Retinyl oligopeptide derivative with anti-aging and wrinkle fading functions and application thereof

Abstract

The invention provides a retinyl oligopeptide derivative with anti-aging and wrinkle fading functions and application thereof, and belongs to the technical field of polypeptide derivative application, the retinyl oligopeptide derivative is obtained by connecting a carboxyl terminal of retinoic acid and an amino terminal of oligopeptide, and has a structure as shown in a formula I. In the formula I, n is 1 or 2; l is oligopeptide. The retinoic acid modified oligopeptide derivative provided by the invention has good solubility in both a water phase and an oil phase, is beneficial to product formula design in an application process, has good antioxidant activity and skin cell injury protection activity, can reduce MMP2 and TNF alpha expression increased by ROS in skin fibroblasts, and has a good application prospect. The composition has excellent activity of promoting collagen expression, and has good effects of resisting oxidation, repairing skin injury and smoothing wrinkles when being used for cosmetics or skin care products.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide derivative application technology, and in particular relates to a class of retinyl oligopeptide derivatives with anti-aging and wrinkle-reducing functions and their applications. Background Technology

[0002] Skin aging is a complex physiological process driven by both internal and external mechanisms, leading to multifaceted changes in skin structure and physiological function. Internal aging is primarily related to endogenous oxidative stress, programmed aging caused by cell damage, and cellular senescence. External aging is induced by environmental factors such as ultraviolet radiation and pollution, which promote the production of reactive oxygen species, resulting in DNA damage and cellular dysfunction. In aging skin, senescent cells accumulate, accelerating the degradation of the extracellular matrix, creating a vicious cycle that further exacerbates the aging process. Therefore, consumers have an urgent need for innovative anti-aging skincare products with more effective and safer ingredients.

[0003] Retinoic acid, specifically all-trans retinoic acid (ATRA), has had its anti-aging effects widely recognized since the 1980s. ATRA works by activating the retinoic acid receptor family (RAR). This family of receptors binds to retinoic acid response elements (RAREs) in DNA, regulating gene transcription. Specifically, it increases the expression of type I procollagen, inhibits the degradation of collagen by matrix metalloproteinases in the dermis, thereby reducing wrinkles. Simultaneously, it promotes epidermal proliferation and differentiation, compacts the stratum corneum, thickens the granular layer, and increases the deposition of glycosaminoglycans in the epidermis and dermis, improving skin texture. Retinol-based active ingredients (including retinol and retinal) need to be converted into ATRA in the body to exert their anti-aging effects. However, retinol and ATRA, as retinoids, suffer from photochemical instability, skin irritation, and potential toxicity, which greatly limits their application in cosmetics.

[0004] Peptides are another important active ingredient in anti-aging skincare formulations, primarily functioning to reduce wrinkles, improve hyperpigmentation, and stimulate collagen production. Due to their high activity and low irritation, peptides are gradually becoming a substitute for traditional anti-aging ingredients. However, peptides face significant difficulties in penetrating the stratum corneum, severely hindering their biological activity. This is mainly because peptides have high hydrophilicity and are highly sensitive to enzymatic degradation, thus requiring formulation technology to improve their stability in the skin and optimize their release pattern.

[0005] Both ATRA and peptides possess significant anti-skin aging capabilities, but their mechanisms of action differ, and each has its own advantages and disadvantages. From an efficacy perspective, combining the two can intervene in the skin aging process through a dual pathway, thus producing a synergistic effect of "1+1>2". However, simple physical mixing cannot solve the problems of ATRA's skin irritation and peptides' poor skin penetration. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a class of retinyl oligopeptide derivatives with anti-aging and wrinkle-reducing functions and their applications. The retinyl acid modified oligopeptide derivatives provided by the present invention have good solubility in both aqueous and oil phases, good antioxidant activity and skin cell damage protection activity, can reduce the expression of MMP2 and TNFα in skin fibroblasts induced by ROS, and have excellent collagen expression-promoting activity.

[0007] This invention provides a class of retinoic acid-modified oligopeptide derivatives having the structure shown in Formula I: Formula I; In formula I, n is 1 or 2; L stands for oligopeptide.

[0008] Preferably, L is β-alanyl-histidine, valine-tryptophan, arginyl-tyrosine, arginyl-alanine, lysyl-threonine, lysyl-serine, phenylalanyl-tryptophan, glycyl-histyl-lysine, alanyl-histyl-lysine, leucyl-histyl-lysine, isoleucyl-histyl-lysine, prolyl-leucyl-glycine, valine-tyrosyl-valine, lysyl-valine-valine, histyl-phenylalanyl-arginine, or cysteine. Any one of acyl-alanyl-serine, glycyl-glutamyl-lysyl-glycine, glycyl-glutamyl-prolyl-arginine, prolycyl-lysyl-glutamyl-lysine, lysyl-glycyl-histyl-lysine, lysyl-threonyl-threonyl-lysyl-serine, tyrosyl-glycyl-glycyl-phenylalanyl-methionine, glycyl-valine-glycyl-valine-proline, and valine-glycyl-valine-alanyl-prolyl-glycine.

[0009] Preferably, when n=1, retinoic acid is linked to the amino terminus of the oligopeptide via amidation at the carboxyl terminus.

[0010] Preferably, when n=2, the retinoic acid is linked to the amino terminus of the oligopeptide via a linker arm consisting of glyceric acid, lysine, serine, threonine, or ornithine.

[0011] Preferably, the C-terminus of the oligopeptide is any one of a free carboxyl group, a carboxyl group, an ester group, or an amide group.

[0012] Preferably, when the C-terminus of the oligopeptide is a carboxylate, the cation is any one of lithium, potassium, sodium, magnesium, zinc, calcium, copper, matrine, chitosan, arginine, lysine, triethanolamine, saturated straight-chain fatty amine, saturated branched-chain fatty amine, unsaturated straight-chain fatty amine, and unsaturated branched-chain fatty amine.

[0013] Preferably, when the C-terminus of the oligopeptide is an ester group, the alcohol forming the ester bond is any one of benzyl alcohol, phenethyl alcohol, menthol, saturated straight-chain fatty alcohol with a carbon chain length of 1-20, saturated branched-chain fatty alcohol with a carbon chain length of 1-20, unsaturated straight-chain fatty alcohol with a carbon chain length of 1-20, and unsaturated branched-chain fatty alcohol with a carbon chain length of 1-20.

[0014] Preferably, when the C-terminus of the oligopeptide is an amide group, the amine forming the amide bond is any one of benzylamine, phenethylamine, dimethylamine, diethylamine, a saturated straight-chain fatty amine with a carbon chain length of 1-20, a saturated branched-chain fatty amine with a carbon chain length of 1-20, an unsaturated straight-chain fatty amine with a carbon chain length of 1-20, or an unsaturated branched-chain fatty amine with a carbon chain length of 1-20.

[0015] This invention provides a method for preparing the aforementioned retinoic acid-modified oligopeptide derivative, comprising the following steps: 1) Prepare NH2-exposed polypeptide-resin using a polypeptide solid-phase synthesis method; 2) The NH2 naked polypeptide-resin prepared in step 1) is condensed with retinoic acid, and the resin is removed to obtain retinoic acid modified oligopeptide derivatives.

[0016] This invention provides the application of the aforementioned retinoic acid-modified oligopeptide derivative in the preparation of daily chemical products.

[0017] Preferably, the daily chemical products include skin care products and / or cosmetics, and the effects of the daily chemical products include anti-oxidation, anti-aging, and repair of skin damage.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a class of retinoic acid modified oligopeptide derivatives with the structure shown in Formula I; retinoic acid is linked to the amino terminus of the oligopeptide via a carboxyl terminus; the retinoic acid modified oligopeptide derivatives provided by the present invention have good solubility in both aqueous and oil phases, which is beneficial for product formulation design during application. They have good antioxidant activity and skin cell damage protection activity, can reduce the expression of MMP2 and TNFα in skin fibroblasts increased by ROS, and have excellent collagen expression-promoting activity. When used in cosmetics or skin care products, they have good antioxidant, skin damage repair, and wrinkle-reducing effects. Attached Figure Description

[0019] Figure 1This is a schematic diagram illustrating the mechanism of action of the compound; Figure 2 The preparation process of NH2-exposed peptide-resin is shown, with pink circles representing resin; Figure 3 The process involves condensing NH2-exposed peptides with ATRA resin and removing the resin to obtain the target compound. Detailed Implementation

[0020] This invention provides a class of retinoic acid-modified oligopeptide derivatives having the structure shown in Formula I: Formula I; in Formula I, n is 1 or 2; L is an oligopeptide.

[0021] In this invention, the structure shown in Formula I is obtained by linking retinoic acid to the amino terminus of an oligopeptide via a carboxyl terminus; the retinoic acid is all-trans retinoic acid or 9-cis retinoic acid.

[0022] The preferred L-oligopeptides are β-alanyl-histidine (βAH), valine-tryptophan (VW), arginyl-tyrosine (RY), arginyl-alanine (RA), lysyl-threonine (KT), lysyl-serine (KS), phenylalanyl-tryptophan (FW), glycyl-histyl-lysine (GHK), alanyl-histyl-lysine (AHK), leucyl-histyl-lysine (LHK), isoleucyl-histyl-lysine (IHK), prolyl-leucyl-glycine (PLG), valine-tyrosyl-valine (VYV), lysyl-valine-lysine (KVK), and histyl-phenylalanyl-arginine (HF). Any one of the following: R), cysteyl-alanyl-serine (CAS), glycyl-glutamyl-lysyl-glycine (GEKG), glycyl-glutamyl-prolyl-arginine (GQPR), prolycyl-lysyl-glutamyl-lysine (PKEK), lysyl-glycyl-histyl-lysine (KGHK), lysyl-threonyl-threonyl-lysyl-serine (KTTKS), tyrosyl-glycyl-glycyl-phenylalanyl-methionine (YGGFM), glycyl-valine-glycyl-valine-proline (GVGVP), and valine-glycyl-valine-alanyl-prolycine (VGVAPG).

[0023] Preferably, when n=1, retinoic acid is linked to the amino terminus of the oligopeptide via amidation at the carboxyl terminus.

[0024] When n=2, the retinoic acid is linked to the amino terminus of the oligopeptide by a linker of glyceric acid, lysine, serine, threonine or ornithine.

[0025] In this invention, the C-terminus of the oligopeptide is preferably any one of a free carboxyl group, a carboxyl salt, an ester group, or an amide group.

[0026] When the C-terminus of the oligopeptide is a carboxylate, the cation is preferably any one of lithium, potassium, sodium, magnesium, zinc, calcium, copper, matrine, chitosan, arginine, lysine, triethanolamine, saturated straight-chain fatty amine, saturated branched-chain fatty amine, unsaturated straight-chain fatty amine, and unsaturated branched-chain fatty amine.

[0027] When the C-terminus of the oligopeptide is an ester group, the alcohol that forms the ester bond is preferably any one of benzyl alcohol, phenethyl alcohol, menthol, saturated straight-chain fatty alcohol with a carbon chain length of 1-20, saturated branched-chain fatty alcohol with a carbon chain length of 1-20, unsaturated straight-chain fatty alcohol with a carbon chain length of 1-20, and unsaturated branched-chain fatty alcohol with a carbon chain length of 1-20.

[0028] When the C-terminus of the oligopeptide is an amide group, the amine forming the amide bond is preferably any one of benzylamine, phenethylamine, dimethylamine, diethylamine, a saturated straight-chain fatty amine with a carbon chain length of 1-20, a saturated branched-chain fatty amine with a carbon chain length of 1-20, an unsaturated straight-chain fatty amine with a carbon chain length of 1-20, or an unsaturated branched-chain fatty amine with a carbon chain length of 1-20.

[0029] This invention also provides a method for preparing the aforementioned retinoic acid-modified oligopeptide derivative, comprising the following steps: 1) Prepare NH2-exposed polypeptide-resin using a polypeptide solid-phase synthesis method; 2) The NH2 naked polypeptide-resin prepared in step 1) is condensed with ATRA, and the resin is removed to obtain retinoic acid modified oligopeptide derivatives.

[0030] In this invention, the process of step 1) is as follows: Figure 2 As shown, the preparation of NH2-exposed peptide-resin specifically involves two steps: a condensation reaction and a Fmoc protecting group removal reaction. In the presence of a condensing agent, the Fmoc-AA-OH group of the first amino acid at the carboxyl terminus of the target oligopeptide undergoes a condensation reaction with the NH2-exposed resin to obtain the first condensation product. The first condensation product undergoes a Fmoc protecting group removal reaction in the presence of an alkaline substance; then, in the presence of the condensing agent, it undergoes a condensation reaction with the Fmoc-AA-OH group of the second amino acid at the carboxyl terminus of the target oligopeptide to obtain the second condensation product. Alternatively, a resin containing one amino acid can be directly used to condense with the Fmoc-AA-OH group of the second amino acid at the carboxyl terminus of the target oligopeptide, and so on, to obtain... Figure 2 The NH2-exposed polypeptide-resin is shown.

[0031] In this invention, the condensing agent is preferably 1-hydroxybenzotriazole (HOBT) and benzotriazole-1-tetramethylhexafluorophosphate (HBTU), the resin is preferably Wang resin, the alkaline substance is preferably piperidine, and the molar ratio of Fmoc-AA-OH to HOBT, HBTU, and NH2 exposed resin is preferably from 1:1:1:0.1 to 5:1.5:1.5:1.

[0032] In this invention, the mass ratio of the exposed NH2 resin to the alkaline substance is preferably 1:1 to 1:50.

[0033] In this invention, the organic solvent used in the condensation reaction is preferably DMF; the temperature of the condensation reaction is preferably 0~35℃, and the time of the condensation reaction is preferably 0.5~4h.

[0034] In this invention, after the condensation reaction, the resulting condensation reaction solution is preferably post-treated. The post-treatment preferably includes the following steps: removing the condensation solvent by vacuum filtration, and then washing the resin with DMF, repeating 3 times.

[0035] In this invention, the organic solvent used in the Fmoc protecting group removal reaction is preferably DMF, and the DMF is mixed with an alkaline substance as a reagent for removing the Fmoc protecting agent; the ratio of DMF to alkaline substance is preferably 1:1 to 20:1.

[0036] In this invention, the preferred temperature for the Fmoc protecting group removal reaction is 0~35℃, and the preferred time is 0.01~4h. After the Fmoc protecting group removal reaction, the present invention preferably performs post-treatment on the obtained reaction solution, which preferably includes the following steps: removing the condensation solvent by vacuum filtration, and then washing the resin sequentially with DMF, dichloromethane (DCM), and DMF, each repeated 3 times.

[0037] In this invention, condensation and Fmoc protecting group removal reactions are performed sequentially according to the sequence of the target oligopeptide, ultimately yielding... Figure 2 The NH2-exposed polypeptide-resin is shown.

[0038] In this invention, after preparing NH2-exposed polypeptide-resin, the NH2-exposed polypeptide-resin is condensed with retinoic acid, and the resin is removed to obtain retinoic acid-modified oligopeptide derivatives.

[0039] In this invention, in the presence of a condensing agent, the NH2-exposed polypeptide-resin undergoes a condensation reaction with retinoic acid to obtain the final condensation product. In the presence of a cleavage solvent, the final condensation product undergoes an acid hydrolysis reaction to obtain… Figure 3 The structure shown is a retinoic acid-modified oligopeptide derivative.

[0040] In this invention, the molar ratio of retinoic acid to HOBT, HBTU, and NH2 naked polypeptide-resin is preferably from 1:1:1:0.1 to 1:1.5:1.5:1.

[0041] In this invention, the organic solvent, temperature, time and post-treatment used in obtaining the final condensation product are the same as those in step 1) above, and will not be repeated here.

[0042] In this invention, the cutting solvent is preferably trifluoroacetic acid (TFA), phenol, water, and sodium tris(1-methylethyl)naphthalenesulfonate (TIPS). In this invention, the mass ratio of the condensation product to TFA, phenol, water, and TIPS is preferably from 1:4.4:0.25:0.25:0.1 to 1:8.8:0.5:0.5:0.2. In this invention, the resin ablation reaction temperature is preferably 0-35°C, and the resin ablation reaction time is preferably 0.5-4 hours.

[0043] Following the resin excision reaction, the present invention preferably performs post-treatment on the resulting reaction solution. This post-treatment preferably includes the following steps: removing the resin by vacuum filtration, then concentrating under reduced pressure to remove the excision solvent, and washing the residue three times with diethyl ether to obtain a crude yellow target product. Further, the present invention dissolves the crude target product in a small amount of water and filters to remove insoluble matter. The pH of the aqueous solution is adjusted to between 7 and 8 with ammonia, and then extracted three times with ethyl acetate. The extracted aqueous solution is freeze-dried to obtain a pure retinoic acid-modified oligopeptide derivative.

[0044] This invention also provides the application of the above-mentioned retinoic acid-modified oligopeptide derivatives in the preparation of daily chemical products. In this invention, the daily chemical products include skincare and / or cosmetic products, and the efficacy of these products includes anti-oxidation, anti-aging, and skin damage repair. In this invention, the retinoic acid-modified oligopeptide derivatives possess excellent antioxidant activity and skin cell damage protection activity, can reduce the expression of MMP2 and TNFα in skin fibroblasts increased by ROS, and have excellent collagen-promoting activity. When used in cosmetics or skincare products, they exhibit good antioxidant, skin damage repair, and wrinkle-reducing effects.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] The specific steps for preparing retinoic acid-modified oligopeptide derivatives are as follows: 1.1 Preparation of retinyl-KT Weigh 2 g of Fmoc-Thr(tBu)-Wang resin (0.37 mmol / g) and transfer it to a 100 mL peptide solid-phase synthesis tube. Then add 30 mL of DMF and allow it to swell at room temperature for 2 h. After swelling, remove the DMF under reduced pressure, add 16 mL of a reagent for removing the Fmoc protecting agent (DMF:piperidine = 4:1, volume ratio) to the reaction tube, shake and react for 5 min. After removing the reaction solution under reduced pressure, add another 16 mL of the reagent for removing the Fmoc protecting agent and shake and react for 10 min. Then remove the reaction solution under reduced pressure, and wash the resin sequentially with DMF, DCM, and DMF, repeating each washing three times, with each washing using 30 mL of solvent. Weigh 0.56 g of HBTU, 0.2 g of HOBt, and 0.69 g of Fmoc-Lys(Boc)-OH and add them to the reaction tube, followed by 10 mL of DMF, and shake and react for 1 h. After 1 hour, the reaction solution was removed under reduced pressure. The resin was then washed sequentially with 15 mL of DMF, DCM, and DMF, repeating the washing process three times with each solvent. 16 mL of a reagent for removing the Fmoc protecting agent was added to the reaction tube again, and the mixture was shaken for 5 minutes. After removing the reaction solution under reduced pressure, another 16 mL of the Fmoc protecting agent was added, and the mixture was shaken for 10 minutes. The reaction solution was then removed under reduced pressure, and the resin was washed sequentially with 30 mL of DMF, DCM, and DMF, repeating the washing process three times with each solvent. 0.56 g of HBTU, 0.2 g of HOBt, and 0.45 g of ATRA were weighed and added to the reaction tube, followed by 10 mL of DMF. The mixture was shaken and reacted for 1 hour. After 1 hour, the reaction solution was removed under reduced pressure, and the resin was washed sequentially with 15 mL of DMF, DCM, and DMF, repeating the washing process three times with each solvent. The resin was then washed with 15 mL of methanol, repeating the washing process three times, and finally, the methanol was removed under reduced pressure. After condensation, the resin was transferred to a 50 mL flask, and 10 mL of a cleavage reagent (TFA: phenol: water: TIPS = 88:5:5:2 by mass) was added. The mixture was stirred in an ice bath for 2.5 h, then filtered to remove the resin, followed by removal of the cleavage reagent under reduced pressure. 10 mL of diethyl ether was added to the residue, and the mixture was ultrasonically dispersed and allowed to stand. The supernatant was discarded, and this process was repeated three times. The ether was then dried under reduced pressure to obtain a yellow solid, which was the crude target product. The crude target product was dissolved in 10 mL of ultrapure water, filtered to remove insoluble impurities, and the pH was adjusted to 7-8 with ammonia. The aqueous solution was extracted with 30 mL of ethyl acetate, repeated three times. The resulting aqueous solution was transferred to a 50 mL centrifuge tube and freeze-dried to obtain 0.333 g of the target compound (RA-KT), with a yield of 85.1% and an ESI of 0.333 g. MS (m / z): 530.41 [M+H] + .

[0048] 1.2 Preparation of retinyl-PR

[0049] Following the procedure described in Example 1.1, 2 g of Fmoc-Thr(tBu)-Wang resin and 0.69 g of Fmoc-Lys(Boc)-OH were replaced with 2 g of Fmoc-Arg(pbf)-Wang resin and 0.50 g of Fmoc-Pro-OH, respectively. The final yield was 0.387 g of the target compound (RA-PR), with a yield of 94.6% and an ESI of [missing value]. MS (m / z): 554.36 [M+H] + .

[0050] 1.3 Preparation of retinyl-GHK

[0051] Following the procedure described in Example 1.1, only the peptide sequence was altered. The final yield was 0.413 g of the target compound (RA-GHK), with a yield of 89.7% and an ESI of [missing value]. MS (m / z): 623.37 [M+H] + .

[0052] 1.4 Preparation of retinyl-KVK

[0053] Following the procedure described in Example 1.1, only the peptide sequence was altered. The final yield was 0.425 g of the target compound (RA-KVK), with a yield of 87.7% and an ESI of [missing value]. MS (m / z): 656.49 [M+H] + .

[0054] 1.5 Preparation of retinyl-HFR

[0055] Following the procedure described in Example 1.1, only the peptide sequence was altered. The final yield was 0.503 g of the target compound (RA-HFR), with a yield of 90.1% and ESI. MS (m / z): 741.40 [M+H] + .

[0056] 1.6 Preparation of retinyl-GEKG

[0057] Following the procedure described in Example 1.1, only the peptide sequence was modified. The final yield was 0.433 g of the target compound (RA-GEKG), with a yield of 87.2% and an ESI of [missing value]. MS (m / z): 672.39 [M+H] + .

[0058] 1.7 Preparation of retinyl-GPQR

[0059] Following the procedure described in Example 1.1, only the peptide sequence was modified. The final yield was 0.471 g of the target compound (RA-GPQR), with a yield of 86.2% and an ESI of [missing value]. MS (m / z): 739.42 [M+H] + .

[0060] 1.8 Preparation of retinyl-KTTKS

[0061] Following the procedure described in Example 1.1, only the peptide sequence was altered. The final yield was 0.473 g of the target compound (RA-KTTKS), with a yield of 74.4% and an ESI of [missing value]. MS (m / z): 846.49 [M+H] + .

[0062] 1.9 Preparation of retinyl-YGGFM

[0063] Following the procedure described in Example 1.1, only the peptide sequence was altered. The final yield was 0.442 g of the target compound (RA-YGGFM), with a yield of 68.7% and an ESI of [missing value]. MS (m / z): 856.42 [M+H] + .

[0064] 1.10 Preparation of retinyl-VGVAPG

[0065] Following the procedure described in Example 1.1, only the peptide sequence was altered. The final yield was 0.416 g of the target compound (RA-VGVAPG), with a yield of 72.1% and an ESI of [missing value]. MS (m / z): 781.51 [M+H] + .

[0066] 1.11 Retinyl-GHK·Na + Preparation

[0067] Weigh 0.12 g of retinyl-GHK, dissolve it in 2 mL of distilled water, add 8 mg of NaOH solid, stir well, and freeze-dry the aqueous solution to obtain the target compound with a yield of 100%.

[0068] 1.12 Preparation of retinyl-PR-OBzl

[0069] Weigh 0.11 g of retinyl-PR and dissolve it in 5 mL of DMF. Add 50 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 30 mg of HOBt sequentially. Stir at room temperature for 30 minutes, then add 0.2 mL of benzyl alcohol and stir overnight at room temperature. After the reaction is complete, add 50 mL of saturated NaHCO3 solution to the reaction mixture. After stirring evenly, a white solid precipitates. Filter and dry the filter cake to obtain 0.087 g of the target product, with a yield of 67.4%. (ESI) MS (m / z): 644.40 [M+H] + .

[0070] 1.13 Preparation of retinyl-PR-NH-Bn

[0071] Weigh 0.11 g of retinyl-PR and dissolve it in 5 mL of DMF. Add 50 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 30 mg of HOBt sequentially. Stir at room temperature for 30 minutes, then add 0.1 mL of benzylamine and stir overnight at room temperature. After the reaction is complete, add 50 mL of saturated NaHCO3 solution to the reaction mixture. After stirring evenly, a white solid precipitates. Filter and dry the filter cake to obtain 0.103 g of the target product, with a yield of 80.2%. (ESI) MS(m / z): 643.39[M+H]+.

[0072] 1.14 Preparation of retinyl-K (retinyl)-PR

[0073] 1.2 g of retinoic acid, 0.6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.7 g of HOSu were added sequentially to 30 mL of dichloromethane, and the mixture was stirred at room temperature. After 4 h, the reaction solution was extracted with 50 mL of saturated NaHCO3 solution, the organic phase was collected, and the dichloromethane was removed by vacuum distillation. The residue was dissolved in 20 mL of tetrahydrofuran. 0.28 g of lysine and 0.12 g of NaHCO3 were weighed and dissolved in 10 mL of distilled water. The aqueous solution was added to the above tetrahydrofuran solution, and the mixture was stirred at room temperature. After 1 h, the tetrahydrofuran in the solution was removed by vacuum concentration. 2 mL of saturated potassium hydrogen sulfate aqueous solution was added to the residual aqueous solution, and the aqueous solution was extracted with 50 mL of ethyl acetate. The ethyl acetate solution was collected and the residual water was removed with about 10 g of anhydrous sodium sulfate. After filtering to remove sodium sulfate, the ethyl acetate was removed by vacuum distillation to obtain 1.18 g of retinyl-K(retinoyl)-OH. Following the procedure described in Example 1.2, 1 g of retinoyl-K(retinoyl)-OH was weighed to replace 0.45 g of retinoic acid, ultimately yielding 0.47 g of the target compound RA-K(RA)-PR, with a yield of 69.2% and an ESI of 0.47 g. MS (m / z): 964.68 [M+H] + .

[0074] Test Example 1

[0075] ROS-induced cell damage protection assay (MTT assay)

[0076] Human keratinocytes (HaCaT): purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number: C6282, and used continuously in laboratory passages.

[0077] Human keratinocytes were cultured in a cell culture incubator at 37°C and 5% CO2. Cells passaged 3–10 times were used for subsequent cell viability assays. The culture medium was DMEM containing 10% FBS and 1% P / S.

[0078] When preparing the test sample, the test compound was prepared into a stock solution with a concentration of 10 mg / mL using DMSO. 10 μL of this stock solution was then added to 190 μL of culture medium (5 times the final concentration of the test compound solution), mixed thoroughly, and set aside. The IC50 of the compound against the cell line was then determined again using the MTT assay. 50 The value was determined and repeated at least three times under the same experimental conditions.

[0079] Normal control group: without TBHP treatment; negative control group: culture medium containing 1% DMSO; treatment group: retinoic acid modified oligopeptide derivative, retinoic acid (ATRA), and oligopeptide GHK alone; the final concentration of retinoic acid modified oligopeptide derivative was 100 ppm, the final concentrations of retinoic acid ATRA were 100 ppm, 10 ppm and 1 ppm, and the concentration of oligopeptide GHK alone was 100 ppm.

[0080] Cell viability = [(mean OD value of negative control group - mean OD value of test compound group) / mean OD value of negative control group - mean OD value of Blank group] × 100% Equation II.

[0081] The specific method was as follows: 24 hours after cell seeding, except for the normal control group, TBHP with a final concentration of 400 μM was added to all wells to induce cell damage through ROS generation. One hour after TBHP addition, all culture medium was removed, and then 200 μL of PBS was added to each well to wash away residual TBHP, for a total of three washes. After washing, all PBS was removed, and 100 μL of fresh culture medium was added to each well, followed by 25 μL of test sample solution at different concentrations. The OD values ​​of each group were then obtained according to the above method. The protective effects of different concentrations of the test compound against ROS-induced cell damage in each cell line are shown in Table 2.

[0082] Table 1. Protective effect of different concentrations of the tested compounds against ROS-induced cell damage in various cell lines (Mean ± SD)

[0083] As shown in Table 1, the target compound of this invention has a significant protective effect against reactive oxygen species-induced cell damage. At concentrations of 100 and 10 ppm, retinoic acid (ATRA) itself exhibits severe cytotoxicity, while the target compound of this invention did not exhibit ATRA cytotoxicity at a concentration of 100 ppm; instead, it promoted the proliferation of human keratinocytes. Meanwhile, the control oligopeptide GHK (i.e., tripeptide-1) showed no protective activity against cells at a concentration of 100 ppm, while the retinoic acid-modified oligopeptide derivative RA-GHK achieved a cell viability of 146.6 ± 6.2%, significantly higher than the control oligopeptide GHK. This suggests that the target compound has better safety and skin damage modification effects.

[0084] Test Example 2

[0085] Experiment on the effects of MMP2 and TNFα expression in human keratinocytes (HaCaT)

[0086] Human keratinocytes (HaCaT): purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number: C6282, and used continuously in laboratory passages.

[0087] Human immortalized fibroblasts (HSF): purchased from Xiamen Yimo Biotechnology Co., Ltd., product number: IM-H032, and continuously used in the laboratory.

[0088] Human keratinocytes (HaCaT) and human immortalized fibroblasts (HSF) were cultured in a cell culture incubator at 37°C and 5% CO2. Cells passaged 3–10 times could be used for subsequent cell viability assays. The culture medium was DMEM containing 10% FBS and 1% P / S.

[0089] When preparing the test sample, the test compound was prepared into a sample solution with a concentration of 10 mg / mL using DMSO.

[0090] Normal control group: not receiving TBHP stimulation; negative control group: culture medium containing 1% DMSO. Treatment group: retinoic acid modified oligopeptide derivative, retinoic acid (ATRA), and oligopeptide GHK alone; the final concentration of retinoic acid modified oligopeptide derivative was 100 ppm, the final concentration of retinoic acid ATRA was 1 ppm, and the final concentration of oligopeptide GHK alone was 100 ppm.

[0091] The specific method is as follows: 1) Cell seeding: Dilute cells in good growth condition and in the logarithmic growth phase with culture medium to 1×10⁻⁶. 6 Cells were seeded at a concentration of 2 cells / mL in 6-well plates, with 2 mL per well.

[0092] 2) ROS-induced inflammation: Observe cell growth and adhesion. When the adhesion rate reaches more than 80%, add 10 μL of TBHP solution (80mM) to each well, gently tap to disperse the sample solution evenly, and incubate in a cell culture incubator for 1 hour. Then remove the culture medium from the 6-well plate, wash 3 times with PBS at a volume of 2 mL per well, and then add fresh culture medium.

[0093] 3) Drug administration: Administer 20 μL of the test sample solution to each pre-set replicate well, gently tap to disperse the sample solution evenly, and incubate in a cell culture incubator for 6 h.

[0094] 4) Post-processing: Remove the culture medium from the 6-well plate, add 1 mL of TRIzol reagent to each well, gently pipette the bottom of the plate to lyse all cells, then transfer all solutions to 1.5 mL centrifuge tubes, extract total RNA according to the TRIzol reagent instructions (Beyotime, R0016), and determine the RNA concentration.

[0095] 5) cDNA synthesis: Using the BeyoRT II cDNA synthesis kit (with gDNA Eraser), follow the instructions in the manual. Add gDNA Eraser to 1 μg of RNA and incubate at 37°C for 2 minutes. Then add 4 μL of 5x buffer, 1 μL of M-mLV reverse transcriptase, and 2 μL of mixed primers. Add an appropriate amount of DEPC water to a final volume of 20 μL. Incubate at 2°C for 60 minutes for reverse transcription. Then incubate at 80°C for 10 minutes to inactivate the reverse transcriptase and place on ice.

[0096] 6) Real-time quantitative PCR analysis (qPCR): Using the cDNA from the above steps as a template, and employing BeyoFastSYBR Green qPCR Mix (2X) reagent (Beyotime, D7260), follow the instructions in the manufacturer's manual. Add 10 μL of BeyoFast™ SYBR Green qPCR Mix (2X), 2 μL of GAPDH, MMP2, or TNFα forward / reverse primer mixture (3 μM), and 6 μL of DEPC water to an 8-tube PCR apparatus. Gently pipette or lightly Vortex to mix. Centrifuge at room temperature for a few seconds to allow the liquid to accumulate at the bottom of the tube. Use the following PCR program: a. Pre-denaturation: 95℃ for 2 min; b. Denaturation: 95℃ for 15 seconds; c. Annealing / Extension: 60℃ for 15-30 seconds; d. Repeat steps b and c for a total of 40 cycles; e. Analyze the results using the software provided by the real-time PCR instrument.

[0097] The effect of the test compound on the expression levels of MMP2 and TNFα was calculated according to Formula III, and each experiment was repeated at least three times.

[0098] Gene expression rate = [2^-(Ct value of target gene in test compound group - Ct value of GAPDH in test compound group)] / [2^-(Ct value of target gene in negative control group - Ct value of GAPDH in negative control group)] Equation III.

[0099] The expression rates of MMP2 and TNFα are shown in Table 2.

[0100] Table 2. Relative expression rates of MMP2 and TNFα under different treatments

[0101] As shown in Table 2, the target compound of this invention can significantly downregulate the high expression of reactive oxygen species-induced matrix metalloproteinases (MMP2) and inflammatory factors (TNFα). Due to the cytotoxicity of ATRA, its maximum dosing concentration can only reach 1 ppm. At this concentration, ATRA can reduce the expression of MMP2 and TNFα, but its maximum efficacy is far lower than that of the target compound. The control oligopeptide GHK (i.e., tripeptide-1) did not reduce the activity of MMP2 and TNFα at a concentration of 100 ppm, indicating that oligopeptide treatment alone does not have anti-inflammatory and anti-wrinkle effects. After modifying the control oligopeptide GHK with retinoic acid, the expression of MMP2 and TNFα at a concentration of 100 ppm was much lower than that of retinoic acid treatment alone, suggesting that the target compound has superior anti-inflammatory and anti-wrinkle effects.

[0102] Test Example 3

[0103] Experiment on the effect of human immortalized fibroblasts (HSF) on collagen (COL1A1) production

[0104] Human immortalized fibroblasts (HSF) were cultured in a cell culture incubator at 37°C and 5% CO2. Cells passaged 3–10 times could be used for subsequent cell viability assays. The culture medium was DMEM containing 10% FBS and 1% P / S.

[0105] When preparing the test sample, the test compound was prepared into a sample solution with a concentration of 10 mg / mL using DMSO.

[0106] Positive control group: 10 ng / mL TGFβ; Negative control group: Culture medium containing 1% DMSO. Treatment group: Retinoic acid modified oligopeptide derivative, retinoic acid (ATRA), and control oligopeptide GHK alone; the final concentration of retinoic acid modified oligopeptide derivative was 100 ppm, the final concentration of retinoic acid ATRA was 1 ppm, and the final concentration of control oligopeptide GHK alone was 100 ppm.

[0107] The specific method is as follows: 1) Cell seeding: Dilute cells in good growth condition and in the logarithmic growth phase with culture medium to 1×10⁻⁶. 6 Cells were seeded at a concentration of 2 cells / mL in 6-well plates, with 2 mL per well.

[0108] 2) Drug administration: Observe cell growth and adhesion. When the adhesion rate reaches more than 80%, administer 20 μL of the test sample solution to each pre-set replicate well. Gently tap the well to disperse the sample solution evenly and incubate in a cell culture incubator for 48 hours.

[0109] 3) Post-processing: Collect the culture medium from the 6-well plate, centrifuge at 1,3000 g at 4°C for 10 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0110] 4) Using the Human Pro-Collagen I alpha 1 ELISA kit (catalog number: 97044ES96), the content of type I collagen secreted in the cell culture medium was detected according to the instructions in the kit.

[0111] The content of type I collagen secreted in the cell culture medium is shown in Table 3.

[0112] Table 3. Content of Type I collagen secreted in cell culture medium

[0113] Table 3 shows the total amount of collagen secreted into the culture medium by HSF cells 48 hours after compound treatment, as determined by enzyme-linked immunosorbent assay (ELISA). The results indicate that the target compound significantly increases collagen expression in human fibroblasts. Under the test conditions, the target compound's efficacy in promoting collagen expression was comparable to that of TGFβ at a concentration of 10 ng / mL, superior to ATRA at 1 ppm, and 3-4 times higher than the control oligopeptide GHK (tripeptide-1) at a concentration of 100 ppm. This suggests that the target compound has superior activity in inducing collagen expression.

[0114] Test Example 4: Solubility Detection of Retinoic Acid Modified Oligopeptide Derivatives

[0115] Take 1.5 mL centrifuge tubes and weigh 10 mg of retinoic acid-modified oligopeptide derivatives into each tube. Weigh out 6 portions of each sample. After labeling, divide the centrifuge tubes of each compound into two groups. Add 0.1 mL of n-octanol to each of the three centrifuge tubes in group 1, and add 0.1 mL of ultrapure water to each of the three centrifuge tubes in group 2. Vortex at room temperature for 3 min, and then centrifuge at 12000 rpm for 5 min at room temperature. After centrifugation, take the supernatant and dilute it with anhydrous ethanol at a ratio of 1:1000 (volume ratio). Then, measure the UV absorption intensity of the ethanol solution at 351 nm in different groups.

[0116] Standard curve preparation: Accurately weigh 50 mg of the target compound and add 1 mL of anhydrous ethanol. Vortex at room temperature to completely dissolve the target compound. Then, serially dilute the sample with anhydrous ethanol to obtain standard solutions with concentrations of 50 mg / mL, 10 mg / mL, 2 mg / mL, 0.4 mg / mL, 0.08 mg / mL, and 0.016 mg / mL. Perform linear regression between the UV absorbance of the standard solution at 292 nm and its concentration to obtain the standard curve equation y = ax + b, where y is the absorbance, a is the equation coefficient, x is the compound concentration, and b is the equation intercept.

[0117] Solubility = (OD of test group) 351 -b) / a Formula 1.

[0118] The solubility of the retinoic acid-modified oligopeptide derivatives prepared in Examples 1-10 in the lipid and aqueous phases was determined in this test example, as shown in Table 1.

[0119] Table 4. Solubility of retinoic acid-modified oligopeptide derivatives in lipid and aqueous phases.

[0120] As can be seen from Table 1, the retinoic acid-modified oligopeptide derivatives prepared in this invention have good solubility in n-octanol. Introducing benzyl ester groups at the ends of oligopeptides can further increase the solubility of the compounds in the lipid phase.

[0121] In summary, the retinoic acid-modified oligopeptide derivatives provided by this invention have good antioxidant activity and skin cell damage protection activity. They can reduce the expression of MMP2 and TNFα in skin fibroblasts caused by ROS and have excellent collagen expression-promoting activity, showing broad application prospects.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A class of retinoic acid-modified oligopeptide derivatives, characterized in that, It has the structure shown in Equation I: Formula I; In formula I, n is 1 or 2; L stands for oligopeptide.

2. The retinoic acid-modified oligopeptide derivative according to claim 1, characterized in that, L represents β-alanyl-histidine, valine-tryptophan, arginyl-tyrosine, arginyl-alanine, lysyl-threonine, lysyl-serine, phenylalanyl-tryptophan, glycyl-histyl-lysine, alanyl-histyl-lysine, leucyl-histyl-lysine, isoleucyl-histyl-lysine, prolyl-leucyl-glycine, valine-tyrosyl-valine, lysyl-valine-valine, histyl-phenylalanyl-arginine, cysteine- Any one of alanyl-serine, glycyl-glutamyl-lysyl-glycine, glycyl-glutamyl-prolyl-arginine, prolycyl-lysyl-glutamyl-lysine, lysyl-glycyl-histyl-lysine, lysyl-threonyl-threonyl-lysyl-serine, tyrosyl-glycyl-glycyl-phenylalanyl-methionine, glycyl-valine-glycyl-valine-proline, and valine-glycyl-valine-alanyl-prolyl-glycine.

3. The retinoic acid-modified oligopeptide derivative according to claim 1, characterized in that, When n=1, retinoic acid is linked to the amino terminus of the oligopeptide via amidation at the carboxyl terminus. When n=2, the retinoic acid is linked to the amino terminus of the oligopeptide by a linker of glyceric acid, lysine, serine, threonine or ornithine.

4. The retinoic acid-modified oligopeptide derivative according to claim 1, characterized in that, The C-terminus of the oligopeptide is any one of a free carboxyl group, a carboxyl group, an ester group, or an amide group.

5. The retinoic acid-modified oligopeptide derivative according to claim 4, characterized in that, When the C-terminus of the oligopeptide is a carboxylate, the cation is any one of lithium, potassium, sodium, magnesium, zinc, calcium, copper, matrine, chitosan, arginine, lysine, triethanolamine, saturated straight-chain fatty amine, saturated branched-chain fatty amine, unsaturated straight-chain fatty amine, and unsaturated branched-chain fatty amine.

6. The retinoic acid-modified oligopeptide derivative according to claim 4, characterized in that, When the C-terminus of the oligopeptide is an ester group, the alcohol forming the ester bond is any one of benzyl alcohol, phenethyl alcohol, menthol, saturated straight-chain fatty alcohol with a carbon chain length of 1-20, saturated branched-chain fatty alcohol with a carbon chain length of 1-20, unsaturated straight-chain fatty alcohol with a carbon chain length of 1-20, and unsaturated branched-chain fatty alcohol with a carbon chain length of 1-20.

7. The retinoic acid-modified oligopeptide derivative according to claim 4, characterized in that, When the C-terminus of the oligopeptide is an amide group, the amine forming the amide bond is any one of benzylamine, phenethylamine, dimethylamine, diethylamine, saturated straight-chain fatty amine with a carbon chain length of 1-20, saturated branched fatty amine with a carbon chain length of 1-20, unsaturated straight-chain fatty amine with a carbon chain length of 1-20, and unsaturated branched fatty amine with a carbon chain length of 1-20.

8. The method for preparing the retinoic acid-modified oligopeptide derivative according to any one of claims 1 to 7, characterized in that, Includes the following steps: 1) Prepare NH2-exposed polypeptide-resin using a polypeptide solid-phase synthesis method; 2) The NH2 naked polypeptide-resin prepared in step 1) is condensed with retinoic acid, and the resin is removed to obtain retinoic acid modified oligopeptide derivatives.

9. The use of the retinoic acid-modified oligopeptide derivative according to any one of claims 1 to 7 in the preparation of daily chemical products.

10. The application according to claim 9, characterized in that, The daily chemical products include skin care products and / or cosmetics, and the effects of the daily chemical products include anti-oxidation, anti-aging, and repair of skin damage.

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