibit part of unnecessary inflammatory reaction, can also inhibit the generation of interleukin when skin is irradiated by ultraviolet rays, and has more excellent anti-radiation effect; can further promote the generation of collagen and obviously improve the skin elasticity; has higher hyaluronidase inhibition activity, and has more obvious anti-allergy effect when being used alone. In addition, the derivative prepared by the invention can exist in beauty cosmetics in an alcogel form, isovaleryl shikonin is added in the preparation process of the alcogel, so that the effect of skin transdermal absorption is enhanced, and the effect of functional peptide is better exerted; the prepared alcogel has more excellent stability; meanwhile, when isovaleryl shikonin and the peptide conjugated derivative based on palmitoyl tetrapeptide-7 are compounded for use, the effect of the peptide conjugated derivative can be obviously improved. The palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention can be widely used as a raw material of a functional beauty skin care product, the functional activity is obviously improved, the transdermal absorption activity is better, and the addition usage amount of the palmitoyl tetrapeptide-7-based peptide conjugate derivative can be obviously reduced during application; can be applied to sensitive skin and has good anti-allergy repairing effect on the skin.

Therefore, the invention provides a peptide conjugated derivative based on palmitoyl tetrapeptide-7, a preparation method and application, wherein the peptide conjugated derivative has more excellent anti-inflammatory and anti-radiation activities, further increases the generation of collagen, and improves the skin elasticity; and has more obvious anti-allergy effect, promotes self-rapid transdermal absorption, and generates better repairing effect on the skin.

Drawings

FIG. 1 is a chemical structural formula of a palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared in example 1 of the present invention;

FIG. 2 is a mass spectrum of a palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared in example 1 of the present invention.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:

example 1:

preparation of palmitoyl tetrapeptide-7-based peptide conjugate derivatives:

dissolving palmitoyl tetrapeptide-7 in a methanol solution (feed-to-liquid ratio of 1 g: 6 mL), adding DCC (molar ratio of 1.2: 1 to palmitoyl tetrapeptide-7) to the solution, reacting at room temperature for 2.5 h, adding probesultiamine (molar ratio of 1.25: 1 to palmitoyl tetrapeptide-7) and triethylamine (molar ratio of 2.2: 1 to palmitoyl tetrapeptide-7), and reacting at room temperature for 18 h with stirring; then, the solvent was distilled off under reduced pressure, a saturated sodium bicarbonate solution (equal volume to the methanol/triethylamine mixed solution) was added, and then it was washed twice with diethyl ether (equal volume to the methanol/triethylamine mixed solution), the aqueous phase was adjusted to pH 2.5 by adding 1M aqueous sodium hydrogen sulfate solution, extracted three times with ethyl acetate (2 times volume amount of methanol/triethylamine mixed solution), and the organic phase was washed successively with distilled water (2 times volume amount of methanol/triethylamine mixed solution) and saturated aqueous sodium chloride solution (2 times volume amount of methanol/triethylamine mixed solution), and finally dried with anhydrous sodium sulfate, concentrated under reduced pressure to obtain a palmitoyl tetrapeptide-7-based peptide conjugate derivative in a yield of 87.1%. The structural formula is shown in figure 1.

¹H NMR(400 MHz, CDCl₃) δ_ppm：9.08(s, 1H, O=C-H), 8.74(s, 1H, Py-H), 5.10(s, 2H, Py-CH ₂), 4.68(t, 2H, CH-CH₂-CH₂-O=C-NH₂& H₂N-C=NH-NH-CH₂-CH₂-CH₂-CH), 4.52(dd, 1H, O=C-CH-N-C=O), 4.16(s, 2H, O=C-NH-CH ₂-O=C-NH₂), 3.70(t, 2H, -CH ₂-CH₂-C=C), 3.67、3.50 (m, 2H, O=C-N-CH ₂-CH₂-CH₂-CH-C=O), 2.71(t, 2H, H₂N-C=NH-NH-CH ₂-CH₂-CH₂-CH), 2.64(t, 2H, S-S-CH ₂-CH₂), 2.59(s, 3H, Py-CH ₃), 2.36(s, 3H, C=C-CH ₃), 2.45、2.22 (m, 2H, O=C-N-CH₂-CH₂-CH ₂-CH-C=O), 2.28(t, 2H, -CH ₂-C=C), 2.18(m, 2H, CH ₂-CH₂-O=C-NH), 2.14(t, 2H, CH ₂-O=C-NH₂), 2.11、1.95(m, 2H, O=C-N-CH₂-CH ₂-CH₂-CH-C=O), 2.25、1.68、1.48~1.54(28H, -CH ₂), 1.89(m, 2H, H₂N-C=NH-NH-CH₂-CH₂-CH ₂-CH), 1.64(m, 2H, H₂N-C=NH-NH-CH₂-CH ₂-CH₂-CH), 1.38(m, 2H, S-S-CH₂-CH ₂), 0.98(t, 3H, S-S-CH₂-CH₂-CH ₃), 0.90(t, 3H, -CH ₃)。

HRMS（ESI）：Calcd for C₄₉H₈₄N₁₂O₈S₂，m/z [M+H]⁺,1032.60. As shown in fig. 2.

Example 2:

preparation of palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel:

mixing the peptide conjugate derivative based on palmitoyl tetrapeptide-7 with alcohol, stirring, heating to dissolve, placing at 45 ℃, standing and aging for 6.5 hours to obtain alcohol gel; wherein the concentration of the palmitoyl tetrapeptide-7-based peptide conjugate derivative is 6.2 mg/mL.

The alcohol used in this example was a mixture of ethanol and sorbitol in a volume ratio of 2.5: 1.

Example 3:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 2 in that: the concentration of the palmitoyl tetrapeptide-7-based peptide conjugate derivative was 3.6 mg/mL; the alcohol is a mixture of propanol and panthenol at a volume ratio of 3: 1.

Example 4:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 2 in that: the concentration of the palmitoyl tetrapeptide-7-based peptide conjugate derivative was 8.4 mg/mL; the alcohol is a mixture of tert-butyl alcohol and propylene glycol, and the volume ratio of the tert-butyl alcohol to the propylene glycol is 1: 1.

Example 5:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 2 in that: the concentration of the palmitoyl tetrapeptide-7-based peptide conjugate derivative was 2.1 mg/mL; the alcohol used was a mixture of hexanol and maltitol in a volume ratio of 2: 1.

Example 6:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 2 in that: isovaleryl shikonin is added into the alcohol, and the addition amount is 1.2 mg/mL.

Example 7:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 6 in that: the alcohol used was also added with (2S,5R) -5-fluorocytosin-1-yl- [1,3] -oxathiolane in an amount of 0.9 mg/mL.

Example 8:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 2 in that: the alcohol used was also added with (2S,5R) -5-fluorocytosin-1-yl- [1,3] -oxathiolane in an amount of 0.9 mg/mL.

Example 9:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 6 in that: palmitoyl tetrapeptide-7 was used instead of the palmitoyl tetrapeptide-7 based peptide conjugate derivative.

Example 10:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 7 in that: palmitoyl tetrapeptide-7 was used instead of the palmitoyl tetrapeptide-7 based peptide conjugate derivative.

Example 11:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 8 in that: palmitoyl tetrapeptide-7 was used instead of the palmitoyl tetrapeptide-7 based peptide conjugate derivative.

Comparative example 1:

the preparation of the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel differs from example 2 in that: palmitoyl tetrapeptide-7 was used instead of the palmitoyl tetrapeptide-7 based peptide conjugate derivative.

Test example 1:

interleukin inhibition Activity assay

Test subjects human keratinocytes (HaCat).

The test method comprises the following steps: HaCat cells were grown at a density of 2.5X 10⁵Inoculating into 12-well plate at one/mL/well, and culturing in 10% FBS-containing MEM at 4 deg.C for 2 weeks for 24 hr; then taking out the culture plate, discarding the original culture solution, adding 1 mL of test sample solution into each hole, namely preparing the test sample solution with the culture medium into 1.0 wt% concentration, and stimulating by LPS with the final concentration of 10 mug/mL; and simultaneously setting a model group and a blank control group, wherein the model group is stimulated by only LPS with the final concentration of 10 mug/mL, and the blank control group is stimulated by adding 1 mL of complete culture medium without adding LPS. After 24 h of culture, the original culture medium was removed, 1 mL of fresh culture medium was added, and the culture was continued for 24 h. After the completion of the assay, the supernatant was collected and stored at-80 ℃ and the cytokine was measured by ELISA kit to examine the release of inflammatory factors (IL-6 and IL-8). The test conditions were: temperature 37 + -0.5 deg.C, humidity 92% and 5% CO₂And (4) concentration.

Grouping experiments: experimental group, M1 group: palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared in example 1; group M2: isovalerylshikonin; group M3: palmitoyl tetrapeptide-7-based peptide conjugate derivative + isovalerylshikonin (5.17: 1 mass ratio) prepared in example 1. Control group: palmitoyl tetrapeptide-7.

The test results are shown in table 1:

table 1 anti-inflammatory activity test results

As can be seen from the data in Table 1, the blank group shows that the relative content of IL-6 and IL-8 inflammatory factors in normal cells is 100%, and the IL-6 and IL-8 levels in the model group are obviously increased compared with the blank group after the keratinocytes are stimulated by LPS, which indicates successful modeling. The levels of IL-6 and IL-8 in the cells after the treatment of the M1 group are obviously lower than those of the control group, which shows that the carnosine derivative prepared by the invention has more excellent anti-inflammatory response activity. The results of group M3 were significantly better than those of groups M1 and M2, indicating that isovalerylshikonin and palmitoyl tetrapeptide-7-based peptide conjugate derivatives were used in combination, and that the anti-inflammatory effect was better, and the presence of isovalerylshikonin had a gain effect on the derivatives.

Test example 2:

collagen production promoting Activity test

Subject: human neonatal skin fibroblasts were seeded in 24-well plates in DMEM medium containing 10% fetal bovine serum at a concentration of 5X 10⁴One/well, placing at 37 ℃ and 5% CO₂Culturing for 24 h under the condition; then, the original culture medium is discarded, serum-free DMEM medium is added for replacement, after the culture is continued for 24 hours, the sample solution is added for treatment, and the final concentration of the added sample solution reaches 5 mu g/mL. Following 48 h of culture, the cell culture medium was collected and the amount of procollagen in the medium was measured using a collagen measurement kit (Takara Shuzo co., ltd., Japan). The specific test operation comprises the following steps: adding the collected cell culture medium into a 96-well plate uniformly coated with the primary collagen antibody, and performing antigen-antibody reaction at 37 ℃ for 3 hours; the cell culture medium was then removed and washed four times with PBS per well; then adding a chromogenic substance, incubating for 15 min at room temperature, and adding 1N sulfuric acid solution to stop reaction; finally, the absorbance at 450 nm was measured with a spectrophotometer. Wherein a standard curve is prepared using a standard solution, and then the amount of procollagen produced is calculated.

Grouping experiments: experimental group, M1 group: palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared in example 1; group M2: isovalerylshikonin; group M3: palmitoyl tetrapeptide-7-based peptide conjugate derivative + isovalerylshikonin (5.17: 1 mass ratio) prepared in example 1. Control, N1 group: no sample solution was added; group N2: palmitoyl tetrapeptide-7.

Analysis of results

The test results are shown in table 2:

TABLE 2 results of the test for promoting collagen production

As can be seen from the data in Table 2, the procollagen content after the treatment of the sample solution of group M1 is significantly higher than that after the treatment of group N2, which indicates that the palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention has higher collagen production promoting activity and can better promote the self healing and regeneration of skin. The results of the group M3 are obviously better than those of the group M1 and the group M2, which shows that the isovaleryl shikonin and the peptide conjugate derivative based on palmitoyl tetrapeptide-7 are compounded for use, so that the generation of collagen in skin is further increased, and the isovaleryl shikonin has a synergistic effect.

Test example 3:

hyaluronidase inhibitory Activity test

0.1 mL of 0.25M CaCl was taken₂Reacting the solution with 0.5 mL of hyaluronidase solution (dissolved with acetic acid buffer solution with pH of 5.6, 1250U/mL) in water bath at 37 deg.C for 20 min; adding 0.5 mL of test sample solution (diluted to 1 wt% by acetic acid buffer solution with pH of 5.6), and keeping the temperature for 20 min; adding 0.5 mL sodium hyaluronate solution (dissolved with acetic acid buffer solution with pH of 5.6, 0.5 mg/mL), reacting in water bath at 37 deg.C for 30 min, taking out, and standing at room temperature for 5 min; then adding 0.1 mL of 0.4M NaOH solution and 0.5 mL of acetylacetone solution, heating in a boiling water bath for 15 min, and immediately transferring to an ice water bath for cooling for 5 min; 1.0 mL of Ellisib reagent is added dropwise, and diluted with 3.0 mL of absolute ethanol, the mixture is placed at room temperature for 20 min for color development, and the absorbance value at the wavelength of 540 nm is measured by a spectrophotometer. The hyaluronidase inhibition was calculated according to the following formula:

hyaluronidase inhibition = [ (A-B) - (C-D) ]/(A-B) × 100%

Wherein A is the absorbance value of the control solution (the sample solution is replaced by acetic acid buffer solution); b is the absorbance value of the control blank solution (the sample solution and the enzyme solution are replaced by acetic acid buffer solution); c is the absorbance value of the sample solution; d is the absorbance value of the sample blank solution (the enzyme solution is replaced by acetic acid buffer solution).

Grouping experiments: experimental group, M1 group: palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared in example 1; group M2: isovalerylshikonin; group M3: palmitoyl tetrapeptide-7-based peptide conjugate derivative + isovalerylshikonin (5.17: 1 mass ratio) prepared in example 1. Control, palmitoyl tetrapeptide-7.

Analysis of results

The test results are shown in table 3:

TABLE 3 Hyaluronidase inhibitory Activity test results

As can be seen from the data in Table 3, after the M1 group sample solution is treated, the hyaluronidase inhibition rate is obviously higher than that after the control group is treated, which shows that the palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention has higher hyaluronidase inhibition activity and more obvious anti-allergy effect. The results of the group M3 are obviously better than those of the group M1 and the group M2, which shows that the isovaleryl shikonin and the peptide conjugate derivative based on palmitoyl tetrapeptide-7 are compounded for use, so that the inhibition effect on hyaluronidase is further enhanced, and the isovaleryl shikonin has a synergistic effect. In addition, the experimental result of the M2 group shows that isovalerylshikonin also has a certain inhibition effect on hyaluronidase.

Test example 4:

test for radiation resistance Activity

Female ICR mice were randomly divided into 6 groups of a model group, a normal group, a control group and each experimental group (experimental groups were the same as in experimental example 1), and 5 mice were each group, and the model group and the sham-operated group (control group) were treated with distilled water of the same volume. Except the normal group, the hair on the back of the other groups is shaved off; the experimental group was smeared with 2 mL of a sample solution (concentration 25 mg/mL). Mice were photoaged by daily exposure to UVA light for 8 weeks. Animals were sacrificed at 8 weeks, and the whole skin of the depilated area of the back was removed and assayed for SOD activity.

TABLE 4 test results of anti-radiation Activity

As can be seen from the data in Table 4, the SOD activity of the treated M1 group is obviously higher than that of the control group, which indicates that the carnosine derivative prepared by the invention has good radiation-resistant activity. The results of the M3 group are obviously better than those of the M1 group and the M2 group, which shows that the compound use of isovaleryl shikonin and the peptide conjugated derivative based on palmitoyl tetrapeptide-7 has better radiation resistant effect, and the existence of isovaleryl shikonin has gain effect on the derivative.

Test example 5:

evaluation of transdermal absorption Activity of alcohol gel

Test object

Sprague Dawley male rats (purchased from commercial vendors) weighing 180-200 g. Taking a rat, killing cervical vertebrae, shaving with an electric shaver, cutting off abdominal skin, removing subcutaneous fat layer, blood vessels and other residues, cleaning with physiological saline to ensure the integrity, rinsing with physiological saline, and freezing and storing in a refrigerator at-20 deg.C for use.

Experimental test method

Experiments were performed using a modified horizontal Franz diffusion apparatus. The operation is as follows: rat skin was held between the feeding chamber and the receiving chamber with the stratum corneum facing the feeding chamber and an effective permeation area of 1.20 cm²(ii) a Then 4.5 mL of sample is added into the supply chamber, 4.5 mL of PBS buffer solution is added into the receiving chamber, and the liquid level of the supply chamber and the liquid level of the receiving chamber are ensured to be completely submerged at the uppermost edge of the skin; and then stirring continuously at the constant temperature of 32 ℃ and the rotating speed of 310 r/min, sucking 0.1 mL of solution in the receiving chamber by a liquid-moving machine at 10 th, 20 th, 40 th, 60 th, 80 th, 100 th and 120 th min to serve as test solution, simultaneously supplementing the same amount of PBS, and measuring the content of the active substances by an ultraviolet spectrophotometer. The transdermal experiment was repeated 5 times for each sample. And calculating the transdermal rate of the sample according to the fitting of the cumulative transdermal permeation quantity-time curve.

Grouping experiments: experimental group, P1 group: palmitoyl tetrapeptide-7-based peptide conjugate derivatives prepared in example 1 (added in the same amount as that contained in the group P2 alcohol gel); p2 group: the alcogel prepared in example 2; p3 group: the alcogel prepared in example 3; p4 group: the alcogel prepared in example 4; p5 group: the alcogel prepared in example 5; p6 group: the alcogel prepared in example 6; p7 group: the alcogel prepared in example 7; p8 group: the alcogel prepared in example 8; p9 group: the alcogel prepared in example 9; p10 group: the alcogel prepared in example 10; p11 group: the alcogel prepared in example 11. Control, group D1: palmitoyl tetrapeptide-7; d2: the alcogel prepared in comparative example 1.

The results of the transdermal rate measurements are shown in table 5:

TABLE 5 results of the percutaneous absorption Performance test

As can be seen from the data in Table 5, the transdermal rate of the treatment of the P1 group is obviously better than that of the treatment of the D1 group, which indicates that the palmitoyl tetrapeptide-7-based peptide conjugate derivative prepared by the invention has more excellent transdermal absorption effect and enhances the skin care effect. The effect of group P2 is better than that of group P1, which shows that the alcohol gel prepared by compounding the peptide derivative and the alcohol can effectively improve the transdermal absorption effect of the bioactive substances. The effect of P6 is obviously better than that of P2 group, which shows that the existence of isovalerylshikonin in alcogel can further improve the transdermal absorption effect. In addition, the effect of the P7 group is better than that of the P6 group and the P8 group, and the effect of the P10 group is obviously better than that of the P9 group and the P11 group, which shows that the simultaneous existence of isovalerylshikonin and (2S,5R) -5-fluorocytosin-1-yl- [1,3] -oxathiolane has better effect on improving the transdermal absorption effect of the bioactive substances.

Test example 6:

thermal stability test

The test method comprises the following steps: the samples were stored in a sealed state under severe conditions of a temperature of (40 ± 1) ° c and a relative humidity of (75 ± 0.5)% and the percentages of the active ingredients in the respective samples after 7 d, 14 d, and 28 d were obtained by analyzing the initial value with respect to the content of the active ingredient at 0 d by high performance liquid chromatography.

The results of the above tests on the alcogels obtained in comparative example 1 and examples 2 to 11 are shown in Table 6:

TABLE 6 hydrothermal stability test results

As can be seen from the data in Table 6, the alcogel prepared in example 6 still has excellent activity after being stored for 28 days under severe conditions, which is better than that of example 1, and the existence of isovaleryl shikonin in the alcogel can effectively enhance the storage stability of the alcogel. Furthermore, the effect of example 7 is better than that of examples 6 and 8, and the effect of example 10 is significantly better than that of examples 9 and 11, indicating that the simultaneous presence of isovalerylshikonin and (2S,5R) -5-fluorocytosin-1-yl- [1,3] -oxathiolane has a better effect of enhancing the stability of the alcogel.

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A palmitoyl tetrapeptide-7-based peptide conjugate derivative having the amino acid sequence: Pal-Gly-Gln-Pro-Arg-R;

said R comprises probenazole;

the chemical structure of the palmitoyl tetrapeptide-7-based peptide conjugate derivative is shown as follows:

；

the preparation method of the palmitoyl tetrapeptide-7-based peptide conjugate derivative comprises the following steps: the palmitoyl tetrapeptide-7 and the probenazole are subjected to condensation reaction under the action of a condensing agent.

2. The palmitoyl tetrapeptide-7-based peptide conjugate derivative of claim 1, wherein: the condensing agent is DCC; reagents used in conjunction with the condensing agent include triethylamine; the solvent used in the condensation reaction was methanol.

3. A cosmetic product comprising a palmitoyl tetrapeptide-7-based peptide conjugate derivative of claim 1.

4. The product of claim 3, wherein: the palmitoyl tetrapeptide-7-based peptide conjugate derivative is also present in the form of a cosmetically acceptable salt or an alcogel thereof.

5. The product of claim 4, wherein: the palmitoyl tetrapeptide-7-based peptide conjugate derivative alcogel is a gel with alcohol as a dispersion system, and comprises the following components:

palmitoyl tetrapeptide-7-based peptide conjugate derivatives, and

at least one lower alcohol and/or polyol.

6. The product of claim 5, wherein: the lower alcohol comprises one or more of ethanol, propanol, hexanol or tert-butanol;

the polyalcohol comprises one or more of ethylene glycol, propylene glycol, 1, 2-butanediol, glycerol, maltitol, sorbitol, and panthenol.

7. The product of claim 5, wherein: isovaleryl shikonin is also added into the polyalcohol.

8. Use of a peptide conjugate derivative according to claim 1 for the preparation of a cosmetic product for increasing the elasticity and radiation resistance of the skin.