CN113956244B

CN113956244B - Carnosine derivative for skin rejuvenation and application thereof

Info

Publication number: CN113956244B
Application number: CN202111566537.0A
Authority: CN
Inventors: 刘慧敏; 黄毅; 周永兵; 钱令页
Original assignee: Zhejiang Pai Peptide Biology Co ltd Shenzhen Branch
Current assignee: Shenzhen Branch Of Zhejiang Peptide Biology Co ltd
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2022-03-29
Anticipated expiration: 2041-12-21
Also published as: CN113956244A

Abstract

The invention discloses a carnosine derivative for skin rejuvenation and application thereof, and relates to the technical field of polypeptide derivative synthesis. The chemical structure of the carnosine derivative is shown as follows:

(ii) a Is a derivative of carnosine and glycitin. The carnosine derivative prepared by the invention has more excellent antioxidant activity, further enhances the synthesis of collagen, and improves the skin elasticity; and has better anti-aging effect, can directly remove aged skin cells and stimulate the regeneration of skin cells, and can be widely applied to raw materials of functional cosmetics.

Description

Carnosine derivative for skin rejuvenation and application thereof

Technical Field

The invention belongs to the technical field of synthesis of polypeptide derivatives, and particularly relates to a carnosine derivative for skin rejuvenation and application thereof.

Background

During natural or photoaging, the skin, and in particular the dermis, is subject to many variations and various types of damage, with many adverse changes occurring on the skin. These adverse changes are caused by a dysfunction of the homeostasis of the skin, in particular a dysfunction of the metabolism of fibroblasts. At the cellular level, adverse changes in the physiology or metabolism of the major cell types of the dermis, particularly fibroblasts, and of the epidermis, such as keratinocytes, can lead to aging. At the tissue level, aging is manifested by a disruption of the framework of the epidermis, the blood transport and innervation systems, and a slowing down of various types of metabolism, such as metabolic processes involved in the balance of barrier functions. This abnormality of the metabolism of skin cells, in particular fibroblasts, can be reflected by an undesirable change in the mechanical properties of the skin, such as an undesirable change in the microtopography, the appearance of wrinkles and fine lines, a decrease in the firmness and elasticity, an undesirable change in the skin tone and the surface age of the skin.

It is therefore of great importance to develop available products which are effective on skin cells, in particular fibroblasts, to limit the adverse changes of the skin and to prevent, reduce and/or delay the signs of skin ageing.

Disclosure of Invention

The invention aims to provide a carnosine derivative for skin rejuvenation and application thereof, wherein the carnosine derivative has more excellent antioxidant activity, further increases the synthesis of collagen, and improves the skin elasticity; and has better anti-aging effect, can directly remove aged skin cells and stimulate the regeneration of skin cells, and can be widely applied to raw materials of functional cosmetics.

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

a compound of formula I:

I；

which is a derivative of carnosine and glycitin carnosic acid methyl shown in a formula II,

II。

the compound shown in the formula I is a carnosine derivative subjected to chemical modification, wherein the amino terminal (-NH) of the carnosine₂) The N-substituted amide carnosine derivative is obtained by amidation reaction with carboxyl in a structure of the glycil carnosic acid A, and the amino terminal change provides obviously different chemical properties, metabolic stability and biological properties, so that higher efficacy than that of carnosine can be obtained. The carnosine derivative prepared by the invention has higher antioxidant activity; can stimulate more effectivelyThe formation of extracellular matrix components promotes the expression of procollagen C endopeptidase enhancer, and remarkably improves skin wrinkles and elasticity by enhancing the synthesis of collagen. Meanwhile, the fibroblast is effectively caused to shrink, so that the densification of the dermis is enhanced, the generation of skin wrinkles is prevented or reduced, and the loss of the skin compactness is reduced, so that the skin is stimulated to be updated, and the skin aging is effectively resisted; the carnosine derivative prepared by the invention not only can enhance the supervision function of macrophages on aged cells, but also can directly remove aged skin cells, and simultaneously can effectively inhibit the expression of SASP factors, thereby having more excellent anti-aging effect. The carnosine derivative prepared by the invention can be widely used as a raw material of functional cosmetics, and is beneficial to preventing or improving the functional disorder related to skin aging, namely chronobiological related skin aging or photobiological related aging; and can improve the condition of skin or mucosa, rejuvenate skin, treat damaged skin or mucosa, and repair atrophic tissues.

A process for preparing a compound of formula I, comprising: derivatization of the above compounds is achieved by formation of an amide bond between the amino group of carnosine and the carboxylic acid group of carnosic acid methyl ester.

Furthermore, the preparation method of the carnosine derivative shown in the formula I is the prior art disclosed, the carnosine and the carnosine are taken as raw materials, the carnosine derivative is synthesized by a two-step one-pot method of carboxyl activation and amide formation, and a reaction product is purified by a methanol/dichloromethane column chromatography to obtain the carnosine derivative.

Preferably, CDI is used as the carboxyl activating reagent.

Preferably, the molar ratio of the narcosis carnosic acid A to the carnosine is 1: 0.9-1.2; the molar ratio of the narcosis carnosic acid A to the carboxyl activating reagent is 1: 1.2-1.5.

A cosmetic composition comprising a compound of formula I or a cosmetically acceptable salt or excipient or adjuvant thereof.

Preferably, the cosmetic composition further comprises vanillin.

Preferably, the mass ratio of the compound shown in the formula I in the cosmetic composition to the vanillin is 1: 0.6-1.2.

Preferably, the composition comprises the following effects:

-oxidation resistance;

-procollagen synthesis activity;

-anti-ageing activity.

Preferably, the composition is used by external topical route for stimulating, repairing or modulating the metabolism of skin cells and semi-mucosal cells.

Preferably, the method for producing the composition for external application to skin containing the carnosine derivative represented by formula I is not limited to the above production method, and a person having ordinary knowledge in the art to which the present invention pertains can produce a composition for external application to skin containing the carnosine derivative by partially modifying the above production method.

It is a further object of the present invention to provide the use of a compound of formula I and vanillin for the preparation of a cosmetic product capable of stimulating the formation of one or more extracellular matrix components in the skin or mucous membranes. The invention adopts the vanillin and the carnosine derivative to be compounded for use, can effectively enhance the efficacy of the carnosine derivative prepared by the invention, further promotes the antioxidant activity, the anti-aging activity and the skin rejuvenation activity of the carnosine derivative, and better plays the skin repairing function of the carnosine derivative.

The invention also discloses an application of vanillin in enhancing the skin repair effect of the compound shown in the formula I.

Preferably, vanillin is replaced by a compound of formula III,

III。

according to the invention, the vanillin is chemically modified by the cordycepin to obtain the vanillin derivative, so that the bioactivity of the vanillin derivative can be effectively enhanced, the inhibition of the vanillin derivative on tyrosinase is further enhanced, the formation of melanin is reduced, and the vanillin derivative has a more excellent whitening effect on skin; meanwhile, the composition also has certain inhibitory activity on the expression of SASP factors, and can prevent or improve skin aging phenomena. The carnosine derivative is compounded with the carnosine derivative prepared by the invention, so that the carnosine derivative has more excellent skin repairing and anti-aging effects.

Further, a process for preparing vanillin derivatives of formula III, comprising:

dissolving cordycepin in ethanol/water (v/v, 1: 0.6-1), adding vanillin, and heating, refluxing and stirring for reacting for 8-12 h; after the reaction is finished, carrying out reduced pressure filtration, washing a filter cake with ethanol, and drying to obtain an intermediate M; dissolving the mixture in methanol, adding sodium borohydride at room temperature, and stirring for reaction for 1.5-3 h; and after the reaction is finished, decompressing and desolventizing, adding water to dissolve the residue, then adjusting the pH value to 3-3.5 by using 0.1M hydrochloric acid, then adding ethyl acetate for separating and extracting, drying by anhydrous sodium sulfate, and separating and purifying by silica gel column chromatography to obtain the vanillin derivative.

Preferably, the molar ratio of the cordycepin to the vanillin is 1: 0.92-1.10; the molar ratio of the intermediate M to the sodium borohydride is 1: 2-3.

Preferably, the compounds of formula I and the compounds of formula III are used to combat withered, flabby, sunken and/or skinned and/or to enhance and/or restore the elasticity or firmness of the skin.

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

the carnosine is modified by the Gancisella carnosic acid A to obtain the N-substituted amide carnosine derivative, so that the N-substituted amide carnosine derivative has higher antioxidant activity; can more effectively stimulate the formation of extracellular matrix components, promote the expression of a procollagen C endopeptidase enhancer, and obviously improve skin wrinkles and elasticity by increasing the synthesis of collagen; effectively causing the contraction of fibroblasts, further enhancing the densification of dermis, thereby stimulating the regeneration of skin and effectively resisting skin aging; the carnosine derivative prepared by the invention can directly remove aged skin cells, can effectively inhibit the expression of SASP factors, and has more excellent anti-aging effect. In addition, the vanillin is chemically modified by the cordycepin to obtain the vanillin derivative, so that the inhibition on tyrosinase can be further enhanced, and the formation of melanin is reduced; meanwhile, the composition also has certain inhibitory activity on the expression of SASP factors, and can prevent or improve skin aging; the carnosine derivative is compounded with the carnosine derivative prepared by the invention, so that the carnosine derivative has more excellent skin repairing and anti-aging effects. The carnosine derivative prepared by the invention can be widely applied to raw materials of functional cosmetics.

Therefore, the invention provides a carnosine derivative for skin rejuvenation and application thereof, wherein the carnosine derivative has more excellent antioxidant activity, can further enhance the synthesis of collagen and improve the skin elasticity; and has better anti-aging effect, can directly remove aged skin cells, stimulates the regeneration of skin cells, and can be widely used as a raw material of functional cosmetics.

Drawings

FIG. 1 shows the results of the measurement of the ultraviolet absorption spectrum in example 1 of the present invention;

FIG. 2 is a graph showing the mRNA expression levels of SASP factors IL-6 and IL-8 in HaCaT cells in example 5 of the present invention;

FIG. 3 is the mRNA expression levels of MMP-1 and MMP-3 of the SASP factors in HFF-1 cells in example 5 of the present invention;

FIG. 4 is a graph showing the mRNA expression level of the procollagen C endopeptidase enhancer in example 6 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:

human skin fibroblast cell line (HFF-1) and human monocyte (THP-1) used in the examples of the present invention were purchased from cell banks of the Chinese academy of sciences; the human epidermal keratinocyte cell line (HaCaT) was purchased from ScienCell, USA.

Reference is made to the literature for the synthesis of carnosine derivatives used in the examples of the invention (Sun, Q.; Burke, J. P.; Phan J.; Burns, M. C.; Olejniczak, E. T.; Waterson, A. G.; Lee, T.; Rossanese, O. W.; Fesik, S. W.).Angew. Chem., Int. Ed2012, 51(25), 6140-:

the method comprises the following steps of taking the carnosic acid A and carnosine as raw materials, taking CDI as a carboxyl activating reagent, synthesizing by a two-step one-pot method of carboxyl activation and amide formation, and purifying a reaction product by a methanol/dichloromethane column chromatography to obtain a carnosine derivative; wherein the molar ratio of the narcosis carnosic acid A to the carnosine is 1: 0.96; the molar ratio of the narcosis carnosic acid A to the carboxyl activating reagent is 1: 1.36; the yield was 68.3%.

Example 1:

the structural formula of the carnosine derivative prepared by the invention is shown as a formula I:

I

¹H NMR (400 MHz, D₂O), δ_ppm: 8.91 (s, 1H, IMI-H), 7.70 (s, 1H, IMI-H), 8.16、6.44 (d, 2H, CH=CH), 6.80~6.86、6.95、8.07 (5H, Ph-H), 5.71、4.69 (dd, 2H, Ph-CH), 4.83 (dd, 1H, N-CH), 3.29、3.01 (m, 2H, IMI-CH₂), 3.55 (t, 2H, N-CH₂), 2.61 (t, 2H, O=C-CH₂)。HRMS (ESI): Calcd for C₂₇H₂₆N₄O₁₀, m/z [M+H]⁺, 566.43。

the ultraviolet absorption spectrum of the carnosine derivative shown in the formula I is shown in figure 1, and the ultraviolet absorption peaks of the carnosine derivative prepared by the invention at 258 nm and 312 nm can be seen from the figure.

Example 2:

preparation of vanillin derivatives of formula III:

dissolving cordycepin in ethanol/water (v/v, 1: 0.7), adding vanillin, heating under reflux, stirring, and reacting for 10.5 hr; after the reaction is finished, carrying out reduced pressure filtration, washing a filter cake with ethanol, and drying to obtain an intermediate M; dissolving the mixture in methanol, adding sodium borohydride at room temperature, and stirring for reaction for 2.5 hours; after the reaction is finished, decompression is carried out for desolventizing, water is added for dissolving the residue, then 0.1M hydrochloric acid is used for adjusting the pH value to 3.4, then ethyl acetate is added for separating and extracting, anhydrous sodium sulfate is used for drying, and silica gel column chromatography is used for separating and purifying to obtain the vanillin derivative. Wherein the molar ratio of the cordycepin to the vanillin is 1: 1.05; the molar ratio of the intermediate M to the sodium borohydride is 1: 2.4. The chemical structure of the product is shown below:

III

¹H NMR (400 MHz, DMSO-d6), δ_ppm: 8.41 (s, 1H, IMI-H), 8.27 (s, 1H, pyr-H), 7.20 (d, 1H, Ph-H), 6.94、6.89 (dd, 2H, Ph-H), 6.19 (d, 1H, Ep-H), 5.48、3.79、3.64 (s, 3H, -OH), 4.42 (s, 2H, Ph-CH₂), 4.09 (s, 1H, N-H), 3.97 (s, 3H, O-CH₃), 3.92 (m, 1H, Ep-H), 3.71 (m, 1H, Ep-H), 3.86、3.61 (s, 2H, Ep-CH₂), 2.24、1.93 (m, 2H, Ep-H)。HRMS (ESI): Calcd for C₁₈H₂₁N₅O₅, m/z [M+H]+, 387.09。

¹³C NMR (150 MHz, DMSO-d6), δ_ppm: 157.6、155.1、152.5、150.2、148.7、142.3、136.9、125.0、122.1、117.4、111.5、101.6、83.3、76.1、64.2、58.7、45.0、36.9。

example 3: cytotoxicity assays for carnosine derivatives

Human keratinocytes (HaCaT cells) were treated with 10⁶24 well for 24 h, and replaced with FBS-free medium for 24 h starvation. Adding bioactive substances with final concentration of 25 mM, wherein carnosine derivative obtained in example 1 is added in group K1, and vanillin is added in group K2; k3 group, adding carnosine derivative obtained in example 1 and vanillin (mass ratio of the carnosine derivative to the vanillin is 1: 1); k4 group, adding vanillin derivatives obtained in example 2; in group K5, the carnosine derivative obtained in example 1 and the vanillin derivative obtained in example 2 were added (mass ratio of the carnosine derivative to the vanillin derivative is 1: 1). After 24 h of drug treatment, the medium was removed, 40. mu.L of 5 mg/mL MTT solution was added to each well for 4 h, the medium was removed, 1 mL DMSO (Amresco, 0231-500 mL) was added thereto and the mixture was shaken for 12 min, 200. mu.L of each well was used to measure the absorbance at 540 nm with a spectrophotometer, and a solvent containing no bioactive substance was used as a control. Cell viability was calculated according to the following formula:

cell survival% = (absorbance of drug-treated group/absorbance of control group) × 100%

The test results are shown in table 1:

table 1 results of cytotoxicity test of derivatives

Experimental group	Cell viability/%
		Group K1	106
Group K2	110
		Group K3	108
Group K4	101
		Group K5	100

As can be seen from the data in Table 1, neither carnosine derivatives nor vanillin derivatives produced by the present invention are toxic to cells.

Example 4: antioxidant activity (DPPH-stable free radical scavenging action) of carnosine derivatives

Experimental groups: 2 mL of 0.2 mM DPPH ethanol solution and a final concentration of 25 mM bioactive substance were added to the reaction tube, mixed well, reacted at room temperature in the dark for 40 min, and the absorbance was read at 517 nm. Taking the absorbance of the mixture of 2 mL of DPPH and 2 mL of PBS buffer solution as a control group, taking the absorbance of the mixture of 2 mL of double distilled water and 2 mL of ethanol as a normal group, and calculating the clearance rate according to the following formula:

clearance% = [ 1- (a)_{517 Experimental group}－A_{517 Normal group})/A_{517 control group}]×100%

The bioactive substances are divided into groups: carnosine derivatives obtained in example 1 were added to group K1, and vanillin was added to group K2; k3 group, adding carnosine derivative obtained in example 1 and vanillin (mass ratio of the carnosine derivative to the vanillin is 1: 1); carnosine was added in group D1.

The test results are shown in table 2:

table 2 results of radical scavenging rate test of derivatives

Experimental group	Clearance rate/%)
		Group K1	56.8
Group K2	19.7
		Group K3	81.4
D1 group	24.5

As can be seen from the data in Table 2, the DPPH free radical clearance rate after the treatment of the K1 group is obviously higher than that of the D1 group, which indicates that the carnosine derivative prepared by the invention has more excellent antioxidation. The effect of the group K3 is obviously better than that of the group K2 and the group K1, which shows that the antioxidant effect is better by using vanillin and carnosine derivatives in a compounding way, and the existence of vanillin has a synergistic effect on the development of the antioxidant activity of the carnosine derivatives.

Example 5: anti-aging Activity assay for carnosine derivatives

The recovery and subculture of the HaCaT cell and the HFF-1 cell are all conventional operations;

macrophage induced differentiation: THP-1 cells at 1X 10⁶Inoculating the cells/mL into a 6-well plate, adding PMA with the final concentration of 200 ng/mL for induction for 6 h, discarding the culture medium, washing with PBS, and adding a fresh culture medium to obtain the macrophage.

Experimental groups: carnosine derivatives obtained in example 1 were added to group K1, and vanillin was added to group K2; the carnosine derivative obtained in example 1 and vanillin (mass ratio of 1: 1) were added to group K3, the vanillin derivative obtained in example 2 was added to group K4, and the carnosine derivative obtained in example 1 and vanillin derivative obtained in example 2 were added to group K5 (mass ratio of 1: 1). Control group: carnosine was added in group D1 and no active substance was added in group D2.

a. Scavenging of aged cells in macrophage-HaCaT cell co-culture model

HaCaT cells at 4X 10⁴Inoculating the seeds/wells into a 48-well plate, culturing for 24 h, then changing to a serum-free culture medium containing 250 mu M t-BHP, incubating for 2 h to induce cell aging, and then changing to a complete culture medium containing no t-BHP for culturing for 12 h; macrophages are added according to the effective target ratio of 5: 1, active substances with the final concentration of 25 mM are cultured for 24 h, SA-beta-gal positive aged cells are marked by SPiDER-beta gal (green fluorescence), cell nuclei are marked by Hoechst (blue fluorescence), and the number of the aged cells with the green fluorescence is observed under a fluorescence inverted microscope.

b. Scavenging effect on aged cells in HaCaT cell single culture model

HaCaT cells at 4X 10⁴Inoculating the seeds/wells into a 48-well plate, culturing for 24 h, then changing to a serum-free culture medium containing 250 mu M t-BHP, incubating for 2 h to induce cell aging, and then changing to a complete culture medium containing no t-BHP for culturing for 12 h; adding active substance with final concentration of 25 mM, culturing for 24 h, labeling SA-beta-gal positive aged cells with spiDER-beta-gal (green fluorescence), labeling nuclei with Hoechst (blue fluorescence), and developing by fluorescence inversionThe number of aged cells with green fluorescence was observed under a microscope.

c. Scavenging of aged cells in macrophage-HFF-1 cell co-culture model

HFF-1 cells were cultured at 4.5X 10³Inoculating the seeds/wells into a 48-well plate, culturing for 48 h, then changing to a serum-free culture medium containing 300 mu M t-BHP, incubating for 2 h to induce cell aging, and then changing to a complete culture medium containing no t-BHP for culturing for 12 h; macrophages are added according to the effective target ratio of 5: 1, active substances with the final concentration of 25 mM are cultured for 24 h, SA-beta-gal positive aged cells are marked by SPiDER-beta gal (green fluorescence), cell nuclei are marked by Hoechst (blue fluorescence), and the number of the aged cells with the green fluorescence is observed under a fluorescence inverted microscope.

d. Effect on the elimination of aged cells in a model of HFF-1 cell culture alone

HFF-1 cells were cultured at 4.5X 10³Inoculating the seeds/wells into a 48-well plate, culturing for 48 h, then changing to a serum-free culture medium containing 300 mu M t-BHP, incubating for 2 h to induce cell aging, and then changing to a complete culture medium containing no t-BHP for culturing for 12 h; after 24 h incubation with 25 mM final active substance, SA-. beta. -gal-positive aged cells were labeled with SPiDER-. beta.gal (green fluorescence), nuclei were labeled with Hoechst (blue fluorescence), and the number of aged cells with green fluorescence was observed under a fluorescence inverted microscope.

The above test results on the elimination of aged cells showed that carnosine has a very strong effect of eliminating aged cells in the macrophage-HaCaT cell or macrophage-HFF-1 cell co-culture model, but fails to show the activity of eliminating aged cells in the HaCaT cell or HFF-1 cell single culture model; the carnosine derivative prepared by the invention has extremely strong effect of eliminating aged cells in a co-culture model of macrophage-HaCaT cells or macrophage-HFF-1 cells, and also shows good activity of eliminating aged cells in a single culture model of HaCaT cells or HFF-1 cells, which indicates that the carnosine derivative prepared by the invention can enhance the aging monitoring function of macrophages and can directly eliminate aged skin cells.

e. Effect of carnosine derivatives on the expression level of SASP factor in aged HaCaT cells/HFF-1 cells

HaCaT cells at 3.5X 10⁵Inoculating the seeds/wells into a 6-well plate, adding a serum-free culture medium containing 250 mu M t-BHP, incubating for 2 h to induce cell aging, and then changing to a complete culture medium containing no t-BHP for culturing for 12 h; then adding 25 mM active substance to culture for 12 h, lysing cells to extract total RNA, and detecting the mRNA expression level of IL-6 and IL-8 by qPCR method.

HFF-1 cells at 2X 10⁵Inoculating each well into a 6-well plate, adding a serum-free culture medium containing 300 mu M t-BHP, incubating for 2 h to induce cell aging, and then changing to a complete culture medium containing no t-BHP for culturing for 12 h; then adding active substances with a final concentration of 25 mM for culturing for 12 h, cracking cells to extract total RNA, and detecting the mRNA expression level of MMP-1 and MMP-3 in the cells by a qPCR method.

RNA extraction and quantitative detection: collecting cells, and extracting RNA by using a Thermo K0731 RNA extraction kit; adding 2 mu L ddH into a micropore plate of a multifunctional microplate reader₂And O calibration, and adding 2 mu L of extracted RNA into a microplate to detect the RNA concentration and the A260/A280 ratio. The result shows that the A260/A280 ratio of the extracted RNA sample is in the range of 1.9-2.1, and the RNA sample meets the experimental requirements.

Reverse transcription: according to PrimeScript^TMReverse transcription reaction was performed in the reverse transcription kit instructions, 20. mu.L total, 5 XPrimeScript RT Master Mix 4. mu.L, 1. mu.g RNA sample, RNase Free ddH₂The content of O is filled to 20 mu L. The reaction condition is 37 ℃ and 15 min; at 85 ℃ for 5 s; after the reaction is finished, the mixture is stored at 4 ℃ for standby.

qRCR amplification reaction: the reaction system is shown below.

PowerUp^TM SYBR^TM Green Master Mix 5 μL

Forward Primer（10 μM） 0.4 μL

Reverse Primer（10 μM） 0.4 μL

cDNA template 0.8. mu.L

RNase Free ddH₂O 3.4 μL

Total 10 μL

The amplification procedure was: UDG activation at 50 ℃ for 55 s; pre-denaturation at 95 ℃ for 2 min; PCR amplification cycles 40 times: 95 deg.C, 14 s, 60 deg.C, 65 s; and (4) analyzing a dissolution curve.

And (4) analyzing results: using relative quantities 2^-ΔΔCt(RQ value) represents the difference in fold expression of the gene of interest between the experimental and blank groups. Wherein Δ Ct = Ct_{Target gene}－Ct_{Internal reference gene}，ΔΔCt=ΔCt_{Experimental group}－ΔCt_{Blank group}Blank RQ values were 1.

TABLE 3 primer sequences for qPCR

The test results are shown in fig. 2 and 3.

FIG. 2 shows the effect of bioactive substances on the mRNA expression levels of SASP factors IL-6 and IL-8 in aged HaCaT cells, and it can be seen from the analysis in the figure that the carnosine derivatives prepared by the present invention in group K1 can significantly reduce the expression levels of IL-6 and IL-8, and the vanillin derivatives in group K4 also have a certain down-regulation effect on the expression levels of IL-6 and IL-8, but the vanillin in group K2 and the carnosine in group D1 obviously do not affect the expression of SASP factors in aged HaCaT cells, indicating that both the carnosine derivatives and the vanillin derivatives prepared by the present invention have the effect of inhibiting the expression of some SASP factors in aged HaCaT cells. In addition, the effect of group K3 is better than that of group K1, which shows that the compound of the derivative prepared by the invention and vanillin plays a synergistic role in carnosine derivatives, and can further enhance the inhibition effect on the expression of part of SASP factors in aged HaCaT cells. The effect of the K5 group is far better than that of other groups, which shows that the carnosine derivative and the vanillin derivative are compounded for use, the inhibition effect on the partial SASP factor expression in aged HaCaT cells is better, and the existence of the carnosine derivative has a synergistic effect on the SASP factor expression inhibition activity of the vanillin derivative.

FIG. 3 shows the effect of bioactive substances on the mRNA expression levels of SASP factors MMP-1 and MMP-3 in aged HFF-1 cells, and it can be analyzed from the figure that the carnosine derivatives prepared by the present invention in group K1 can significantly reduce the expression levels of MMP-1 and MMP-3, and the vanillin derivatives in group K4 also have a certain down-regulation effect on the expression levels of MMP-1 and MMP-3, but the vanillin in group K2 and the carnosine in group D1 obviously have no effect on the expression of SASP factors in aged HFF-1 cells, indicating that both the carnosine derivatives and the vanillin derivatives prepared by the present invention have the effect of inhibiting the expression of SASP factors in aged HFF-1 cells. In addition, the effect of group K3 is better than that of group K1, which shows that the compound of the derivative prepared by the invention and vanillin plays a synergistic role in carnosine derivatives, and can further enhance the inhibition effect on the SASP factor expression in aged HFF-1 cells. The effect of the K5 group is far better than that of other groups, which shows that the carnosine derivative and the vanillin derivative are compounded for use, and the inhibition effect on the SASP factor expression of part of the aging HFF-1 cell is better.

In conclusion, the carnosine derivative prepared by the invention not only can enhance the aging supervision function of macrophages, but also can directly remove aged skin cells, and simultaneously can effectively inhibit the expression of SASP factors, thereby having more excellent anti-aging effect. And the prepared vanillin derivative also has certain inhibitory activity on the expression of SASP factor.

Example 6: effect of carnosine derivatives on expression of the Pro-collagen C endopeptidase enhancer (PCOLCE)

Normal generation 8 human skin fibroblasts were cultured in Dulbecco's modified eagle's medium DMEM, 10% fetal bovine serum, 1% antibacterial-antifungal agent (antibacterial-antifungal, GIBCO, Cat. #15240-₂Culturing under the condition that when the coverage rate of human skin fibroblasts reaches more than 80%, the medium is replaced by DMEM without FBS at 4 × 10⁵The wells are inoculated into 96-well plates, a bioactive substance is added to the plates at a final concentration of 25 mM (no bioactive substance is added to the control), and the culture is continued for 6 h; the collected cells were washed with PBS solution, and mRNA was extracted and analyzed for marker expression by qPCR method. The details of the processing, grouping, and detection of the bioactive substances are described in example 3.

The test results are shown in fig. 4. From the analysis in the figure, it is known that the carnosine derivative prepared by the invention in the K1 group can obviously improve the mRNA expression level of PCOLCE compared with the carnosine in the D1 group, and the vanillin derivative in the K4 group and the vanillin derivative in the K2 group obviously have no influence on the mRNA expression of PCOLCE, which indicates that the carnosine derivative prepared by the invention can promote the expression of procollagen C endopeptidase enhancer. In addition, the effect of the group K3 is better than that of the group K1, which shows that the prepared carnosine derivative and vanillin are compounded for use, wherein the vanillin can play a synergistic effect on the carnosine derivative, and further enhances the activity of the carnosine derivative for promoting the expression of a procollagen C endopeptidase enhancer. The K5 group has better effect than other groups, which shows that the carnosine derivative and vanillin derivative are compounded for use, and the promotion effect on the expression of procollagen C endopeptidase enhancer is better.

Example 7: effect of carnosine derivatives on fibroblast contraction

Dermis equivalent model: constructed by transplantation of normal human fibroblasts in a collagen matrix (type I collagen, commercially available solution).

Dermal fibroblasts were isolated from human skin explants and cultured to passage 8 in DMEM medium containing 10% fetal bovine serum, 2 mM L-glutamine, 50U/mL penicillin and 50. mu.g/mL streptomycin.

Reference to Asselineau et al in Exp. Cell. Dermal equivalents were prepared as described by Res., 1985, 159, 536-539 and by Models in Dermatology, 1987, vol. 3, 1-7:

to MEM medium, 1% glutamine, 1% nonessential amino acid, 1% sodium pyruvate, 1% amphotericin B, 1% penicillin, 10% fetal calf serum, and 5% NaOH were added, and the mixture was introduced into a 50 mL centrifugal tube in an ice bath, and the mixture was centrifuged at 3X 10⁵Fibroblasts were added per mL. A mixture of collagen and acetic acid at a volume ratio of 1/1000 was slowly added along the tube wall and a whitish cloud was observed; carefully mixed and dispensed into 12-well plates at a ratio of 2 mL/well to give 6X 10⁴Dermal equivalent of individual cells/well. Plates were incubated at 37 ℃ with 5% CO₂Incubation under conditions to form collagen aggregates, 3 d detachment of the dermal equivalent from the support to initiate contraction, and image capture to measure surface area. Replacing the culture medium and addingThe bioactive substances were treated and cultured for 5 days, and images were taken. The surface area of the dermal equivalent was measured using NIS-Elements software (base version 3.10) and the percent shrinkage was calculated according to the following formula:

shrinkage% (% S)_b－S_t)/S_b×100

In the formula, S_bIs the surface area of the culture well in the culture plate, corresponding to the total surface area before the dermal equivalent is contracted; s_tThe surface area of the dermal equivalent at time t of the contraction kinetics.

Treatment of bioactive substances: the biologically active substance was added to MEM medium at a final concentration of 25 mM (control no biologically active substance added), and experimental groups: the carnosine derivative obtained in example 1 is added in group K1, vanillin is added in group K2, the carnosine derivative obtained in example 1 and vanillin are added in group K3 (the mass ratio of the carnosine derivative to vanillin is 1: 1), the vanillin derivative obtained in example 2 is added in group K4, the carnosine derivative obtained in example 1 and vanillin derivative obtained in example 2 are added in group K5 (the mass ratio of the carnosine derivative to vanillin is 1: 1); control group: carnosine was added in group D1 and no active substance was added in group D2.

The test results are shown in table 4:

TABLE 4 results of related shrinkage test of derivatives

Experimental group	Shrinkage/%
		K1	86.8
K2	100
		K3	78.4
K4	100
		K5	85.7
D1	99.3
		D2	100

As can be seen from the data in table 4, the contraction effect of the dermal equivalent treated by the carnosine derivative prepared by the invention in group K1 is better than that of group D1, which indicates that the carnosine derivative prepared by the invention has excellent effect of promoting dermal contraction, and the effect lasts at least for more than 5 days; the activity of carnosine derivatives in stimulating, repairing/regulating the metabolism of aged cells of the skin and preventing/treating the signs of aging skin is effectively demonstrated. In addition, the effect of the group K3 is better than that of the group K1, which shows that the combination of the carnosine derivative prepared by the invention and vanillin can play a role in synergy of the carnosine derivative, thereby further enhancing the effect of the vanillin derivative on promoting the dermis contraction.

Example 8: inhibition of tyrosinase by carnosine derivatives

Weighing 4 g of acetone powder, adding into 80 mL of PBS buffer solution (pH = 6.5), magnetically stirring and homogenizing at-2 ℃ for 45 min, centrifuging at-2 ℃ and 12500 r/min for 35 min, and taking supernatant to obtain the tyrosinase crude extract. The tyrosinase has maximum absorption wavelength of 425 nm when catechol is used as a substrate.

25 mM of the bioactive substance solution was prepared in PBS buffer (pH = 6.5), and 0.1 mL of the tyrosinase crude extract, 0.1 mL of the bioactive substance solution, and 2.8 mL of catechol (0.2M) were added to the cuvette in this order, and the OD value was immediately scanned at 425 nm (0.2 mL of double distilled water and 2.8 mL of catechol (0.2M) as a control). The inhibition rate of tyrosinase was calculated according to the following formula:

inhibition% = (A-B)/A × 100%

Wherein A is the OD value of the control group at 425 nm; b is the OD value at 425 nm of the bioactive substance treated group.

Experimental groups: the bioactive substance prepared in group K1 is carnosine derivative obtained in example 1, and group K2 is vanillin; the carnosine derivative obtained in example 1 and vanillin are taken as a K3 group (the mass ratio of the carnosine derivative to the vanillin is 1: 1), the vanillin derivative obtained in example 2 is taken as a K4 group, the carnosine derivative obtained in example 1 and the vanillin derivative obtained in example 2 are taken as a K5 group (the mass ratio of the carnosine derivative to the vanillin is 1: 1), and cordycepin is taken as a K6 group; control group: group D1 is carnosine.

The test results are shown in table 5:

TABLE 5 results of the related inhibition test of derivatives

Experimental group	Inhibition rate/%)
		K1	84.8
K2	4.6
		K3	90.9
K4	82.1
		K5	96.7
K6	60.5
		D1	84.3

As can be seen from the data in table 5, the inhibitory rate of the carnosine derivative prepared by the present invention on tyrosinase in the K1 group is not significantly different from that in the D1 group, indicating that the carnosine derivative prepared by the present invention has no significant inhibitory and enhancing effects on melanin. In addition, the effect of the group K3 is better than that of the group K1, which shows that the compounding of vanillin and carnosine derivatives can play a synergistic role and enhance the inhibiting effect of the carnosine derivatives on melanin. The inhibition rate of K4 group on tyrosinase is obviously higher than that of K2 group and K6 group, which shows that the vanillin derivative prepared by the invention can effectively enhance the inhibitory activity on melanin.

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 compound of formula I:

I；

II。

2. a process for the preparation of a compound of formula I as claimed in claim 1, comprising: the compounds are prepared by amide bond formation between the amino group of carnosine and the carboxylic acid group of carnosic acid methyl ester.