Acetylferulic acid-modified blue copper peptide derivatives and use thereof

Abstract

The application discloses an acetyl ferulic acid modified blue copper peptide derivative with the effects of repairing, wrinkle removing, anti-aging and the like and application thereof. The structure of the acetyl ferulic acid modified blue copper peptide derivative is shown as formula I: R1 is selected from the group consisting of ; and R2 is selected from the group consisting of. The acetyl ferulic acid modified blue copper peptide derivative has the effects of promoting cell proliferation, promoting collagen synthesis, promoting barrier repair, promoting wound healing and reducing the degree of skin wrinkle, and has a lower effective concentration than the original blue copper peptide. The acetyl ferulic acid modified blue copper peptide derivative can be used as a main active ingredient in the fields of medicine and daily chemical products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemistry, biotechnology, daily chemical products, and specifically relates to a kind of blue copper peptide derivative and its application. BACKGROUND

[0002] Skin is the largest organ of the human body, not only protects the internal organs of the human body from the invasion of harmful factors from the outside world, but also plays a sensing function and metabolic function. Whether for the needs of skin health or the pursuit of aesthetics, skin care products should, to some extent, have the integrity of improving the structure and function of the skin, or delaying the skin problems caused by aging. Therefore, the development of new skin care active ingredients should meet the needs of consumers for more efficient and safer skin care products.

[0003] The cosmetic polypeptides used in the past cosmetics are mainly small molecule oligopeptides composed of two to ten amino acids, which can promote cell growth and differentiation. Repairing skin damage, it has significant wrinkle removal, anti-aging and other effects, has become an important component of functional cosmetics, and has extremely high safety in use. As a kind of biological active regulator, blue copper peptide (GHK-Cu) is considered to be able to stimulate the metabolism of skin cells and promote the synthesis of collagen and elastin, which are the key components to maintain skin elasticity and firmness.

[0004] Blue copper peptide is a compound of glycyl histidine tripeptide (Glycyl histidine tripeptide, GHK) combined with copper, named blue copper peptide / blue copper peptide because its aqueous solution is blue, and the structure is as follows: Blue copper peptide naturally exists in human plasma, urine, saliva and cerebrospinal fluid, and its content decreases with age. Usually GHK is in the form of a complex with 2-valent copper ions to play a role in promoting healing and repair. GHK can regulate the synthesis and decomposition of collagen and glycosaminoglycans, and regulate the activity of metalloproteinase and its inhibitor. Therefore, the application of blue copper peptide in cosmetics can inhibit the activity of matrix metalloproteinase, regulate the synthesis of collagen and elastin, so as to promote the regeneration of skin epithelial tissue and increase the elasticity and toughness of the skin. However, the side effect of GHK-Cu making the skin black has been known in the industry. The study of the effect of GHK-Cu on the synthesis of melanin in melanocytes and its mechanism (Zhang Yunfei, Master's thesis, Dalian Medical University, 2013) shows that GHK-Cu can increase the activity of tyrosinase in melanocytes and the content of melanin, and has a dose-dependent effect within a certain concentration range.

[0005] At present, the market price of blue copper peptide is high, and the addition amount in cosmetics is generally low due to cost control. At present, researchers are committed to improving the cost-effectiveness of blue copper peptide, that is, to reduce the effective concentration or to improve the product experience under the same addition cost. SUMMARY

[0006] This invention addresses the shortcomings of existing GHK-Cu technologies, such as skin darkening and low cost-effectiveness. It modifies the uncomplexed free amino groups in GHK with skin-care-enhancing active small molecules, developing a class of blue copper peptide derivatives. These derivatives exhibit excellent abilities to promote cell growth, wound healing, and collagen and elastin production, thereby achieving anti-aging effects such as smoothing fine lines and enhancing skin barrier function. Furthermore, these derivatives have a lower effective concentration, reducing copper ion intake and thus minimizing tyrosinase activation, lowering the risk of skin darkening with long-term use.

[0007] The specific technical solution of this invention is as follows:

[0008] A blue copper peptide derivative, the structure of which is shown in Formula I:

[0009] R1 is selected from: R2 is selected from: C1 to C20 alkyl groups (preferably C1 to C18 alkyl groups, more preferably C1 to C16 alkyl groups) or -COCH2R3, where R3 represents NH2 or C1 to C20 alkyl groups (preferably C1 to C18 alkyl groups, more preferably C14 to C18 alkyl groups).

[0010] Preferably, R2 is selected from: C1 to C6 alkyl groups (more preferably methyl, ethyl, propyl, isopropyl) or C15 to C18 alkyl groups (such as -C 15 H 31 -C 16 H 33 -C 17 H 35 -C 18 H 37 () or -COCH2R3, where R3 represents NH2, -C 14 H 29 -C 15 H 31 -C 16 H 33 -C 17 H 35 or -C 18 H 37 .

[0011] Specifically, for the above derivatives, when R1 is selected from... This compound serves as a precursor for the preparation of bioactive copper peptide derivatives. It can be reacted with copper acetate to generate bioactive copper peptide derivatives (R1 is...). ).

[0012] In a specific embodiment of the present invention, the structure of the bioactive blue copper peptide derivative is as follows:

[0013]

[0014]

[0015] Another object of the present invention is to provide the application of the aforementioned copper peptide derivative in the preparation of pharmaceuticals or daily chemical products that promote collagen production or cell proliferation.

[0016] Preferably, the drug or daily chemical product is a topical skin product.

[0017] Furthermore, the daily chemical products mentioned are cosmetics or skin care products.

[0018] The content of the blue copper peptide derivative described in this invention in daily chemical products is 0.01 to 1.0% wt.

[0019] Advantages of this invention:

[0020] 1. The present invention reacts the free amino group of blue copper peptide with acetylferuloyl acid, acetylsalicylic acid, tranexamic acid, glycine, propionic acid, palmitic acid, n-propionaldehyde, and n-hexadecaldehyde. The resulting derivatives showed varying degrees of improvement compared to the original peptide in MMP-1 inhibition rate test, Col-1 generation promotion test, and cell migration promotion test, achieving unexpected results.

[0021] 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 on improving cell activity, promoting cell proliferation, promoting collagen synthesis, increasing skin elasticity, and improving skin firmness, and has anti-aging and repair effects.

[0022] 3. In contrast to the activating effect of copper peptide on tyrosinase, the copper peptide derivatives described in this invention have the effect of inhibiting tyrosinase activity, thus avoiding the problem of skin darkening during the use of copper peptide. Attached Figure Description

[0023] Figure 1 These are the results of the blue copper peptide cell scratch assay. The blank group represents a control group containing no active ingredient; the EGF group represents a positive control group containing recombinant human epidermal growth factor.

[0024] Figure 2 The results of cell scratch assays for the tranexamic acid-copper peptide derivative, acetylsalicylic acid-copper peptide derivative, acetylsferulic acid-copper peptide derivative, and glycine-copper peptide derivative described in this invention are as follows.

[0025] Figure 3The results of cell scratch assays for the palmitic acid-copper peptide derivative, n-hexadecyl-copper peptide derivative, propyl-copper peptide derivative, and propionic acid-copper peptide derivative described in this invention are as follows.

[0026] Figure 4 This invention relates to the effect of the blue copper peptide derivative on the migration level of human keratinocytes. Detailed Implementation

[0027] The specific steps of the present invention are illustrated below through examples, but are not limited to these examples.

[0028] Unless otherwise stated, the terms used in this invention generally have the meanings commonly understood by those skilled in the art.

[0029] The present invention will now be described in further detail with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention in any way.

[0030] In the following embodiments, various processes and methods, not described in detail, are conventional methods known in the art.

[0031] Example 1: Preparation of intermediate M6, a blue copper peptide derivative

[0032]

[0033] 1. Compound M2 (70.50 g, 113 mmol, 1.0 eq.) was placed in a 500 mL double-necked flask, and 200 mL of dry DMF was added. The mixture was cooled 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 sequentially. The reaction was allowed to proceed 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 mixture. The reaction was allowed to proceed at -10 °C for 1.5 h. The reaction was monitored by TLC (5% MeOH / DCM v / v%, Rf = 0.5). The reaction mixture was poured into 200 mL of ice water, allowed to settle for 1 h, and then filtered. The filter cake was collected, redissolved in ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. Compound M3 (105.00 g, yellow viscous liquid, yield 99%) was obtained.

[0034] 2. Add M3 (105.00 g, 112 mmol, 1.0 eq.) to a 2.0 L three-necked round-bottom flask, dissolve and clarify with 1.4 L of DMF, and cool to -10 °C under argon protection. Add piperidine (50 mL, 481 mmol, 4.3 eq.) and DBU (10 mL, 67 mmol, 0.6 eq.). After the addition is complete, 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 of ice water, and extract the aqueous phase twice with EA 1.0 L each time. The organic phases were combined, washed twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a crude product. The crude product was purified by column chromatography (stationary phase: 200-300 mesh silica gel; mobile phase: 50% EA / PE v / v% for impurities, 5% MeOH / DCM + 0.1% triethylamine v / v% for the target product) to give compound M4 (63.68 g, yellow viscous liquid, yield 78%). Calcd. For [M+H] + =716.3812found:716.3812.

[0035] 3. Add Boc-glycine (18.54 g, 104 mmol, 1.2 eq.) to a 500 mL double-necked flask, add 200 mL of dry DMF, cool to -10 °C, and add HATU (40.03 g, 104 mmol, 1.2 eq.) and DIPEA (48 mL, 259 mmol, 3.0 eq.) sequentially. React for 10 min, then dissolve M4 (62.31 g, 87 mmol, 1.0 eq.) in 160 mL of DMF and add it 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 to settle, and then filter. The filter cake was collected, redissolved in ethyl acetate, dried with anhydrous sodium sulfate, and concentrated to dryness 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% for impurities, 2% MeOH / DCM + 0.1% triethylamine v / v% for the target product) to give compound M5 (58.05 g, yellow foamy solid, yield 78%). Calcd. For [M+H] + =873.4551found:873.4551.

[0036] 4. Add M5 (15.44 g, 18 mmol, 1.0 eq.) dissolved in 35 mL of methanol to a 500 mL single-necked round-bottom flask, and add 1.60 g of palladium on carbon. Replace with hydrogen three times at room temperature. Stir the reaction overnight at room temperature (25 °C). Monitor the reaction by TLC (5% MeOH / DCM v / v%, Rf = 0.2). Filter the reaction mixture, collect the filtrate, and wash the filter cake several times with methanol; combine the washings with the filtrate. Concentrate the filtrate under reduced pressure to obtain the crude product. Purify the crude product 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.

[0037] Example 2: Preparation of compound SL-PP-032:

[0038]

[0039] 1. Boc-tranexamic acid (2.0 g, 2.84 mmol, 1.05 eq.) was added to a 100 mL double-necked flask, along with 20 mL of dry DMF. The mixture was cooled to -10 °C, followed by the sequential addition of HATU (1.13 g, 2.98 mmol, 1.1 eq.) and DIPEA (700.0 mg, 5.41 mmol, 2.0 eq.), and the reaction was allowed to proceed for 10 min. M6 (2.0 g, 2.71 mmol, 1.0 eq.) was dissolved in 10 mL of DMF and added dropwise to the reaction mixture. The reaction was allowed to proceed at -10 °C for 1.5 h, and the reaction was monitored by TLC (5% MeOH / DCM v / v%, Rf = 0.5). The reaction mixture was poured into 200 mL of ice water, allowed to settle for 1 h, and then filtered. The filter cake was collected, redissolved in ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain compound M7 (2.13 g, yellow foamy solid, yield 80%).

[0040] 2. Under argon protection, M7 (2.13 g, 2.18 mmol, 1.0 eq) was dissolved in a 100 mL single-necked flask containing a pre-prepared TFA:EDT:Tis:H2O 95:1:2:2 (v / v%). The mixture was stirred in an ice bath for 2 h. The reaction was considered complete by mass spectrometry. The reaction mixture was poured into chilled diethyl ether, and a large amount of solid precipitated. The solid was filtered. After dissolving the solid in purified water, it was lyophilized to give product M8 (1.02 g, pale yellow powder, yield 97%). Calcd.For [M+H] + =480.2934found:480.2915.

[0041] 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).

[0042] 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 lyophilized to obtain the product SL-PP-032 (981.1 mg, blue powder, yield 84%).

[0043] Example 3: Preparation of compound SL-PP-033:

[0044]

[0045] 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%).

[0046] 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).

[0047] Example 4: Preparation of compound SL-PP-031:

[0048]

[0049] Referring 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%).

[0050] 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).

[0051] Example 5: Preparation of compound SL-PP-043:

[0052]

[0053] 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%).

[0054] 1 ¹H NMR (500MHz, methanol-d⁴) δ 8.80 (s, ¹H), 7.41 (s, ¹H), 4.83 (t, J = 6.5Hz, ¹H), 4.38 (dd, J = 9.4, 4.6Hz, ¹H), 3.77 (s, 2H), 3.69 (s, 2H), 3.33 (d, J = 3.3Hz, 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).

[0055] Example 6: Preparation of compound SL-PP-044:

[0056]

[0057] 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%).

[0058] 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=15.0,7.1Hz,2H), 1.41(tt,J=14.4,6.4Hz,2H), 1.12(t,J=7.6Hz,3H).

[0059] Example 7: Preparation of compound SL-PP-045:

[0060]

[0061] Referring to Example 2, palmitic acid was used as the raw material to obtain compound SL-PP-045 (864 mg, blue-green powder, 50% yield in three steps).

[0062] 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).

[0063] Example 8: Preparation of compound SL-PP-041:

[0064]

[0065] 1. M6 (2.0 g, 2.71 mmol, 1.0 eq.) was added to a 100 mL double-necked flask, along with 27 mL of dry DCE. At room temperature, 0.5 mL of glacial acetic acid, n-propanal (157 mg, 2.71 mmol, 1.0 eq.), and sodium cyanoborohydride (187.1 mg, 2.98 mmol, 1.1 eq.) were added sequentially. The reaction was allowed to proceed 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 the mixture was stirred for 10 min. The solution was then 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.

[0066] The subsequent steps were the same as in Example 2, and the final product was compound SL-PP-041 (761 mg, blue-green powder, three-step yield 62%).

[0067] 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).

[0068] Example 9: Preparation of compound SL-PP-042:

[0069]

[0070] Referring to Example 9, the raw material used was hexadecaldehyde, to obtain compound SL-PP-042 (942 mg, blue-green powder, three-step yield 55%).

[0071] 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).

[0072] Example 10: The inhibitory effect of the copper peptide derivatives of the present invention on MMP-1 and their effect on promoting type I collagen production. The present invention evaluates the anti-wrinkle activity of each copper peptide derivative by inhibiting the matrix metalloproteinase MMP-1 and promoting the production of type I collagen.

[0073] 1. Effect of the derivative of the present invention on the expression level of matrix metalloproteinase MMP-1 in human foreskin fibroblasts (HFF-1).

[0074] Test method: After resuscitation, HFF-1 cells were cultured in DMEM high-glucose medium containing 10% FBS and incubated at 37°C in a 5% CO2 incubator, and passaged at a ratio of 1:3. HFF-1 cells in the logarithmic growth phase were digested, resuspended, and counted; cells were then passaged at a ratio of 2 × 10⁻⁶ cells / year. 4 Cells were seeded per well in 96-well plates and cultured overnight at 37°C with 5% CO2 in DMEM high-glucose medium containing 10% FBS to allow adherence. The old medium was discarded, and the cells were gently washed once with 200 μL PBS. Three control, model, and test sample groups were set up. The control group was replaced with 200 μL 1% FBS medium and cultured at 37°C with 5% CO2 for 24 h. The model and test sample groups were replaced with 50 μL PBS. The model and test sample groups were exposed to 30 mJ UVB radiation in a UV crosslinker. The PBS was discarded. 200 μL of 1% FBS medium was added to each well of the model group, and 200 μL of the test sample solution prepared with the above medium to a final concentration of 0.05 mg / mL was added to each well of the test sample group. The cells were cultured at 37°C with 5% CO2 for 24 h. The cell supernatant was collected, and the expression level of MMP-1 was detected using a human matrix metalloproteinase 1 (MMP-1) ELISA kit.

[0075] Calculation formula: MMP-1 expression inhibition rate = (UM) / U × 100%;

[0076] Where: M - the average expression level of MMP-1 in the test sample; U - the average expression level of MMP-1 in the model group.

[0077] Experimental groups: Blank group: cultured in medium containing 1% FBS. Model group: cultured in medium containing 30mJ UVB + 1% FBS. Test sample group: cultured in medium containing 30mJ UVB + test sample with a final concentration of 0.05mg / mL + 1% FBS. For physically mixed test samples, the preparation ratio is as follows: weigh and 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.

[0078] 2. The effect of the derivative of this invention on the production of human type I collagen in human foreskin fibroblasts (HFF-1).

[0079] Test method: After resuscitation, HFF-1 cells were cultured in DMEM high-glucose medium containing 10% FBS and incubated at 37°C in a 5% CO2 incubator, and passaged at a ratio of 1:3. HFF-1 cells in the logarithmic growth phase were digested, resuspended, and counted; cells were then passaged at a ratio of 2 × 10⁻⁶ cells / year. 4 Cells were seeded per well in 96-well plates and incubated overnight at 37°C with 5% CO2 in DMEM high-glucose medium containing 10% FBS to allow adherence. The old medium was discarded, and the cells were gently washed once with 200 μL PBS. Three groups were set up: a control group, a model group, and a test sample group. 200 μL of culture medium containing 0.05 mg / mL of the test sample was added to the wells of the test sample group, while normal cell culture medium was added to the wells of the control group. After administration, the 96-well plates were incubated in a CO2 incubator for 24 h. After incubation, 200 μL of cell culture supernatant was collected from each well into a 1.5 mL sterile centrifuge tube, and the type I collagen content was measured according to the instructions of the Human Type I Collagen Enzyme-Linked Immunosorbent Assay Kit.

[0080] Calculation formula: Upward adjustment rate = (TC) / C × 100%;

[0081] Where: T - average type I collagen content of the test sample; C - average type I collagen content of the blank control.

[0082] Experimental groups: Blank group: cultured in normal cell culture medium. Test sample group: cultured in culture medium with a final concentration of 0.05 mg / mL test sample. For physically mixed test samples, the preparation ratio is 1:1 molar ratio of the two samples, weighed and mixed, and then prepared to a final concentration of 0.05 mg / mL.

[0083] Test results: See Tables 1 and 2. As shown in Tables 1 and 2, the copper peptide derivatives described in this invention all possess certain MMP-1 inhibitory activity and type I collagen-promoting effects. Among them, the anti-wrinkle activity of tranexamic acid-copper peptide derivatives, acetylsalicylic acid-copper peptide derivatives, acetylsulonic acid-copper peptide derivatives, glycine-copper peptide derivatives, palmitic acid-copper peptide derivatives, and n-hexadecyl-copper peptide derivatives is significantly better than that of copper peptides, and even more significantly better than that of active small molecules (tranexamic acid, acetylsalicylic acid, acetylsulonic acid, glycine, and palmitic acid), achieving unexpected results.

[0084] Table 1

[0085] Experimental group M (ng / mL) Inhibition rate Blank group 1.821489 / Model group 2.325111 0.00％ Blue copper peptide 2.034665 12.49％ Omeprazole-blue copper peptide derivative 0.706575 69.61％ Blue copper peptide + omeprazole 2.094692 9.91％ Omeprazole 2.299911 1.08％ Acetyl ferulic acid-blue copper peptide derivative 1.478417 36.42％ Blue copper peptide + acetyl ferulic acid 2.588546 -11.33％ Acetyl ferulic 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 derivative 1.719187 26.06％ Blue copper peptide + glycine 1.887060 18.84％ Glycine 2.064466 11.21％ Propyl-blue copper peptide derivative 1.998433 14.05％ N-hexadecyl-blue copper peptide derivative 1.867994 19.66％ Propionic acid-blue copper peptide derivative 2.122361 8.72％ Palmitic acid-blue copper peptide derivative 1.831490 21.23％

[0086] Table 2

[0087] Experimental group T (Col-I) (ng / mL) Up-regulation rate Blank group 12.001000 0.00％ Blue copper peptide 16.015306 33.45％ Omeprazole-blue copper peptide derivative 20.859752 73.82％ Blue copper peptide + omeprazole 14.698825 22.48％ Omeprazole 11.822185 -1.49％ Acetyl ferulic acid-blue copper peptide derivative 19.286166 60.70％ Blue copper peptide + acetyl ferulic acid 13.184299 9.86％ Acetyl ferulic 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 derivative 18.216318 51.79％ Blue copper peptide + glycine 14.481607 20.67％ Glycine 12.707859 5.89％ Propyl-blue copper peptide derivative 15.307276 27.55％ N-hexadecyl-blue copper peptide derivative 16.855405 40.45％ Propionic acid-blue copper peptide derivative 14.384399 19.86％ Palmitic acid-blue copper peptide derivative 17.726677 47.71％

[0088] Example 11: Evaluation of the tyrosinase inhibitory activity of the blue copper peptide derivative of the present invention

[0089] Test subject: Primary human melanocytes.

[0090] Basic Principle: Melanocytes are specialized cells in the skin, located in the basal layer of the epidermis. Melanosomes within these cells synthesize melanin, which is then transferred to surrounding keratinocytes to provide color to the skin. Tyrosinase is an oxidase and the rate-limiting enzyme regulating melanin production. This enzyme participates in two reactions in melanin synthesis: first, the hydroxylation of monophenols to diphenols; second, the oxidation of o-diphenols to o-diquinones. O-diquinones then undergo several further reactions to become melanin. Tyrosinase can be found in the melanosomes of skin melanocytes. Primary human melanocytes can be used to evaluate the skin-whitening efficacy. The skin-whitening effect of the test product is evaluated by comparing the levels of tyrosinase expressed in melanocytes with and without the test product.

[0091] Test methods: (1) After primary human melanocytes were revived, they were cultured in DMEM high-glucose medium containing 10% FBS and placed in a 37℃, 5% CO2 incubator, and passaged at a ratio of 1:3. (2) Primary human melanocytes in the logarithmic growth phase were digested, resuspended, and counted. (3) According to 2×10 4Cells were seeded per well in a 96-well plate, and cultured overnight at 37°C with 5% CO2 in DMEM high-glucose medium containing 10% FBS to allow them to adhere to the plate. (4) The old medium was discarded, and the cells were gently rinsed once with 200 μL PBS. (5) A blank group and a test sample group were set up separately. (6) 200 μL of medium containing 0.2% (wt%) of the test sample was added to the wells of the test sample group, and normal cell culture medium was added to the wells of the blank control group. After the drug administration was completed, the 96-well plate was placed in a CO2 incubator for 24 h. (7) After the incubation was completed, 200 μL of cell culture supernatant was collected from each well into a 1.5 mL sterile centrifuge tube, and the tyrosinase content was detected according to the instructions of the human tyrosinase ELISA kit. (8) The tyrosinase inhibition rate was calculated.

[0092] The formula for calculating the tyrosinase inhibition rate is: Tyrosinase inhibition rate (%) = (CT) / C × 100%.

[0093] In the formula:

[0094] T—average tyrosinase content of the test sample;

[0095] C—Negative tyrosinase content (blank control) Experimental groups: Blank group: cultured in normal cell culture medium. Positive control group: cultured in culture medium with a final concentration of 0.2% phenylethyl resorcinol. Test sample group: cultured in culture medium with a final concentration of 0.2% test sample. For physically mixed test samples, the preparation ratio is 1:1 molar ratio of the two components, weighed and mixed, and then prepared to a final concentration of 0.2%. Each group uses a three-well parallel experiment.

[0096] Test results: See Table 3. Table 3 shows that the copper peptide has a certain enhancing effect on tyrosinase activity, and all the copper peptide derivatives described in this invention have certain tyrosinase inhibitory activity. Among them, the peptide derivatives modified with tranexamic acid, acetylsalicylic acid, acetylsulonic acid, and glycine have superior tyrosinase inhibitory activity, significantly better than the active small molecules tranexamic acid, acetylsalicylic acid, acetylsulonic acid, and glycine. The inhibitory activity of the peptide derivatives of tranexamic acid, acetylsalicylic acid, and acetylsulonic acid is close to that of the positive control, indicating potential skin whitening activity. Unexpected results were achieved.

[0097] Table 3

[0098]

[0099] Example 12: The skin barrier repair effect of the blue copper peptide derivative described in this invention.

[0100] Test subject: Human immortalized keratinocytes (HaCat).

[0101] Test methods: (1) After HaCat cells were revived, they were cultured in DMEM high-glucose medium containing 10% FBS and placed in a 37℃, 5% CO2 incubator. They were passaged at a ratio of 1:3. (2) HaCat cells in the logarithmic growth phase were digested, resuspended, and counted. (3) They were passaged at a ratio of 6×10⁻⁶. 5 (3) Seed cells / well in 12-well plates, add DMEM high-glucose medium containing 10% FBS, and incubate overnight at 37°C with 5% CO2 to allow them to adhere to the walls; (4) After the cells have grown to confluence, use a 200μL pipette tip (yellow tip) to make a cross mark in each well along a ruler perpendicular to the plate. Wash the cells twice with PBS to remove the marked cells, making the gaps clearly visible to the naked eye; (5) Set up blank group, positive control group and test sample group respectively; (6) Add 1% FBS medium to the blank group, add 1% FBS medium containing 0.001% EGF to the positive control group, and add 1% FBS medium containing 0.001% (wt%) blue copper peptide derivative to the test sample group. Take pictures under a microscope with 4× objective lens and 10× eyepiece lens, and record it as 0h. Incubate at 37°C with 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 a microscope with 4× and 10× eyepieces and record the 24 hours. (8) Use ImageJ software to measure the width of the scratch area and calculate the cell migration rate.

[0102] Calculation formula: Cell migration rate (%) = (0h scratch width - 24h scratch width) / (0h scratch width) × 100%

[0103] Experimental groups: Blank group: cultured in medium containing 1% FBS. Positive control group: cultured in medium containing 0.001% EGF in 1% FBS. Test sample group: cultured in medium containing 0.001% sample in 1% FBS.

[0104] Experimental results: see Figure 1 , Figure 2 and Figure 3 . Figure 1 , Figure 2 , Figure 3 The attached images are of cell scratch assay results. The blank group represents a blank control without any active ingredients; the EGF group represents a positive control with EGF (EGF refers to recombinant human epidermal growth factor).

[0105] Conclusion: When a cross-shaped injury was caused by external force to a cell layer uniformly grown in the wells, the difference in repair before and after treatment with the blue copper peptide derivative described in this invention or the positive control was evident, both showing varying degrees of repair-promoting effects. The average scratch width before and after sample treatment, and the quantitative data on cell migration levels calculated by software, are shown below. Figure 4 .Depend onFigure 4 It is evident that the peptide derivatives modified with tranexamic acid, acetylsalicylic acid, acetylsuronic acid, glycine, palmitic acid, and hexadecaldehyde exhibit superior cell proliferation-promoting activity, achieving unexpected results.

Claims

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

2. The use of the acetylferulic acid-modified copper peptide derivative as described in claim 1 in the preparation of anti-wrinkle or skin barrier repair drugs or daily chemical products.

3. The application as described in claim 2, characterized in that... The medicine or daily chemical product mentioned is a topical skin product.

4. The application as described in claim 3, characterized in that... The daily chemical products mentioned are cosmetics or skin care products.

5. The application as described in 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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