Glutamic acid derivative, preparation method and application of glutamic acid derivative in skin care products

Abstract

The invention relates to the technical field of beauty skin care products, in particular to a glutamic acid derivative, a preparation method and application of the glutamic acid derivative in skin care products. The invention provides a compound with a structure as shown in a formula (I), which not only has rhythm protein up-regulation expression and oxidation resistance, but also has a good effect on up-regulation of aquaporin expression, silk protein expression and hypoxia inducible factor expression. The potential application prospects are realized on the effects of moisturizing the skin, repairing the barrier, promoting the ruddy skin and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetic skin care technology, in particular to N-acetyl-L-glutamine ethyl phenol, a preparation method and its application in skin care products. BACKGROUND

[0002] With the development of society, the improvement of living standards and the enrichment of material, the diversification of entertainment activities, the number of people who stay up late has also increased with the development of the economy, which has become a common phenomenon in society, and young people account for the majority of people who stay up late. However, it is undeniable that the potential harm to the body caused by staying up late is foreseeable. As the largest organ of the human body, the skin is naturally the first to suffer from the adverse effects of long-term staying up late. Staying up late can lead to circadian rhythm disorders, and specifically to the skin, long-term staying up late is accompanied by the disorder of skin cell rhythm regulation, and the disorder of body and skin rhythm brings a series of skin problems, including but not limited to oxidative stress, barrier damage, increased skin water loss, collagen loss, etc. In addition, on the skin surface, further to the facial skin, it shows the phenomena of dark circles, acne, increased acne, dull skin and increased skin wrinkles, etc.

[0003] Instantly changing the personal work and rest schedule is the main path to solve the adverse effects of staying up late on the body. However, in addition to long-term staying up late caused by personal living habits, staying up late due to work attributes may be difficult to adjust in a short period of time. Therefore, by applying external factors to alleviate the adverse effects of staying up late on the skin, especially the facial skin, is a realistic and effective way. Specifically, by developing an effective active ingredient, it is applied to the skin to alleviate a series of problems caused by staying up late in the skin and on the surface.

[0004] Patent CN 120081881 A discloses a glutamic acid derivative and its preparation method and application in rhythm gene regulation and antioxidant. The compound glutamine ethyl phenol disclosed in the patent has an up-regulating expression effect on Clock and Per3 rhythm proteins, and the up-regulating expression of rhythm proteins helps to correct the rhythm disorder of the skin and return it to the normal rhythm rhythm of the skin. At the same time, the glutamic acid derivative disclosed in the patent has a good inhibitory effect on cellular reactive oxygen species ROS, indicating that it has good antioxidant effect and helps to remove the oxidative factors produced in the skin cells of people who stay up late, and improves the "antioxidant" ability of the skin.

[0005] In addition to regulating and improving the "anti-oxidation" ability of skin cells, alleviating skin barrier damage and skin dullness caused by staying up late is also an urgent need for people with staying-up-late skin to improve and improve. For skin barrier repair, increasing the expression of filaggrin in keratinocytes is an important strategy. Filaggrin is an important component of the stratum corneum, synthesized by keratinocytes and distributed in different parts of the stratum corneum. Filaggrin plays a crucial role in maintaining the stability of the skin barrier. Filaggrin is an important molecule that connects keratin fibers in the stratum corneum. With the help of FLG monomers, keratin fibers are regularly aggregated to form a solid physical barrier in the outermost layer of the epidermis, which can prevent water loss from the epidermis and invasion of external allergens. In addition, after being degraded by enzymes, it forms small molecular substances such as natural moisturizing factors required by the stratum corneum, which play an important role in moisturizing and barrier integrity. As for the regulation of skin dullness, increasing the expression of hypoxia-inducible factor (HIF) in the skin is an effective solution. HIF-1α is an important transcription factor that plays a key role in regulating hemoglobin synthesis. It can sense changes in oxygen levels and regulate the expression of oxygen-related genes, including the synthesis of erythropoietin (EPO). Staying up late causes skin hypoxia, and in this state, HIF-1α is activated to promote the synthesis of EPO, which in turn promotes the formation of red blood cells, thereby increasing the concentration of hemoglobin in the blood. By further promoting the expression of HIF-1α through external regulatory factors, the concentration of hemoglobin in the skin capillaries can be better improved, thereby making the skin appear more rosy. SUMMARY

[0006] Therefore, the present application provides N-acetyl-L-glutamine ethyl phenol, a preparation method and its application in skin care products. The disclosed compound not only has the ability to up-regulate the expression of rhythm protein and antioxidant capacity, but also shows good effects in up-regulating the expression of water channel protein AQP3, filaggrin (FLG) and hypoxia-inducible factor (HIF-1α). It has potential application prospects in skin moisturizing, barrier repair and promoting skin redness.

[0007] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0008] The present application provides a glutamic acid derivative having the structure shown in formula I:

[0009] Formula (I).

[0010] The present application also provides a preparation method of the above-mentioned glutamic acid derivative, comprising:

[0011] The L-glutamic acid-5-tert-butyl ester and acetic acid-N-succinimidyl ester are subjected to an amidation reaction, the obtained intermediate is subjected to an amidation reaction with tyramine, and after de-tert-butyl ester and purification, a glutamic acid derivative shown in formula (I) is obtained.

[0012] In some embodiments of the present application, the amidation reaction of the above preparation method is added with a condensing agent and an acid-binding agent;

[0013] The condensing agent includes at least one of N-hydroxysuccinimide, 1-hydroxybenzimidazole, diisopropyl carbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide;

[0014] The acid-binding agent can be a basic compound that can be used as an acid-binding agent, which is well known to those skilled in the art, including but not limited to sodium bicarbonate.

[0015] In some embodiments of the present application, the solvent of the reaction of the above preparation method is preferably an aqueous acetonitrile solution.

[0016] In some embodiments of the present application, the reaction temperature of the above preparation method can be 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.

[0017] The present application also provides the use of the above glutamic acid derivative in the preparation of a product for correcting skin rhythm disorder, skin moisturizing, skin barrier repair, and / or promoting skin redness.

[0018] In some embodiments of the present application, the product of the above use includes a skin care product or a cosmetic product.

[0019] In some embodiments of the present application, the skin rhythm disorder of the above use is caused by staying up late.

[0020] The present application also provides a skin care product, which includes the above glutamic acid derivative.

[0021] In some embodiments of the present application, the form of the above skin care product is an aqueous agent, a gel, an emulsion, a cream agent, a film agent, a suspension agent, a spray agent, or a powder agent.

[0022] The present application also provides a cosmetic product, which includes the above glutamic acid derivative.

[0023] In some embodiments of the present application, the form of the above cosmetic product is an aqueous agent, a gel, an emulsion, a cream agent, a film agent, a suspension agent, a spray agent, or a powder agent.

[0024] The present application also provides a skin care method, comprising: administering the above glutamic acid derivative or the above skin care product to a subject.

[0025] The present application also provides a cosmetic method, comprising: administering the above glutamic acid derivative or the above cosmetic product to a subject.

[0026] The present application has the following beneficial effects:

[0027] The application discloses N-acetyl-L-glutamine ethyl phenol and a preparation method and application thereof, and has the following differences compared with a molecular structure protected in patent CN 120081881 A: acetylation is performed on an amino group of L-glutamine ethyl phenol, and through the acetylation, the disclosed compound has good effects on up-regulating expressions of water channel protein AQP3, filaggrin (FLG), and hypoxia-inducible factor (HIF-1 alpha), and has potential application prospects in skin moisturizing, barrier repair and promotion of skin redness. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0029] Figure 1 Results of Clock protein immunofluorescence experiments are shown;

[0030] Figure 2 Results of Per3 protein immunofluorescence experiments are shown;

[0031] Figure 3 Results of ROS inhibition experiments are shown;

[0032] Figure 4 Results of AQP3 protein expression experiments are shown;

[0033] Figure 5 Results of filaggrin (FLG) expression regulation experiments are shown;

[0034] Figure 6 Results of hypoxia-inducible factor (HIF-1 alpha) expression regulation experiments are shown. DETAILED DESCRIPTION

[0035] This invention discloses N-acetyl-L-glutamine ethylphenol, its preparation method, and its application in skincare products. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0036] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0037] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0038] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0039] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0040] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0041] In one embodiment, the glutamic acid derivative has the structure shown in formula (I):

[0042]

[0043] Formula (I)

[0044] In one embodiment, a method for preparing a compound of formula (I) includes:

[0045] Step 1: Weigh L-glutamic acid-5-tert-butyl ester (CAS: 2419-56-9) and N-acetic acid-N-succinimide ester (CAS: 14464-29-0) and disperse them in purified water. Add sodium bicarbonate and stir the reaction for 8-12 h. Add an acid to acidify the reaction solution, precipitate a solid, filter it, wash the filter cake with purified water, and dry it under vacuum to obtain intermediate 1.

[0046] In one example, the preferred acid is citric acid or hydrochloric acid;

[0047] Step 2: Weigh intermediate 1, tyramine, and sodium bicarbonate and disperse them in a mixed solution of acetonitrile and water. Then add an amide condensing agent and stir the mixture at room temperature for 6-8 hours. After the reaction is complete, extract the reaction solution with ethyl acetate, collect the organic phase, and distill it under reduced pressure to obtain intermediate 2.

[0048] In one example, the amide condensing agent may be one or more combinations of N-hydroxysuccinimide (NHS), 1-hydroxybenzimidazole (HOBT), diisopropylcarbodiimide (DIC), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and their hydrochlorides;

[0049] Step 3: Disperse intermediate 2 obtained in step 2 in trifluoroacetic acid solution and stir the reaction at room temperature for 4-6 h.

[0050] After the reaction was complete, trifluoroacetic acid was distilled off under reduced pressure. The resulting viscous solid was then dissolved in 50 mL of aqueous solution and extracted with 20 mL of ethyl acetate. The aqueous phase was collected, and 1 g of activated carbon was added to the aqueous solution and stirred for 30 min. The solution was then filtered and the filtrate was freeze-dried to obtain the target compound N-acetyl-L-glutamine ethylphenol.

[0051] In terms of efficacy, the compounds disclosed in this invention can upregulate the expression of Clock and Per3 proteins, regulate skin rhythm, inhibit cellular ROS, and exhibit antioxidant effects. In addition, in terms of skin barrier repair, they can upregulate the expression of repair factor filaggrin and hypoxia-inducible factor (HIF-1α), and the effects are superior to those of the glutamine-ethylphenol structure disclosed in patent CN 120081881 A.

[0052] In the example, "w" refers to "ten thousand", that is, 10. 4 "w / c" refers to "×10" 4 "Number / hole".

[0053] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0054] The present invention will be further illustrated below with reference to the embodiments.

[0055] Example 1: Preparation of N-acetyl-L-glutamine ethylphenol

[0056] Step 1: Weigh 10 mmol of L-glutamic acid-5-tert-butyl ester (CAS: 2419-56-9) and 12 mmol of acetate-N-succinimide ester (CAS: 14464-29-0) and disperse them in purified water. Add 15 mmol of sodium bicarbonate and stir the reaction for 8-12 h. Then add an acid to acidify the reaction solution, precipitate a solid, filter it, wash the filter cake with purified water, and dry it under vacuum to obtain intermediate 1.

[0057] Step 2: Weigh 1 mmol of intermediate 1 and 1.2-1.5 mmol of tyramine and dissolve them in 50 mL of acetonitrile aqueous solution. Stir at room temperature, then add 1.5 mmol of NHS and 2 mmol of sodium bicarbonate. Stir well and then add 2 mmol of EDCI (1-ethyl-3(3-dimethylpropylamine)carbodiimide). Monitor the reaction by TLC. After the conversion is complete, distill off most of the acetonitrile under reduced pressure, then extract with ethyl acetate and aqueous solution. Collect the organic phase, dry it with anhydrous sodium sulfate, and distill under reduced pressure to obtain intermediate 2.

[0058] Step 3: Dissolve intermediate 2 in 10 mL of trifluoroacetic acid solution, stir at room temperature for 4–6 h, remove trifluoroacetic acid by vacuum distillation, then dissolve the resulting viscous solid in 50 mL of aqueous solution, extract with 20 mL of ethyl acetate, collect the aqueous phase, add 1 g of activated carbon to the resulting aqueous solution, stir for 30 min, filter, and freeze-dry the filtrate to obtain the target compound N-acetyl-L-glutamine ethylphenol. Appearance: Yellowish-white solid powder, yield 51%. 1 H NMR (400MHz, DMSO-d6) δ 9.17 (s, 1H), 8.01 – 7.76 (m, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.62 (d, J = 8.5 Hz, 2H), 4.24 – 4.07 (m, 1H), 3.24 – 3.04 (m, 2H), 2.53 (t,J = 7.3 Hz, 2H), 2.14 (t, J = 7.8 Hz, 2H), 1.80 (s, 4H), 1.62 (d, J = 14.8Hz, 1H).

[0059] Example 2: Preparation of N-acetyl-L-glutamine ethylphenol

[0060] The desired compound was prepared according to the method described in Example 1, but the amide condensing agent in step 2 was replaced with HOBT instead of NHS, while other conditions remained unchanged. Yield: 57%.

[0061] Comparative Example 1: L-Glutamylethylphenol

[0062] Prepared according to the method disclosed in patent CN 120081881 A.

[0063] Example of effect 1

[0064] This example illustrates the effect of N-acetyl-L-glutamine ethylphenol (prepared according to Example 1) on the upregulation of the expression of the rhythm proteins Clock and Per3.

[0065] Experimental principle: By simulating the decrease in Clock or Per3 protein expression in the human body after exposure to blue light, and then restoring gene expression using a regulator sample, the effect of the regulator sample on the CLOCK gene / Per3 protein can be analyzed.

[0066] Brief testing method:

[0067] A. Cell seeding: Adjust the density of the keratinocyte (Hacat) suspension to 1.5 × 10⁻⁶. 4 After inoculating each well with 100 μL of the solution, the cells were seeded into 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h.

[0068] B. Blue light irradiation: Blue light was used for modeling. Specifically, the blue light intensity was adjusted to 90 irradiance and irradiated at a height of 3 cm from the 96-well plate for 45 minutes to obtain the cell model after blue light irradiation.

[0069] C. Raw material efficacy test: Add the sample of the regulator to be tested, 100 μL / well, to the blue light irradiated cell model and place it in a 37℃, 5% CO2 incubator for static repair for 24 h.

[0070] D. Cell immunofluorescence staining: After sample collection, cell fixation, permeabilization, one wash, blocking, primary antibody, secondary antibody, two washes, and image acquisition, fluorescence images of different samples were obtained.

[0071] E. Image Processing: The fluorescence images were analyzed using ImageJ's average fluorescence intensity.

[0072] F. Statistical Analysis: GraphPad Prism 8.0 software was used for data statistical analysis and graph plotting. Quantitative data are expressed as x±s. One-way ANOVA analysis was used to analyze differences between groups, and P<0.05 was considered statistically significant.

[0073] Formula for calculating the relative growth rate of CLOCK / Per3 protein expression:

[0074] C%=(Cx-C0) / C0*100%;

[0075] in:

[0076] C represents the relative growth rate of CLOCK / Per3 protein expression;

[0077] Cx represents the histone expression value of the sample after cell assay;

[0078] C0 represents the gene expression value in the positive group (Blue Light, 45 min) after the cell experiment.

[0079] Table 1

[0080]

[0081] *The test concentration (%) is a mass-volume percentage concentration with the numerator in grams and the denominator in milliliters. For example, 0.05% means that each 100 mL of liquid contains 0.05 grams of solute / solid. The same applies below.

[0082] From Table 1, Figure 1 , Figure 2 As can be seen, within the tested dosage concentration range, the N-acetyl-L-glutamine ethylphenol prepared in Example 1 significantly upregulated the expression of both Clock and Per3 proteins. This indicates that the structure disclosed in this invention is beneficial for enhancing the expression of circadian rhythm proteins (Clock, Per3).

[0083] Example 2

[0084] This example illustrates the verification of the antioxidant effects of N-acetyl-L-glutamine ethylphenol on cells.

[0085] Reactive oxygen species (ROS) refer to the collective term for oxygen-containing free radicals and peroxides that readily form free radicals, all of which are involved in oxygen metabolism within living organisms. In a biological context, ROS formation is a natural byproduct of normal oxygen metabolism and plays an important role in cell signaling and homeostasis. However, during periods of environmental stress (e.g., ultraviolet radiation, lack of sleep), ROS levels can increase dramatically. This can cause severe damage to cell structure, a condition known as oxidative stress.

[0086] ROS assay principle: 2,7'-Dichlorodi-hydrofluoresceindiacetate (DCFH-DA) is non-fluorescent and can freely cross cell membranes. Once inside the cell, it is hydrolyzed by esterases to form DCFH, which, unable to penetrate the cell membrane, accumulates inside the cell. Intracellular reactive oxygen species (ROS) oxidize the non-fluorescent DCFH to produce fluorescent DCF. The intensity of green fluorescence is directly proportional to the level of ROS within the cell. Hydrogen peroxide (H2O2), ultraviolet light, and blue light, as exogenous oxidants, can induce an increase in ROS content in human keratinocytes. By comparing the changes in fluorescence intensity of the intracellular fluorescent dye DCFH-DA after administration of the negative control and the test substance, the inhibitory effect of the raw material on intracellular ROS is reflected.

[0087] Brief steps of the ROS test experiment:

[0088] A. Dilute the cells with cell culture medium to the seeding density (the confluence reaches 45%~60% 24 h after seeding), seed them into well plates, and incubate them in a CO2 incubator for 24 h ± 2 h.

[0089] B. Discard the culture medium in each well of the plate and proceed with the drug administration. Add a certain concentration of the test substance's culture medium to the test substance wells, and add normal culture medium to the negative control, blank control, and naked cell wells. After drug administration, place the plate in a CO2 incubator for 24 h ± 2 h.

[0090] C. After incubation, do not discard the solution. Except for the blank control and naked cell wells, all other wells need to be induced with H2O2. The induction time of H2O2 is 30 min and the induction dose is 250 μM.

[0091] D. The working solution for loading the probe DCFH-DA was prepared by diluting DCFH-DA with PBS at a dilution ratio of 1:1000 to a final concentration of 10 µmol / L.

[0092] E. Except for the wells with naked cells, the culture medium in the other wells should be discarded. After washing three times with PBS, add 1 mL of DCFH-DA working solution to each well and incubate in a CO2 incubator for 30 min. After incubation, wash the cells in each well three times with PBS.

[0093] F. Monitoring with an inverted fluorescence microscope: After the operation, place the well plate on the microscope stage, use the FITC channel, incident light wavelength of 525 nm, excitation light wavelength of 488 nm, 20x objective lens, and set the same exposure time to take pictures. Analyze the fluorescence intensity of the acquired images using ImageJ or Image Pro Plus software.

[0094] Result calculation:

[0095] Formula for calculating ROS inhibition rate:

[0096] ;

[0097] In the formula:

[0098] —The average of three fluorescence intensities of the tested sample;

[0099] —The average of three fluorescence intensities of the negative control;

[0100] —The average of three fluorescence intensities in the naked cell group.

[0101] The experimental results are shown in Table 2. Figure 3 As shown.

[0102] Table 2

[0103]

[0104] The experimental results show that N-acetyl-L-glutamine ethylphenol has a significant scavenging effect on ROS at all three set concentrations.

[0105] Example 3

[0106] This example illustrates the effect of N-acetyl-L-glutamine ethylphenol on the cellular moisturizing factor AQP3.

[0107] Aquaporin-3 (AQP3), a subfamily of the aquaporin family, is the most expressed aquaporin in the skin. It is expressed in the epidermis and basal layer. As a natural moisturizing factor, AQP3 maintains the hydration level of the intercellular matrix and intracellular fluid in the skin and plays a key role in the skin's moisturizing, elasticity and barrier repair function after damage.

[0108] Basic experimental steps:

[0109] A. Cell seeding: Carefully place sterile cell slides into 24-well plates, and seed cells at a rate of 3 × 10⁶ cells / well. 4 Cells were seeded per well, with a cell-negative well included. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h.

[0110] B. Dilution and addition of analytes: Using complete culture medium as the diluent, based on the results of the safety test concentration, set a concentration gradient within the range of analyte concentrations that ensure cell viability of more than 90%, add to the cells at 500 μL / well, add an equal volume of complete culture medium to the cell control wells, and incubate at 37℃ in a 5% CO2 cell culture incubator for 24 h.

[0111] C. Fixation: Carefully remove the cell smear and place it in a new well. Rinse with PBS 3 times, 3 min each time. After removing the PBS, add 4% paraformaldehyde fixative to fix the smear for 20 min. After removing the fixative, add PBS to rinse 3 times, 3 min each time.

[0112] D. Blocking: After removing PBS, add 5% BSA blocking solution and place at room temperature with shaking for 1 h;

[0113] E. Incubation with primary antibody: Cut a small piece of sealing film and place it in a clean culture dish. Dilute the primary antibody 1:50 using primary antibody dilution buffer and carefully add the primary antibody to the sealing film. Carefully remove the cell slide, blot off any remaining liquid with absorbent paper, and then invert it onto the sealing film to ensure full contact between the cells and the primary antibody dilution buffer. Transfer the culture dish to a humidified box protected from light and incubate overnight at 4°C.

[0114] F. Incubation with fluorescent secondary antibody: Carefully remove the cell smear, rinse 3 times with PBS for 3 minutes each time, cut a small piece of sealing film and place it in a clean culture dish, add fluorescent secondary antibody (1:900) to the sealing film, carefully remove the cell smear, blot off any remaining liquid on absorbent paper, and then place it upside down on the sealing film. Incubate in a humidified chamber at room temperature in the dark for 1 hour.

[0115] G. Mounting: Carefully remove the cell slide, rinse it 3 times with PBS for 3 minutes each time, blot dry the remaining liquid on absorbent paper, add a small drop of mounting medium and place it on a glass slide, then invert the cell slide onto the glass slide (carefully tilt and place it slowly).

[0116] H. Photography: Use a fluorescence microscope to take pictures. The excitation wavelength is 492 nm and the emission wavelength is 520 nm.

[0117] Statistical analysis: GraphPad Prism 8.0 software was used for statistical analysis and graph plotting. Quantitative data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used to analyze differences between groups, and P < 0.05 was considered statistically significant.

[0118] Calculation: Percentage of upregulation compared to the negative control group = (mean fluorescence intensity of cells in each group - mean fluorescence intensity of cells in the negative control group) / mean fluorescence intensity of cells in the negative control group × 100%.

[0119] The experimental results are shown in Table 3. Figure 4 As shown.

[0120] Table 3

[0121]

[0122] The experimental results show that N-acetyl-L-glutamine ethylphenol has a good upregulation effect on the expression of aquaporin AQP3.

[0123] Example of effect 4

[0124] This example illustrates the study and comparative test of the cell repair effects of N-acetyl-L-glutamine ethylphenol prepared in Example 1 and the amino acid derivative described in Comparative Example 1.

[0125] In verifying the cell repair efficacy, this example selected the upregulation of filaggrin (FLG) expression for validation and compared its efficacy with that of Comparative Example 1. Flaggrin is an important component of the stratum corneum, synthesized by keratinocytes and distributed in different parts of the stratum corneum. Flaggrin plays a crucial role in maintaining the stability of the skin barrier. It is an important molecule connecting keratin fibers in the stratum corneum of human skin. With the assistance of FLG monomers, keratin fibers aggregate regularly, forming a solid physical barrier on the outermost layer of the epidermis, preventing the loss of epidermal moisture and the invasion of external allergens. Furthermore, after being degraded by enzymes, it forms small molecules needed by the stratum corneum, such as natural moisturizing factors, which play an important role in moisturizing and maintaining barrier integrity.

[0126] Experimental steps:

[0127] A. Cell seeding: After adjusting the density of human immortalized keratinocytes HaCaT suspension to 15,000 cells / well, seed them into 96-well plates at 100 μL / well and incubate at 37°C in a 5% CO2 incubator for 24 h.

[0128] B. Sample collection: Discard the culture medium and wash the cells three times with PBS;

[0129] C. Cell fixation: Discard PBS, add 4% paraformaldehyde fixative to fix cells, and incubate at room temperature for 20 min.

[0130] D. Washing: Wash 5 times with PBS to remove residual paraformaldehyde;

[0131] E. Blocking: Use 5% BSA (or 5% FBS blocking solution prepared with PBS), 100 μL / well, and incubate at room temperature for 1 h;

[0132] F. Applying the primary antibody: Under light protection, dilute the fluorescent primary antibody at a ratio of 1:50 using immunofluorescence diluent. Remove the liquid from the well plate without washing, add the primary antibody diluent, and incubate overnight at 4°C under light protection.

[0133] G. Washing: Warm up for 30 min, remove the fluorescent primary antibody in the dark, and wash the cells 3 times with washing solution for 5 min each time;

[0134] H. Image Acquisition: Leave PBS in each well, observe and photograph under an inverted fluorescence microscope.

[0135] Statistical analysis: GraphPad Prism 8.0 software was used for statistical analysis and graph plotting. Quantitative data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used to analyze differences between groups, and P < 0.05 was considered statistically significant.

[0136] Calculation: Percentage of upregulation compared to the negative control group = (mean fluorescence intensity of cells in each group - mean fluorescence intensity of cells in the negative control group) / mean fluorescence intensity of cells in the negative control group × 100%.

[0137] The experimental results are shown in Table 4. Figure 5 As shown.

[0138] Table 4

[0139]

[0140] The experimental results show that N-acetyl-L-glutamine ethylphenol (Example 1) and L-glutamine ethylphenol (Comparative Example 1) both upregulated the expression of cell filaggrin at the tested concentrations, and were superior to Comparative Example 1, with an overall performance improvement of about 6% to 10%.

[0141] Example 5

[0142] This example compares the upregulation effects of N-acetyl-L-glutamine ethylphenol (Example 1) and L-glutamine ethylphenol (Comparative Example 1) on hypoxia-inducible factor (HIF-1α).

[0143] HIF-1α is an important transcription factor that plays a crucial role in regulating hemoglobin synthesis. It senses changes in oxygen levels and regulates the expression of oxygen-related genes, including the synthesis of erythropoietin (EPO). When you stay up late, your skin becomes oxygen-deficient. In this state, HIF-1α is activated, promoting EPO synthesis, which in turn promotes red blood cell production, thereby increasing the concentration of hemoglobin in the blood. Further promotion of HIF-1α expression through external regulatory factors can further enhance the concentration of hemoglobin in skin capillaries, resulting in a more rosy complexion.

[0144] Experimental steps:

[0145] A. Cell seeding plate

[0146] HFF-1 cells were washed with PBS, digested with trypsin at 37°C, digestion was terminated, the supernatant was removed by centrifugation and resuspended, and cell viability and total number were detected. Cells were seeded into 48-well plates 3 weeks / well by calculation; groups: blank group, siRNA-BMAL1 group, and sample experimental group.

[0147] B. siRNA transfection

[0148] On the second day, cell growth was observed under a microscope. Transfection was performed when cell confluence reached 50%. Transfection working solution preparation: Pre-packaged target gene siRNA and LIP were placed in a biosafety cabinet and the entire process was performed on ice. DMEM medium without serum and antibiotics was used in RNase-free centrifuge tubes to prepare the solution, with a final siRNA concentration of 10 nM. LIP and siRNA were co-incubated for 15 min. The siRNA-Lip mixture was gently added dropwise to the siRNA group and sample experimental group, and incubated for 4–6 h. The blank group and siRNA group were then replaced with complete medium, while the sample experimental group was replaced with sample working solution diluted with complete medium. The plates were then incubated for 48 h.

[0149] C. Cell immunofluorescence staining

[0150] (1) Sample collection: Discard the culture medium and wash the cells 3 times with PBS;

[0151] (2) Cell fixation: Discard PBS, add 4% paraformaldehyde fixative to fix cells, and incubate at room temperature for 20 min;

[0152] (3) Washing: Wash 5 times with PBS to remove residual paraformaldehyde;

[0153] (4) Permeabilization: After removing the liquid, use 0.1% Triton-100 to permeabilize the cell membrane;

[0154] (5) Blocking: Use 5% FBS blocking solution prepared with PBS and incubate at room temperature for 1 hour;

[0155] (6) Apply primary antibody: Dilute the primary antibody at a ratio of 1:100 using immunofluorescence diluent under light protection. Remove the liquid from the well plate without washing, add the primary antibody diluent, and incubate overnight at 4°C under light protection.

[0156] (7) Washing: Warm up for 30 min, remove the primary antibody in the dark, and wash the cells 3 times with washing solution for 5 min each time;

[0157] (8) Applying fluorescent secondary antibody: Dilute the secondary antibody at a ratio of 1:200 with fluorescent diluent under light-protected conditions and incubate at room temperature for 1 hour;

[0158] (9) Image collection: After washing three times, retain liquid in each well and observe and photograph under an inverted fluorescence microscope.

[0159] D. Data Processing

[0160] Image J Mean Fluorescence Intensity (MFI) Analysis and Image Processing:

[0161] (1) After adding the fluorescence image, extract a single channel (Image-Color-Split Channels); after the operation, the original image will be divided into three channels: red, green and blue, each with an 8-bit black and white image. Select the corresponding fluorescence color image that needs to be analyzed, and turn off the other two images.

[0162] (2) Adjust the threshold and select an appropriate area (Image-Adjust-Threshold); manually select the threshold to ensure that the entire area is represented by red;

[0163] (3) Set the parameters to be measured (Analyze-Set Measurements); be sure to check Limit tothreshold, so that the selected target area is measured, otherwise the software will calculate the entire map by default;

[0164] (4) Analyze-Measure; where Mean is the average gray value, which also represents the average fluorescence intensity of red fluorescence in this image.

[0165] Calculation: Percentage of upregulation compared to the siRNA-BMAL1 control group = (mean fluorescence intensity of cells in each group - mean fluorescence intensity of cells in the control group) / mean fluorescence intensity of cells in the negative control group × 100%.

[0166] (5) Statistical analysis

[0167] GraphPad Prism 8.0 software was used for statistical analysis and graph plotting. Quantitative data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used to analyze differences between groups, and p < 0.05 was considered statistically significant.

[0168] The experimental results are shown in Table 5. Figure 6 As shown.

[0169] Table 5

[0170]

[0171] The cell test results above show that hypoxia-inducible factor (HIF) expression was slightly upregulated in the model group compared to the control group, indicating successful model construction. Based on the two drug administration groups of the model group (Example 1 and Comparative Example 1), the drug administration group (Example 1) further significantly upregulated HIF expression. At drug concentrations of 0.1% and 0.5%, HIF expression was upregulated by 13.7% and 7.94%, respectively. The comparative example showed less upregulation at both concentrations than Example 1, with virtually no upregulation at a 0.5% concentration. Therefore, the compound N-acetyl-L-glutamine ethylphenol described in this invention has a good effect on upregulating HIF expression and is superior to the comparative example L-glutamine ethylphenol, showing potential application in promoting a rosy skin condition.

[0172] 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 glutamic acid derivative, characterized by, A glutamic acid derivative having a structure represented by formula (I):

2. The method of producing the glutamic acid derivative according to claim 1, characterized by, The application relates to a glutamic acid derivative and a preparation method thereof. The glutamic acid derivative is prepared by carrying out an amidation reaction on L-glutamic acid-5-tert-butyl ester and acetic acid-N-succinimide ester, carrying out an amidation reaction on the obtained intermediate and tyramine, and carrying out a tert-butyl ester removal and purification.

3. The production method according to claim 2, characterized by, The amidation reaction is added with a condensing agent and an acid-binding agent. The condensing agent comprises at least one of N-hydroxysuccinimide, 1-hydroxybenzimidazole, diisopropyl carbodiimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

4. Application of the glutamic acid derivative of claim 1 in a product for correcting skin rhythm disorder, skin moisturizing, skin barrier repair and / or promoting skin redness.

5. Use according to claim 4, characterized in that, The product comprises a skin care product or a cosmetic product.

6. Use according to claim 4, characterized in that, The skin rhythm disorder is caused by staying up late.

7. A skin care product characterized by, The application further relates to a skin care product containing the glutamic acid derivative of claim 1.

8. The skin care product of claim 7, wherein, The skin care product is in the form of an aqueous agent, a gel, an emulsion, a cream agent, a film agent, a suspension agent, a spray agent or a powder agent.

9. A cosmetic product, characterized by, The application further relates to a cosmetic product containing the glutamic acid derivative of claim 1.

10. The cosmetic product according to claim 9, characterized in that, The cosmetic product is in the form of an aqueous agent, a gel, an emulsion, a cream agent, a film agent, a suspension agent, a spray agent or a powder agent.

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