Acetylferulic acid modified blue copper peptide derivative and application thereof

Abstract

The invention discloses an acetyl ferulic acid modified blue copper peptide derivative with the effects of repairing, removing wrinkles, resisting aging and the like and application of the acetyl ferulic acid modified blue copper peptide derivative. The structure of the acetyl ferulic acid modified blue copper peptide derivative is shown as a formula I: # imgabs0 #, and R1 is selected from # imgabs1 #; and R2 is selected from the group consisting of # imgabs2. Compared with prototype blue copper peptide, the acetylferulic acid modified blue copper peptide derivative disclosed by the invention has better effects of promoting cell proliferation and promoting collagen synthesis, has the effects of promoting barrier repair, promoting wound healing and reducing skin wrinkle degree, and is lower in effective concentration. The compound can be used as a main active component in the fields of medicines and daily chemical products.

Description

Technical Field

[0001] The invention belongs to the fields of chemistry, biotechnology and daily chemical products, and specifically relates to a class of blue copper peptide derivatives and applications thereof. Background Art

[0002] The skin is the largest organ in the human body. It not only protects the internal tissues and organs of the human body from the invasion of harmful external factors, but also plays a role in perception and metabolism. Whether for the need of skin health or the pursuit of beauty, skin care products should, to a certain extent, improve the integrity of skin structure and function, or delay skin problems caused by aging. Therefore, the research and development of new skin care active ingredients should meet consumers' demand for more efficient and safer skin care products.

[0003] In the past, the beauty peptides used in cosmetics were mainly small molecule oligopeptides composed of two to ten amino acids, which can promote cell growth and differentiation. Repair skin damage, have significant wrinkle removal and anti-aging effects, and have become an important component of functional cosmetics, and have extremely high safety of use. For example, blue copper peptide (GHK-Cu), as a bioactive regulatory factor, is believed to stimulate the metabolism of skin cells and promote the synthesis of collagen and elastin, which are the key components for maintaining skin elasticity and firmness.

[0004] Blue copper peptide is a compound of glycyl histidine tripeptide (GHK) and copper. It is named blue copper peptide because its aqueous solution is blue. The structure is as follows: Blue copper peptides are naturally present in human plasma, urine, saliva, and cerebrospinal fluid, and their content decreases with age. GHK is usually used as a complex with divalent copper ions to promote healing and repair. GHK can regulate the synthesis and decomposition of collagen and glycosaminoglycans, and regulate the activity of metalloproteinases and their inhibitors. Therefore, the application of blue copper peptides in cosmetics can inhibit the activity of matrix metalloproteinases and regulate the synthesis of collagen and elastin, thereby promoting the regeneration of skin epithelial tissue and increasing skin elasticity and toughness. However, the side effect of GHK-Cu on darkening the skin has become well known in the industry. "Study on the Effect and Mechanism of GHK-Cu on Regulating Melanocyte Melanin Synthesis" (Zhang Yunfei, Master's Thesis, Dalian Medical University, 2013) shows that GHK-Cu can increase tyrosinase activity and melanin content in melanocytes, and is dose-dependent within a certain concentration range.

[0005] At present, the market price of blue copper peptide is relatively high. Due to cost control, the amount added to cosmetics is generally low. Currently, researchers are committed to improving the cost-effectiveness of blue copper peptide, that is, reducing the effective concentration, or improving the product experience at the same addition cost. Summary of the invention

[0006] In view of the shortcomings of the prior art GHK-Cu that the skin turns darker and the low cost-effectiveness ratio, the present invention modifies the free amino groups in GHK that are not involved in the complexation with active small molecules with skin care effects, and develops a class of blue copper peptide derivatives, which have excellent abilities to promote cell growth, wound healing, collagen and elastin production, thereby achieving the anti-aging effect of smoothing skin fine lines and enhancing skin barrier function. The derivative also has a lower effective concentration, which can reduce the intake of copper ions and thus reduce the activation of tyrosinase, and reduce the risk of skin darkening caused by long-term use.

[0007] The specific technical solutions of the present invention are as follows: A blue copper peptide derivative, the structure of which is shown in Formula I: Wherein, R1 is selected from: R2 is selected from: C1~C20 alkyl group (preferably C1~C18 alkyl group, more preferably C1~C16 alkyl group) or -COCH2R3, R3 represents NH2 or C1~C20 alkyl group (preferably C1~C18 alkyl group, more preferably C14~C18 alkyl group).

[0008] Preferably, R2 is selected from: C1-C6 alkyl (more preferably methyl, ethyl, propyl, isopropyl) or C15-C18 alkyl (such as -C 15 H 31 , -C 16 H 33 , -C 17 H 35 , -C 18 H 37 ) or -COCH2R3, R3 represents NH2, -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , -C 17 H 35 or -C 18 H 37 .

[0009] Specifically, in the above derivatives, when R1 is selected from When the compound is a precursor compound for preparing a blue copper peptide derivative with biological activity, it can be reacted with copper acetate to generate a blue copper peptide derivative with biological activity (R1 is ).

[0010] In a specific embodiment of the present invention, the structure of the biologically active blue copper peptide derivative is as follows:

[0011] Another object of the present invention is to provide the use of the blue copper peptide derivative in the preparation of medicines or daily chemicals that promote the production of collagen or cell proliferation.

[0012] Preferably, the medicine or daily chemical product is a skin external product.

[0013] Furthermore, the daily chemical products are cosmetics or skin care products.

[0014] The content of the blue copper peptide derivative in daily chemical products is 0.01-1.0%wt.

[0015] Advantages of the present invention: 1. The present invention reacts the free amino group of blue copper peptide with acetylferulic acid, acetylsalicylic acid, tranexamic acid, glycine, propionic acid, palmitic acid, propionaldehyde, and hexadecanal. The obtained derivatives are tested for MMP-1 inhibition rate, Col-1 generation promotion, cell migration promotion and other activity tests, and have different degrees of improvement compared with the prototype peptide, achieving unexpected results.

[0016] 2. The blue copper peptide derivative of the present invention has a lower effective concentration than blue copper peptide. At the same addition amount, it has a higher effect of improving cell activity, promoting cell proliferation, promoting collagen synthesis, increasing skin elasticity, and improving skin firmness, and has anti-aging and repair effects.

[0017] 3. In contrast to the activation effect of blue copper peptide on tyrosinase, the blue copper peptide derivatives of the present invention have the effect of inhibiting tyrosinase activity, which can avoid the problem of skin darkening during the use of blue copper peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the blue copper peptide cell scratch test. The blank group refers to the blank control without any active ingredients; the EGF group refers to the addition of recombinant human epidermal growth factor as a positive control.

[0019] Figure 2 These are the results of cell scratch experiments on the tranexamic acid-blue copper peptide derivatives, acetylsalicylic acid-blue copper peptide derivatives, acetylferulic acid-blue copper peptide derivatives, and glycine-blue copper peptide derivatives described in the present invention.

[0020] Figure 3 These are the results of cell scratch experiments on the palmitic acid-blue copper peptide derivatives, n-hexadecyl-blue copper peptide derivatives, propyl-blue copper peptide derivatives, and propionic acid-blue copper peptide derivatives described in the present invention.

[0021] Figure 4This is the effect of the blue copper peptide derivatives of the present invention on the migration level of human keratinocytes. DETAILED DESCRIPTION

[0022] The specific steps of the present invention are described below by way of examples, but are not limited to the examples.

[0023] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0024] The present invention will be further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0025] In the following examples, various processes and methods not exhaustively described are conventional methods well known in the art.

[0026] Example 1: Preparation of blue copper peptide derivative intermediate M6

[0027] 1. Compound M2 (70.50 g, 113 mmol, 1.0 eq.) was placed in a 500 mL two-necked bottle, and 200 mL of dry DMF was added. The temperature was lowered to -10°C, and then HATU (47.28 g, 124 mmol, 1.1 eq.) and DIPEA (40.50 mL, 226 mmol, 2.0 eq.) were added in sequence, and the reaction was continued for 10 min. M1 (40.32 g, 118 mmol, 1.05 eq.) was dissolved in 160 mL of DMF and added dropwise to the reaction system, and the reaction was continued at -10°C for 1.5 h. The reaction was monitored by TLC (5% MeOH / DCM v / v%, Rf=0.5). The reaction solution was poured into 200 mL of ice water, and filtered after standing for 1 h. The filter cake was collected and re-dissolved in ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. Compound M3 (105.00 g, yellow viscous liquid, yield 99%) was obtained.

[0028] 2. Add M3 (105.00 g, 112 mmol, 1.0 eq.) to a 2.0 L three-necked round-bottom flask, add 1.4 L DMF to dissolve, under argon protection, cool to -10 ° C, add piperidine (50 mL, 481 mmol, 4.3 eq.), add DBU (10 mL, 67 mmol, 0.6 eq.) After the addition, react at -5 ° C for 15 min. Monitor the reaction by TLC (5% MeOH / DCM v / v%, Rf = 0.25). Pour the reaction solution into 2 L ice water, and extract the aqueous phase twice with EA 1.0 L / time. The organic phases were combined, washed twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and spin-dried to obtain a crude product, which was purified by column chromatography (stationary phase: 200-300 mesh silica gel, mobile phase: 50% EA / PE v / v% to elute impurities, 5% MeOH / DCM+0.1% triethylamine v / v% to elute the target product) to obtain compound M4 (63.68 g, yellow viscous liquid, yield 78%). Calcd.For [M+H] + =716.3812found:716.3812.

[0029] 3. Add Boc-glycine (18.54 g, 104 mmol, 1.2 eq.) into a 500 mL two-necked flask, add 200 mL of dry DMF, cool to -10°C, add HATU (40.03 g, 104 mmol, 1.2 eq.) and DIPEA (48 mL, 259 mmol, 3.0 eq.) in sequence, and react for 10 min. Dissolve M4 (62.31 g, 87 mmol, 1.0 eq.) in 160 mL of DMF and add dropwise to the reaction system. React at -10°C for 1.5 h, monitor the reaction by TLC (5% MeOH / DCM v / v%, Rf=0.5). Pour the reaction solution into 200 ml of ice water, let it stand for 1 h, and then filter. The filter cake was collected and redissolved with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated for later use. The crude product was purified by column chromatography (stationary phase: 200-300 mesh silica gel, mobile phase: 50% EA / PE v / v% to elute impurities, 2% MeOH / DCM+0.1% triethylamine v / v% to elute the target product) to obtain compound M5 (58.05 g, yellow foamy solid, yield 78%). Calcd.For [M+H] + =873.4551found:873.4551.

[0030] 4. Add M5 (15.44 g, 18 mmol, 1.0 eq.) to a 500 mL single-mouth round-bottom flask and dissolve it in 35 mL of methanol. Add 1.60 g of palladium carbon. Replace hydrogen three times at room temperature. Stir and react overnight at room temperature (25°C). Monitor the reaction by TLC (5% MeOH / DCM v / v%, Rf=0.2). Filter the reaction system, collect the filtrate, wash the filter cake with methanol several times; and combine the washings into the filtrate. After concentrating the filtrate under reduced pressure, a crude product is obtained. The crude product is purified by column chromatography (stationary phase: 200-300 mesh silica gel, mobile phase: 50% EA / PE v / v% to elute impurities, 5% MeOH / DCM+0.1% triethylamine v / v% to elute the target product) to obtain compound M6 (12.05 g, yellow foamy solid, yield 92%), Calcd.For [M+H] + =739.4183found:739.4191.

[0031] Example 2: Preparation of compound SL-PP-032:

[0032] 1. Add 20 mL of dry DMF to Boc-tranexamic acid (2.0 g, 2.84 mmol, 1.05 eq.) in a 100 mL double-necked bottle, cool to -10 °C, then add HATU (1.13 g, 2.98 mmol, 1.1 eq.) and DIPEA (700.0 mg, 5.41 mmol, 2.0 eq.) in sequence, and react for 10 min. Dissolve M6 (2.0 g, 2.71 mmol, 1.0 eq.) in 10 mL of DMF and add dropwise to the reaction system, react at -10 °C for 1.5 h, monitor the reaction by TLC (5% MeOH / DCM v / v%, Rf=0.5). Pour the reaction solution into 200 mL of ice water, let it stand for 1 h, and then filter. Collect the filter cake, redissolve it in ethyl acetate, dry it over anhydrous sodium sulfate, and concentrate it. Compound M7 (2.13 g, yellow foamy solid, yield 80%) was obtained.

[0033] 2. Under argon protection, M7 (2.13 g, 2.18 mmol, 1.0 eq) was dissolved in a 100 mL single-mouth bottle of TFA:EDT:Tis:H2O95:1:2:2 (v / v%) (10 mL). Stir for 2 h in an ice bath. The reaction was completed by mass spectrometry monitoring. The reaction was poured into iced ether, and a large amount of solid precipitated, which was filtered. After the solid was dissolved in pure water, it was freeze-dried to obtain the product M8 (1.02 g, light yellow powder, yield 97%). Calcd.For [M+H] + =480.2934found:480.2915.

[0034] 1 H NMR (500MHz, methanol-d4) δ8.79(d,J=1.4Hz,1H),7.39(s,1H),4.80(t,J=6.5Hz,1H),4.35(dd,J=9.3,4. 7Hz,1H),3.74(s,2H),3.27(dd,J=15.3,6.6Hz,1H),3.21–3.09(m,3H),2.79(d,J=7.0Hz,2H),2.16( tt,J=12.2,3.3Hz,1H),1.93–1.83(m,5H),1.72(dtd,J=14.2,9.2,5.3Hz,1H),1.62(th,J=10.5,3. 4Hz,1H),1.56–1.46(m,4H),1.40(dtt,J=20.2,9.3,5.4Hz,2H),1.07(qd,J=13.5,13.1,3.8Hz,2H).

[0035] 3. Under argon protection, M8 (1.02 g, 2.13 mmol, 1.0 eq.) was dissolved in a precisely prepared and weighed aqueous solution of copper acetate (709 μL, 2.13 mmol, 1.0 eq.), and stirred at room temperature for 30 min. The solution was filtered and freeze-dried to obtain the product SL-PP-032 (981.1 mg, blue powder, yield 84%).

[0036] Example 3: Preparation of compound SL-PP-033:

[0037] Referring to Example 2, acetylferulic acid was used as the raw material to obtain compound SL-PP-033 (1.16 g, blue-green powder, three-step yield 68%).

[0038] 1H NMR (500MHz, methanol-d4) δ8.79(s,1H),7.54(d,J=15.7Hz,1H),7.40(s,1H),7.27(d,J=2.0Hz,1H),7.18 (dd,J=8.2,1.9Hz,1H),7.06(dd,J=8.1,2.1Hz,1H),6.68(d,J=15.8Hz,1H),4.81(t,J=6.5Hz,1H),4 .39(dd,J=9.2,4.8Hz,1H),4.30(t,J=7.6Hz,2H),3.82(s,3H),3.75(s,2H),3.28(d,J=6.1Hz,1H), 3.18(dd,J=15.4,7.0Hz,1H),2.23(s,3H),2.00–1.89(m,1H),1.81–1.64(m,3H),1.57–1.43(m,2H).

[0039] Example 4: Preparation of compound SL-PP-031:

[0040] With reference to Example 2, acetylsalicylic acid was used as the raw material to obtain compound SL-PP-031 (921.1 mg, blue-green powder, three-step yield 60%).

[0041] 1 H NMR(500MHz, methanol-d4)δ8.84(s,1H),7.95(td,J=7.9,7.2,1.9Hz,1H),7.58–7.49( m,1H),7.44(s,1H),7.10–7.03(m,2H),4.86(t,J=6.5Hz,1H),4.44(dd,J=9.2,4 .8Hz,1H),3.75(s,2H),3.49(s,2H),3.33(d,J=6.1Hz,1H),3.22(dd,J=15.4,7. 0Hz,1H),2.88(s,3H),2.04–1.94(m,1H),1.85–1.68(m,3H),1.61–1.47(m,2H).

[0042] Example 5: Preparation of compound SL-PP-043:

[0043] Referring to Example 2, N-Boc glycine was used as the raw material to obtain compound SL-PP-043 (781 mg, blue-green powder, three-step yield 62%).

[0044] 1H NMR (500 MHz, methanol-d4) δ8.80 (s, 1H), 7.41 (s, 1H), 4.83 (t, J = 6.5 Hz, 1H), 4.38 (dd, J = 9.4, 4.6 Hz, 1H), 3.77 (s, 2H), 3.69 (s, 2H), 3.33 (d, J = 3.3 Hz, 1H), 3.24 (ddd, J = 31 .7,15.1,7.7Hz,3H),1.92(ddt,J=13.9,9.1,3.8Hz,1H),1.75(dtd,J=14.3,9.3,5 .2Hz,1H),1.57(dp,J=14.5,6.9Hz,2H),1.51–1.39(m,2H),1.33(t,J=7.5Hz,1H).

[0045] Example 6: Preparation of compound SL-PP-044:

[0046] Referring to Example 2, propionic acid was used as the raw material to obtain compound SL-PP-044 (843 mg, blue-green powder, three-step yield 67%).

[0047] 1 H NMR (500MHz, methanol-d4) δ8.79(s,1H),7.40(s,1H),4.81(t,J=6.5Hz,1H),4.37( dd,J=9.4,4.6Hz,1H),3.75(s,2H),3.32–3.25(m,1H),3.23–3.11(m,3H),2. 24–2.18(m,2H),1.96–1.87(m,1H),1.73(dtd,J=14.2,9.4,5.2Hz,1H),1.52 (tt, J = 15.0, 7.1 Hz, 2H), 1.41 (tt, J = 14.4, 6.4 Hz, 2H), 1.12 (t, J = 7.6 Hz, 3H).

[0048] Example 7: Preparation of compound SL-PP-045:

[0049] Referring to Example 2, palmitic acid was used as the raw material to obtain compound SL-PP-045 (864 mg, blue-green powder, three-step yield 50%).

[0050] 1H NMR (500MHz, methanol-d4) δ8.76(d,J=1.4Hz,1H),7.38(s,1H),4.81(t,J=6.5Hz,1H),4.34(dd,J=9.3,4.6Hz,1H),3.29–3.10(m,6H ), 2.17(t,J=7.6Hz,2H),1.98–1.85(m,1H),1.79–1.68(m,1H),1.64–1.55(m,2H),1.55–1.13(m,28H),0.90(t,J=6.9Hz,3H).

[0051] Example 8: Preparation of compound SL-PP-041:

[0052] 1. M6 (2.0 g, 2.71 mmol, 1.0 eq.) was placed in a 100 mL two-necked flask, and 27 mL of dry DCE was added. At room temperature, 0.5 mL of glacial acetic acid, n-propionaldehyde (157 mg, 2.71 mmol, 1.0 eq.) and sodium cyanoborohydride (187.1 mg, 2.98 mmol, 1.1 eq.) were added in sequence. The reaction was allowed to react for 1 h at room temperature. The reaction was monitored by TLC (5% MeOH / DCM v / v%, Rf=0.4). 20 mL of methanol was added to the reaction solution and stirred for 10 min. The reaction solution was concentrated to dryness. The residue was dissolved in 100 mL of EA and washed once with 100 mL of brine. After separation, the organic phase was dried over anhydrous sodium sulfate and concentrated. Compound M19 (1.86 g, yellow foamy solid, yield 88%) was obtained.

[0053] The subsequent steps were carried out with reference to Example 2, and finally compound SL-PP-041 (761 mg, blue-green powder, three-step yield 62%) was obtained.

[0054] 1 H NMR (500MHz, methanol-d4) δ9.00(s,1H),7.61(s,1H),5.04(d,J=6.5Hz,1H),4.62(dd,J=9.1,4.7Hz,1H),3.96(s,2H),3.53(s,3H),3.42 –3.28(m,2H),2.18(tt,J=9.4,5.1Hz,1H),2.04–1.89(m,5H),1.71(p,J=7.6Hz,2H),1.39(t,J=7.0Hz,1H),1.23(t,J=7.3Hz,3H).

[0055] Example 9: Preparation of compound SL-PP-042:

[0056] Referring to Example 9, the raw material was n-hexadecanal to obtain compound SL-PP-042 (942 mg, blue-green powder, three-step yield 55%).

[0057] 1 H NMR (500MHz, methanol-d4) δ8.78(s,1H),7.04(s,1H),4.86(t,J=6.5Hz,1H),4.64(dd,J=9.2,4.8Hz,1H),4.14(s,2H),3.33 (d,J=6.1Hz,2H),2.55–2.42(m,4H),2.04–1.94(m,2H),1.85–1.68(m,4H),1.34–1.25(m,28H),1.06(t,J=6.5Hz,3H).

[0058] Example 10: The inhibitory effect of the blue copper peptide derivatives of the present invention on MMP-1 and the effect of promoting the production of type I collagen. The present invention evaluates the anti-wrinkle activity of each blue copper peptide derivative based on the inhibitory activity of inhibiting matrix metalloproteinase MMP-1 and the amount of promoting the production of type I collagen.

[0059] 1. Effects of the derivatives of the present invention on the expression of matrix metalloproteinase MMP-1 in human foreskin fibroblasts (HFF-1).

[0060] Test method: After HFF-1 cells were revived, they were cultured in DMEM high-glucose medium containing 10% FBS, placed in a 37°C, 5% CO2 incubator, and passaged at a ratio of 1:3. HFF-1 cells in the logarithmic growth phase were digested, resuspended, and counted; 2×10 4 Cells were inoculated into 96-well plates, and DMEM high-glucose medium containing 10% FBS was added to culture overnight at 37°C and 5% CO2 to allow them to adhere to the wall; the old medium was discarded, and the cells were gently rinsed once with 200μL PBS; blank group, model group and test group were set up respectively; the blank group was replaced with 200μL 1% FBS medium at 37°C and 5% CO2 for 24h, and the model group and test group were replaced with 50μL PBS; the model group and test group were irradiated with 30mJ UVB in a UV crosslinker. PBS was discarded, 200μL 1% FBS medium was added to the model group, and 200μL of the test sample solution prepared with the above medium to a final concentration of 0.05mg / mL was added to each well of the test group, and cultured at 37°C and 5% CO2 for 24h; the cell supernatant was collected, and the expression of MMP-1 was detected using a human matrix metalloproteinase 1 (MMP-1) ELISA kit.

[0061] Calculation formula: MMP-1 expression inhibition rate = (UM) / U × 100%; Wherein: M-the average value of MMP-1 expression in the test sample; U-the average value of MMP-1 expression in the model group.

[0062] Experimental groups: Blank group: cultured in medium containing 1% FBS. Model group: cultured in 30mJ UVB+1% FBS medium. Test group: cultured in 30mJ UVB+test sample with a final concentration of 0.05mg / mL+1% FBS medium. For physically mixed test samples, the preparation ratio is to mix the two samples at a molar ratio of 1:1, and then prepare the test sample with a final concentration of 0.05mg / mL.

[0063] 2. The effect of the derivatives of the present invention on the production of human type I collagen in human foreskin fibroblasts (HFF-1).

[0064] Test method: After HFF-1 cells were revived, they were cultured in DMEM high-glucose medium containing 10% FBS, placed in a 37°C, 5% CO2 incubator, and passaged at a ratio of 1:3. HFF-1 cells in the logarithmic growth phase were digested, resuspended, and counted; 2×10 4 Cells were inoculated into 96-well plates, and DMEM high-glucose medium containing 10% FBS was added to culture overnight at 37°C and 5% CO2 to allow them to adhere to the wall; the old medium was discarded and the cells were gently rinsed once with 200μL PBS; blank group, model group and test group were set up respectively; the test group wells were added with medium containing the test sample at a final concentration of 0.05mg / mL, and the blank control wells were added with normal cell culture medium, 200μL per well. After the administration, the 96-well plate was placed in a CO2 incubator for 24h; after the incubation, 200μL of cell culture supernatant was collected from each well in a 1.5mL sterile centrifuge tube, and the type I collagen content was detected according to the instructions of the human type I collagen enzyme-linked immunosorbent assay kit; Calculation formula: Upward adjustment rate = (TC) / C×100%; Wherein: T-average value of type I collagen content in the test sample; C-average value of type I collagen content in the blank control.

[0065] Experimental groups: Blank group: normal cell culture medium culture. Test group: culture medium culture with a final concentration of 0.05 mg / mL test sample. For physically mixed test samples, the preparation ratio is to mix the two samples at a molar ratio of 1:1, and then prepare the test sample with a final concentration of 0.05 mg / mL.

[0066] Test results: See Table 1 and Table 2. As can be seen from Table 1 and Table 2, the blue copper peptide derivatives described in the present invention all have certain MMP-1 inhibitory activity and promote type I collagen production. Among them, the anti-wrinkle activity of tranexamic acid-blue copper peptide derivatives, acetylsalicylic acid-blue copper peptide derivatives, acetylferulic acid-blue copper peptide derivatives, glycine-blue copper peptide derivatives, palmitic acid-blue copper peptide derivatives and hexadecyl-blue copper peptide derivatives is significantly better than blue copper peptide, and more significantly better than active small molecules (tranexamic acid, acetylsalicylic acid, acetylferulic acid, glycine, palmitic acid), achieving unexpected results.

[0067] Table 1 Experimental Group M (ng / mL) Inhibition rate Blank Group 1.821489 / Model Group 2.325111 0.00％ Blue Copper Peptide 2.034665 12.49％ Tranexamic acid-blue copper peptide derivative 0.706575 69.61％ Blue Copper Peptide + Tranexamic Acid 2.094692 9.91％ Tranexamic acid 2.299911 1.08％ Acetylferulic acid-blue copper peptide derivative 1.478417 36.42％ Blue Copper Peptide + Acetylferulic Acid 2.588546 -11.33％ Acetylferulic acid 2.345946 -0.90％ Acetylsalicylic acid-blue copper peptide derivative 1.670639 28.15％ Blue Copper Peptide + Acetylsalicylic Acid 2.712707 -16.67％ Acetylsalicylic acid 2.473578 -6.39％ Glycine-blue copper peptide derivatives 1.719187 26.06％ Blue Copper Peptide + Glycine 1.887060 18.84％ Glycine 2.064466 11.21％ Propyl-blue copper peptide derivatives 1.998433 14.05％ Hexadecyl-blue copper peptide derivative 1.867994 19.66％ Propionic acid-blue copper peptide derivatives 2.122361 8.72％ Palmitic acid-blue copper peptide derivatives 1.831490 21.23％ Table 2 Experimental Group T(Col-I) (ng / mL) Increase rate Blank Group 12.001000 0.00％ Blue Copper Peptide 16.015306 33.45％ Tranexamic acid-blue copper peptide derivative 20.859752 73.82％ Blue Copper Peptide + Tranexamic Acid 14.698825 22.48％ Tranexamic acid 11.822185 -1.49％ Acetylferulic acid-blue copper peptide derivative 19.286166 60.70％ Blue Copper Peptide + Acetylferulic Acid 13.184299 9.86％ Acetylferulic acid 10.528477 -12.27％ Acetylsalicylic acid-blue copper peptide derivative 20.069441 67.23％ Blue Copper Peptide + Acetylsalicylic Acid 14.533211 21.10％ Acetylsalicylic acid 13.573131 13.10％ Glycine-blue copper peptide derivatives 18.216318 51.79％ Blue Copper Peptide + Glycine 14.481607 20.67％ Glycine 12.707859 5.89％ Propyl-blue copper peptide derivatives 15.307276 27.55％ Hexadecyl-blue copper peptide derivative 16.855405 40.45％ Propionic acid-blue copper peptide derivatives 14.384399 19.86％ Palmitic acid-blue copper peptide derivatives 17.726677 47.71％ Example 11: Evaluation of tyrosinase inhibitory activity of the blue copper peptide derivatives of the present invention Test subjects: primary human melanocytes.

[0068] Basic principle: Melanocytes are a special type of cell in the skin, located in the basal layer of the epidermis. Melanosomes in the cells can synthesize melanin and transfer it to the surrounding keratinocytes to provide color for the skin. Tyrosinase is an oxidase and the rate-limiting enzyme that regulates the production of melanin. This enzyme participates in two reactions in the synthesis of melanin: the first step is to hydroxylate monophenols into diphenols, and the second step is to oxidize o-diphenols into o-diquinones. After several steps of reaction, o-diquinones become melanin. Tyrosinase can be found in the melanosomes of skin melanocytes. Primary human melanocytes can be used to evaluate whitening efficacy. The whitening effect of the test product is evaluated by comparing the tyrosinase levels expressed by melanocytes with and without the test product.

[0069] Test method: (1) After the primary human melanocytes are revived, they are cultured in DMEM high-glucose medium containing 10% FBS, placed in a 37°C, 5% CO2 incubator, and passaged at a ratio of 1:3. (2) Primary human melanocytes in the logarithmic growth phase are digested, resuspended, and counted; (3) 2×10 4Each well was inoculated in a 96-well plate, and DMEM high-glucose medium containing 10% FBS was added to culture overnight at 37°C and 5% CO2 to allow it to adhere to the wall; (4) The old culture medium was discarded and the cells were gently rinsed once with 200 μL PBS; (5) A blank group and a test group were set up respectively; (6) The test group wells were added with a culture medium containing a final concentration of 0.2% (wt%) of the test sample, and the blank control wells were added with normal cell culture medium, 200 μL per well. After the administration, the 96-well plate was placed in a CO2 incubator and cultured for 24 hours; (7) After the incubation and culture, 200 μL of cell culture supernatant was collected from each well in a 1.5 mL sterile centrifuge tube, and the tyrosinase content was detected according to the instruction manual of the human tyrosinase enzyme-linked immunosorbent assay kit; (8) The tyrosinase inhibition rate was calculated.

[0070] Tyrosinase inhibition rate calculation formula Tyrosinase inhibition rate (%) = (CT) / C × 100%; Where: T—average value of tyrosinase content of the test sample; C—Average value of tyrosinase content in blank control Experimental grouping: Blank group: normal cell culture medium. Positive control group: culture medium with a final concentration of 0.2% phenylethyl resorcinol. Test group: culture medium with a final concentration of 0.2% test product. For physically mixed test products, the preparation ratio is to weigh and mix the two in a molar ratio of 1:1, and then prepare the test product with a final concentration of 0.2%. Three parallel wells are used for each group.

[0071] Test results: See Table 3. As can be seen from Table 3, blue copper peptide has a certain enhancing effect on the activity of tyrosinase, and the blue copper peptide derivatives described in the present invention all have certain tyrosinase inhibitory activity. Among them, the peptide derivatives modified with tranexamic acid, acetylsalicylic acid, acetylferulic acid and glycine have excellent tyrosinase inhibitory activity, which is significantly better than the active small molecules tranexamic acid, acetylsalicylic acid, acetylferulic acid and glycine. Among them, the inhibitory activity of the peptide derivatives of tranexamic acid, acetylsalicylic acid and acetylferulic acid is close to that of the positive control, and has potential whitening activity. Unexpected results have been achieved.

[0072] Table 3 Example 12: Repairing effect of the blue copper peptide derivatives of the present invention on the skin barrier Test subject: Human immortalized keratinocytes (HaCat).

[0073] Test method: (1) After HaCat cells are revived, they are cultured in DMEM high-glucose medium containing 10% FBS, placed in a 37°C, 5% CO2 incubator, and passaged at a ratio of 1:3. (2) HaCat cells in the logarithmic growth phase are digested, resuspended, and counted; (3) 6×10 5 Cells were inoculated into 12-well plates, and DMEM high-glucose medium containing 10% FBS was added and cultured overnight at 37°C and 5% CO2 to allow them to adhere to the wall; (4) After the cells are fully grown, a 200μL pipette tip (yellow pipette tip) was used to scratch the culture wells along the ruler perpendicular to the well plate, and a "cross" was scratched in each well. The cells were washed twice with PBS, and the scratched cells were washed away so that the gaps left were clearly visible to the naked eye; (5) A blank group, a positive control group, and a test group were set up respectively; (6) 1% FBS culture medium was added to the blank group, 1% FBS culture medium containing 0.001% EGF was added to the positive control group, and 1% FBS culture medium containing 0.001% (wt%) blue copper peptide derivative was added to the test group. Photos were taken under a microscope with a 4× objective lens and a 10× eyepiece, and recorded as 0h. Cultured in an incubator at 37°C and 5% CO2 for 24h. (7) After 24 hours, discard the old culture medium, wash the cells twice with PBS to remove dead cells, and take pictures under the microscope at 4× and 10× eyepieces and objectives for 24 hours. (8) Use ImageJ software to measure the width of the scratch area and calculate the cell migration rate.

[0074] Calculation formula: Cell migration rate (%) = (0h scratch width - 24h scratch width) / (0h scratch width) × 100% Experimental groups: Blank group: cultured in 1% FBS medium. Positive control group: cultured in 1% FBS medium containing 0.001% EGF. Test group: cultured in 1% FBS medium containing 0.001% sample.

[0075] Experimental results: see Figure 1 , Figure 2 and Figure 3 . Figure 1 , Figure 2 , Figure 3 Attached are photos of the cell scratch experiment, in which the blank group represents the addition of a blank control without any active ingredients; the EGF group represents the addition of a positive control EGF (EGF refers to recombinant human epidermal growth factor).

[0076] Conclusion: The cell layer uniformly grown in the plate wells was damaged by external force in a cross shape. The repair conditions before and after treatment with the blue copper peptide derivative of the present invention or the positive control were different, and both had different degrees of repair promotion effects. The quantitative data of cell migration level calculated by software were compared with the average scratch width before and after sample treatment. Figure 4 .Depend on Figure 4It can be seen that the peptide derivatives modified with tranexamic acid, acetylsalicylic acid, acetylferulic acid, glycine, palmitic acid and hexadecanal in the present invention have more excellent cell proliferation promoting activity and have achieved unexpected effects.

Claims

1. A blue copper peptide derivative modified with acetylferulic acid, characterized in that The structure is shown in Formula I: , wherein R1 is selected from: ; R2 is selected from .

2. Use of the acetylferulic acid-modified blue copper peptide as claimed in claim 1 in the preparation of anti-wrinkle or skin barrier repairing drugs or daily chemicals.

3. The use according to claim 2, characterized in that The medicine or daily chemical product is a skin external product.

4. The use according to claim 3, characterized in that The daily chemical products are cosmetics or skin care products.

5. The use according to claim 4, characterized in that The content of the blue copper peptide derivative in daily chemical products is 0.01%~1.0% wt.

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