Anhydrous essence as well as preparation method and application thereof

Abstract

The invention discloses anhydrous essence as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. The non-aqueous essence comprises the following components in percentage by mass: 50%-80% of a non-aqueous solvent, 1%-20% of a penetration enhancer, 5%-15% of an emollient, 0.5%-2% of an acid substance, 2%-10% of an active substance, 0.5%-10% of a Gly-His-TXA-Cu solution and 0.1%-1.0% of an anti-inflammatory soothing agent. The anhydrous essence disclosed by the invention contains Gly-His-TXA-Cu, when the anhydrous essence and an acid substance jointly act, the acid substance can enable the Gly-His-TXA-Cu to more effectively reach an inflammation part for anti-inflammation, and the anti-inflammation effect of the Gly-His-TXA-Cu can relieve the irritation of the acid substance, so that the anti-inflammation effect of the Gly-His-TXA-Cu is improved, and the anti-inflammation effect of the Gly-His-TXA-Cu is improved. And the tranexamic acid part on the Gly-His-TXA-Cu molecule can also be cooperated with the acid substances to accelerate the removal of the pigmentation on the skin.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to an anhydrous essence, its preparation method, and its application. Background Technology

[0002] Acne is one of the main causes of post-inflammatory hyperpigmentation (PIH). Current technologies generally use acidic ingredients to improve acne symptoms by exfoliating, inhibiting bacterial growth, and reducing sebum secretion. However, acidic ingredients are highly irritating to the skin, potentially causing dryness and redness, and can increase skin sensitivity to ultraviolet radiation, making it more prone to post-sun pigmentation. Furthermore, while acidic ingredients have some effect on improving acne, their effect on improving post-inflammatory hyperpigmentation caused by acne is relatively weak. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide an anhydrous essence, its preparation method and application, to overcome the problems of strong irritation of acidic components and weak effect on improving post-inflammatory hyperpigmentation caused by acne in the prior art.

[0004] In a first aspect, the present invention provides an anhydrous essence, comprising, by weight percentage, the following components: 50%~80% non-aqueous solvent, 1%~20% penetration enhancer, 5%~15% emollient, 0.5%~2% acidic substance, 2%~10% active substance, 0.5%~10% Gly-His-TXA-Cu solution, and 0.1%~1.0% anti-inflammatory soothing agent; The structural formula of the Gly-His-TXA-Cu is shown in formula (I): Formula (I) Compared to existing technologies, the anhydrous essence of this invention contains a Gly-His-TXA-Cu solution. Gly-His-TXA-Cu possesses a unique dual mechanism of anti-inflammatory repair and whitening. When it acts on the skin in conjunction with acidic substances, the acidic substances first cleanse and open the hair follicle channels, paving the way for Gly-His-TXA-Cu to more effectively reach the inflamed area. Simultaneously, the powerful anti-inflammatory effect of Gly-His-TXA-Cu significantly alleviates the irritation of the acidic substances to the skin. Furthermore, the tranexamic acid portion on the Gly-His-TXA-Cu molecule synergizes with the exfoliating effect of the acidic substances, jointly accelerating the removal of pigmentation on the skin. In addition, the anhydrous essence of this invention employs an anhydrous delivery system, which not only prevents the hydrolysis of the peptide complex Gly-His-TXA-Cu but also avoids the ionization of the acidic substances, preventing the reduction in the efficacy of the acidic substances due to ionization.

[0005] Furthermore, based on the mass percentage of the anhydrous essence, it includes the following components: 60%~80% non-aqueous solvent, 5%~15% penetration enhancer, 5%~10% emollient, 1%~2% acidic substances, 2%~8% active substances, 1%~5% Gly-His-TXA-Cu solution, and 0.1%~1.0% anti-inflammatory soothing agent.

[0006] The above technical solution further limits the mass percentage of each component in the anhydrous essence. Under the above ratio, the synergistic effect of Gly-His-TXA-Cu solution and acidic substances can be further enhanced.

[0007] Furthermore, the solvent for the Gly-His-TXA-Cu solution is the aforementioned non-aqueous solvent; and / or, In the Gly-His-TXA-Cu solution, the mass concentration of Gly-His-TXA-Cu is 0.5%~5%.

[0008] The above technical solution pre-dissolves powdered Gly-His-TXA-Cu in a non-aqueous solvent to form a Gly-His-TXA-Cu solution, which makes Gly-His-TXA-Cu easier to disperse when preparing anhydrous essence and avoids the agglomeration of powdered Gly-His-TXA-Cu in the anhydrous system.

[0009] Furthermore, the acidic substance is at least one of salicylic acid, azelaic acid, or mandelic acid.

[0010] The above technical solution further limits the types of acidic substances. All of the above acidic substances can work synergistically with Gly-His-TXA-Cu solution, enabling Gly-His-TXA-Cu to effectively reach the inflamed site to reduce inflammation and remove pigment deposits together with the acidic substances.

[0011] Furthermore, the penetration enhancer is at least one of ethoxydiethylene glycol, isosorbide dimethyl ether, or pentanediol.

[0012] The penetration enhancer in the above technical solution can help the acidic substances, Gly-His-TXA-Cu and other active functional ingredients in the waterless essence penetrate the stratum corneum of the skin and reach the deep layers of the skin, thereby improving the absorption efficiency and efficacy of the active functional ingredients.

[0013] Further, the emollient is at least one of squalane, caprylic / capric triglyceride, or isononyl isononanoate; and / or, The non-aqueous solvent is at least one of propylene glycol, butanediol, or 1,3-propanediol; and / or, The active ingredient is at least one of nicotinamide, retinaldehyde, or bakuchiol; and / or, The anti-inflammatory and soothing agent is at least one of dipotassium glycyrrhizate, bisabolol, or 4-tert-butylcyclohexanol.

[0014] Secondly, the present invention provides a method for preparing anhydrous essence, comprising the following steps: Weigh each component according to the above-mentioned proportions of the anhydrous essence; A phase A is obtained by mixing a non-aqueous solvent, a penetration enhancer, and a skin emollient. The acid, active ingredients, Gly-His-TXA-Cu solution, and anti-inflammatory soothing agent are added to phase A and stirred to dissolve, thus obtaining the anhydrous essence.

[0015] Compared with existing technologies, this invention first mixes a non-aqueous solvent, a penetration enhancer, and a moisturizer to obtain phase A, constructing a basic dispersion system for the anhydrous essence and creating a stable dispersion environment for the subsequent addition of other components. Next, acids, active ingredients, a Gly-His-TXA-Cu solution, and an anti-inflammatory soothing agent are added to phase A. Stirring ensures that these components are fully contacted and mixed with phase A, breaking down any potential interfacial tension and ensuring that all components form a uniform dispersion system. This ultimately yields an anhydrous essence with a uniform texture, no graininess, and good stability.

[0016] Furthermore, the mixing speed of the non-aqueous solvent, penetration enhancer, and emollient is 300 rpm to 600 rpm.

[0017] Further, acidic substances, active substances, Gly-His-TXA-Cu solution, and anti-inflammatory soothing agents are added to phase A and stirred to dissolve at a temperature of 40℃~50℃ and a stirring speed of 300rpm~600rpm.

[0018] The preparation method of Gly-His-TXA-Cu includes the following steps: A recombinant bacterial strain containing the gene encoding TXA-ligase was fermented. After the enzyme activity reached a plateau, a fermentation broth containing TXA-ligase was obtained. The amino acid sequence of TXA-ligase is shown in SEQ ID NO:2.

[0019] The substrates glycyl-L-histidine ethyl ester and tranexamic acid were added to the fermentation broth containing TXA-ligase, and the reaction was carried out at 25℃~40℃ and pH 7.0~8.5 to obtain a fermentation broth containing the peptide ligand Gly-His-TXA. The concentrations of glycyl-L-histidine ethyl ester and tranexamic acid were 10mM~100mM.

[0020] A soluble divalent copper salt was added to the fermentation broth containing the peptide ligand Gly-His-TXA, and the reaction was carried out at 25℃~45℃ and pH 5.5~6.5 to obtain Gly-His-TXA-Cu. The molar ratio of the peptide ligand Gly-His-TXA to the copper ions in the soluble divalent copper salt was (1~1.3):1. The soluble divalent copper salt used in the above reaction was either copper acetate or copper sulfate.

[0021] Thirdly, the present invention provides the application of the above-mentioned anhydrous essence in improving acne-induced post-inflammatory hyperpigmentation.

[0022] Compared with the prior art, the beneficial effects of the anhydrous essence of the present invention in improving acne-induced post-inflammatory hyperpigmentation are the same as those of the above-mentioned anhydrous essence, and will not be repeated here. Detailed Implementation

[0023] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0024] In a first aspect, embodiments of the present invention provide an anhydrous essence, comprising the following components by mass percentage: 50%~80% non-aqueous solvent, 1%~20% penetration enhancer, 5%~15% emollient, 0.5%~2% acidic substance, 2%~10% active substance, 0.5%~10% Gly-His-TXA-Cu solution, and 0.1%~1.0% anti-inflammatory soothing agent.

[0025] For example, the content of non-aqueous solvent in the anhydrous essence of the present invention can be 50%, 60%, 70% or 80%, preferably 60% to 80%; the content of penetration enhancer can be 1%, 5%, 10%, 15% or 20%, preferably 5% to 15%; the content of emollient can be 5%, 8%, 10%, 12% or 15%, preferably 5% to 10%; the content of acidic substances can be 0.5%, 1%, 1.5% or 2%, preferably 1% to 2%; the content of active substance can be 2%, 5%, 8% or 10%, preferably 2% to 8%; the content of Gly-His-TXA-Cu solution can be 0.5%, 1%, 3%, 5%, 7% or 10%, preferably 1% to 5%; the content of anti-inflammatory soothing agent can be 0.1%, 0.5% or 1.0%; or any range of values ​​thereof.

[0026] The structural formula of Gly-His-TXA-Cu is shown in formula (I): Formula (I) The anhydrous essence in the above-mentioned technical solution incorporates a Gly-His-TXA-Cu solution and also contains acidic substances. These acids can unclog blocked pores and hair follicles, allowing Gly-His-TXA-Cu to reach the inflamed area for anti-inflammatory effects. The anti-inflammatory properties of Gly-His-TXA-Cu also alleviate the irritation of the acids. Simultaneously, the tranexamic acid portion of the Gly-His-TXA-Cu molecule synergizes with the exfoliating effect of the acidic substances, further enhancing the removal of pigmentation. To maximize the efficacy and stability of the acidic substances and the peptide complex Gly-His-TXA-Cu, this invention employs an anhydrous delivery system. This system prevents the hydrolysis of the Gly-His-TXA-Cu peptide complex and avoids the ionization of the acidic substances, which would reduce their efficacy. Furthermore, the anhydrous delivery system in this invention's anhydrous essence contains a penetration enhancer, enabling Gly-His-TXA-Cu, acidic substances, and other active ingredients to efficiently penetrate deep into the skin.

[0027] Furthermore, based on the above embodiments, the solvent of the Gly-His-TXA-Cu solution in this embodiment of the invention is a non-aqueous solvent; even further, the solvent of the Gly-His-TXA-Cu solution is an alcohol solvent, including but not limited to at least one of ethanol, ethylene glycol, propanol, propylene glycol, and butanediol; and / or, the mass concentration of Gly-His-TXA-Cu in the Gly-His-TXA-Cu solution is 0.5% to 5%, for example, the mass concentration can be 0.5%, 1%, 3%, or 5%; or any range of values ​​therein.

[0028] The above technical solution uses a non-aqueous solvent to prepare Gly-His-TXA-Cu into a solution, which allows it to dissolve more uniformly in the anhydrous essence, avoiding the precipitation or separation of components and ensuring the stability of the anhydrous essence.

[0029] Furthermore, based on the above embodiments, the embodiments of the present invention further specify that the acidic substance is at least one of salicylic acid, azelaic acid, or mandelic acid.

[0030] The salicylic acid, azelaic acid, or mandelic acid in the above technical solution work synergistically with Gly-His-TXA-Cu to not only further enhance the anti-inflammatory effect on acne-prone skin, but also further remove pigmentation in the skin.

[0031] Furthermore, based on the above embodiments, the embodiments of the present invention further specify that the penetration enhancer is at least one of ethoxydiethylene glycol, isosorbide dimethyl ether, or pentanediol.

[0032] Ethoxydiethylene glycol, isosorbide dimethyl ether, or pentylene glycol in the above technical solutions can all help the active ingredients in the anhydrous essence penetrate the stratum corneum and enter the deep layers of the skin, thereby further enhancing the effectiveness of the active ingredients.

[0033] In some embodiments, the emollient is at least one of squalane, caprylic / capric triglyceride, or isononyl isononanoate.

[0034] The emollient in the above technical solution, together with the non-aqueous solvent and the penetration enhancer, forms an anhydrous delivery system. Furthermore, the emollient can maintain the skin barrier, making it especially suitable for post-inflammatory and skin with a weakened barrier.

[0035] In some embodiments, the non-aqueous solvent is at least one of propylene glycol, butanediol, or 1,3-propanediol.

[0036] In some embodiments, the active substance is at least one of nicotinamide, retinaldehyde, or bakuchiol.

[0037] The active ingredient nicotinamide in the above technical solution can work synergistically with acidic substances to help regulate sebum, improve barrier function, and further reduce inflammation and pigmentation; retinaldehyde can promote keratin metabolism and cell renewal, inhibit abnormal activity of melanocytes, and reduce pigmentation; bakuchiol has significant anti-inflammatory activity, can inhibit the release of skin inflammatory factors, and at the same time promote lipid synthesis in the stratum corneum and enhance the skin's water-locking ability.

[0038] In some embodiments, the anti-inflammatory and soothing agent is at least one of dipotassium glycyrrhizate, bisabolol, or 4-tert-butylcyclohexanol.

[0039] The anti-inflammatory and soothing agents in the above-mentioned technical solutions can work together with other ingredients to further alleviate skin inflammation and maintain the skin barrier.

[0040] Secondly, embodiments of the present invention provide a method for preparing anhydrous essence, comprising the following steps: S1. Weigh each component according to the above-mentioned proportions of the anhydrous essence.

[0041] S2. Mix the non-aqueous solvent, penetration enhancer, and emollient to obtain phase A.

[0042] The above steps can be performed at room temperature, with a mixing speed of 300 rpm to 600 rpm. For example, it can be 300 rpm, 400 rpm, 500 rpm, or 600 rpm; or any range of values ​​within these ranges.

[0043] S3. Add the acidic substances, active substances, Gly-His-TXA-Cu solution and anti-inflammatory soothing agent to phase A and stir to dissolve, thus obtaining anhydrous essence.

[0044] The temperature in the above steps is 40℃~50℃, and the stirring speed is 300rpm~600rpm. For example, the temperature can be 40℃, 45℃ or 50℃; the stirring speed can be 300rpm, 400rpm, 500rpm or 600rpm; or any range of values.

[0045] The temperature of 40℃~50℃ mentioned above can help the active ingredients dissolve better in phase A, and can also prevent thermally unstable components from being deactivated by heat.

[0046] In the above steps, the mixture should be stirred until all components are completely dissolved, forming a clear and transparent homogeneous phase.

[0047] The preparation method of Gly-His-TXA-Cu can refer to the following steps: A targeted mutation was performed on wild-type Bacillus subtilis protease BPN' (amino acid sequence shown in SEQ ID NO:1) derived from Bacillus amyloliquefaciens to obtain TXA-ligase (amino acid sequence shown in SEQ ID NO:2). The mutation sites were S220C, P224A, N76D, L126A, G166S, Y216K, and N217S.

[0048] The optimized TXA-ligase gene (SEQ ID NO:3) was cloned into the expression vector pPICZαA to obtain the recombinant expression vector pPICZαA-TXA. The recombinant expression vector pPICZαA-TXA was then transformed into Pichia pastoris competent cells (strain X-33) via electroporation, resulting in the recombinant strain X-33 / pPICZαA-TXA, which was stored in glycerol tubes.

[0049] The recombinant strain X-33 / pPICZαA-TXA containing the TXA-ligase gene was fermented. After the enzyme activity reached a plateau, a fermentation broth containing TXA-ligase was obtained. At this point, the TXA-ligase activity in the fermentation broth was 500 U / mL to 5000 U / mL, and the amino acid sequence of the TXA-ligase is shown in SEQ ID NO:2.

[0050] The substrates glycyl-L-histidine ethyl ester and tranexamic acid were added to the fermentation broth containing TXA-ligase, and the reaction was carried out at 25℃~40℃ and pH 7.0~8.5 to obtain a fermentation broth containing the peptide ligand Gly-His-TXA. The concentrations of glycyl-L-histidine ethyl ester and tranexamic acid were 10mM~100mM. After adding the substrate, the molar ratio of glycyl-L-histidine ethyl ester to tranexamic acid in the fermentation broth was 1:(1~1.5).

[0051] A soluble divalent copper salt was added to the fermentation broth containing the peptide ligand Gly-His-TXA, and the reaction was carried out at 25℃~45℃ and pH 5.5~6.5 to obtain Gly-His-TXA-Cu. The molar ratio of the peptide ligand Gly-His-TXA to the copper ions in the soluble divalent copper salt was (1~1.3):1. The soluble divalent copper salt used in the above reaction can be either copper acetate or copper sulfate.

[0052] Thirdly, embodiments of the present invention provide the application of the above-mentioned anhydrous essence in improving acne-induced post-inflammatory hyperpigmentation.

[0053] Compared with the prior art, the beneficial effects of the anhydrous essence of the present invention in improving acne-induced post-inflammatory hyperpigmentation are the same as those of the above-mentioned anhydrous essence, and will not be repeated here.

[0054] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0055] Example 1 This invention provides an anhydrous essence, comprising the following components by mass percentage: 71.5% propylene glycol, 10% ethoxydiethylene glycol, 8% squalane, 2% salicylic acid, 5% niacinamide, 3% Gly-His-TXA-Cu solution (1% by mass), and 0.5% dipotassium glycyrrhizate. The solvent for the Gly-His-TXA-Cu solution is propylene glycol.

[0056] The method for preparing the anhydrous essence in this embodiment is as follows: S1. Weigh each component according to the above-mentioned proportions of the anhydrous essence.

[0057] S2. Propylene glycol (a non-aqueous solvent), ethoxydiethylene glycol (a penetration enhancer), and squalane (a skin moisturizer) are stirred and mixed evenly at 500 rpm at room temperature to obtain phase A.

[0058] S3. Add the acidic substance salicylic acid, the active substance nicotinamide, the Gly-His-TXA-Cu solution, and the anti-inflammatory and soothing agent dipotassium glycyrrhizate to phase A. Stir at 500 rpm at 45°C until all components are completely dissolved, forming a clear and transparent homogeneous phase, thus obtaining the anhydrous essence.

[0059] The preparation method of Gly-His-TXA-Cu is as follows: A targeted mutation was performed on wild-type Bacillus subtilis protease BPN' (amino acid sequence shown in SEQ ID NO:1) derived from Bacillus amyloliquefaciens to obtain TXA-ligase (amino acid sequence shown in SEQ ID NO:2). The mutation sites were S220C, P224A, N76D, L126A, G166S, Y216K, and N217S.

[0060] The optimized TXA-ligase gene (SEQ ID NO:3) was cloned into the expression vector pPICZαA to obtain the recombinant expression vector pPICZαA-TXA. The recombinant expression vector pPICZαA-TXA was then transformed into Pichia pastoris competent cells (strain X-33) via electroporation, resulting in the recombinant strain X-33 / pPICZαA-TXA, which was stored in glycerol tubes.

[0061] The recombinant strain X-33 / pPICZαA-TXA was fermented on a large scale using a standard Pichia pastoris high-density fermentation protocol. The specific fermentation process is as follows: Seed activation: The recombinant strain X-33 / pPICZαA-TXA preserved in glycerol tubes was streaked onto YPD solid medium and incubated at 30°C for 48 hours.

[0062] Primary seed culture: Select a single colony from the YPD solid medium and inoculate it into 50 mL of BMGY medium. Incubate at 30°C and 250 rpm for 24 hours. At this time, the recombinant strain will grow to the logarithmic growth phase, and the primary seed culture solution will be obtained.

[0063] High-density fermentation: The entire primary seed culture was inoculated into a 10L fermenter containing 5L of basal salt medium pre-added with 40 g / L glycerol. Fermentation was carried out at 30°C, pH 5.5 (automatically controlled by adding ammonia), and dissolved oxygen (DO) maintained above 20% to accumulate cell biomass. Once the glycerol was depleted, a 50% (w / v) glycerol solution was added at a rate of 18 mL / h / L for 4 hours of feed starvation.

[0064] Methanol-induced expression: Stop adding glycerol and start slowly adding pure methanol to induce TXA-ligase expression and secretion into the fermentation broth. Maintain the methanol concentration in the culture medium at 1% (v / v) and continue induction culture, taking samples regularly to detect enzyme activity until the enzyme activity reaches the plateau phase.

[0065] After the TXA-ligase activity reached a plateau, glycyl-L-histidine ethyl ester and tranexamic acid were added to the fermenter. The reaction was carried out at 30°C and pH 7.5 for 12 hours to synthesize the peptide ligand Gly-His-TXA in situ. At the start of peptide ligand synthesis, the TXA-ligase activity in the fermentation broth was 2000 U / mL, and the concentrations of glycyl-L-histidine ethyl ester and tranexamic acid in the fermentation broth were both 50 mM.

[0066] After the synthesis of the peptide ligand Gly-His-TXA reached a plateau, a sterile aqueous solution of copper acetate was added to the fermenter. Under mild conditions of 30°C and pH 6.0, the reaction was continued with stirring for 1.5 hours, allowing the peptide ligand Gly-His-TXA to chelate with copper ions in the copper acetate, forming the metal-peptide complex Gly-His-TXA-Cu. At the initial stage of metal-peptide complex formation, the concentration of copper acetate in the fermentation broth was 45.7 mM, the concentration of the peptide ligand Gly-His-TXA was 48 mM, and the molar ratio of copper ions to peptide ligand Gly-His-TXA in the fermentation broth was 1:1.05.

[0067] After the above reaction is completed, the Gly-His-TXA-Cu in the reaction solution is purified. The specific purification steps are as follows: Cell isolation and clarification: The reaction solution containing Gly-His-TXA-Cu was centrifuged at 8000 x g for 20 minutes at 4°C using a high-speed refrigerated centrifuge to precipitate yeast cells and most of the insoluble matter. The supernatant was then pre-filtered through a 0.45 µm polyethersulfone (PES) membrane to remove fine particles, and then filtered through a 0.22 µm sterile PES membrane for sterilization to obtain a clear crude extract.

[0068] Ultrafiltration Concentration and Desalination: The crude extract was concentrated to 1 / 15 of its original volume using a tangential flow ultrafiltration system with a molecular weight cutoff of 500 Da. During operation, the inlet pressure was maintained at 2 bar and the outlet pressure at 1 bar to control the transmembrane pressure at 1.0 bar, preventing excessive concentration polarization and gel formation on the membrane surface, and ensuring filtration efficiency. After concentration, constant-volume percolation was performed using 10 times the volume of the concentrate with pure water to ensure that salts and small molecule impurities in the concentrate were fully removed until the conductivity of the filtrate was essentially the same as that of pure water.

[0069] Cation exchange chromatography: SP Sepharose Fast Flow was selected as the chromatography column packing material. Column equilibration: The column was first equilibrated using 10 column volumes (CV) of 20 mM MES buffer (pH 6.0) (i.e., equilibration buffer) at a flow rate of 250 cm / h until the pH and conductivity of the eluent were consistent with the equilibration buffer. Loading: The concentrated solution after ultrafiltration and desalting was loaded onto the equilibrated column at a flow rate of 120 cm / h. The loading volume was determined based on the column's dynamic capacity, and was loaded to 85% of the theoretical capacity. Washing: After loading, the column was washed with 5 CV of equilibration buffer to remove unbound impurities. Elution: NaCl was added to 20 mM MES buffer (pH 6.0) as the eluent. The NaCl concentration in the eluent was linearly increased from 0 M to 1.0 M over a volume of 15 CV at a flow rate of 250 cm / h. Collection: The elution process was monitored using an online ultraviolet detector (220 nm), and the main peak component that was eluted in the range of 0.2 M NaCl to 0.5 M NaCl and showed a deep blue color was collected.

[0070] Reversed-phase high-performance liquid chromatography (RP-HPLC): Preparative HPLC system. Column: Agilent ZORBAX C18 reversed-phase column, 250 mm × 20 mm, 10 µm particle size. Mobile phase: Phase A: Ultrapure aqueous solution containing 0.1% (v / v) trifluoroacetic acid (TFA). Phase B: Acetonitrile solution containing 0.1% (v / v) TFA. Elution gradient: A linear gradient elution was used: 0-5 minutes, 5% volume of phase B and 95% volume of phase A; 5-45 minutes, the volume of phase B linearly increased from 5% to 45%, while the volume of phase A linearly decreased from 95% to 55%; 45-50 minutes, the volume of phase B linearly increased from 45% to 95%, while the volume of phase A linearly decreased from 55% to 5%; 50-55 minutes, maintaining 95% volume of phase B and 5% volume of phase A; 55-60 minutes, the volume of phase B linearly decreased from 95% to 5%, while the volume of phase A linearly increased from 5% to 95%, and this ratio was maintained for a period until the column reached equilibrium. Flow rate: 18 mL / min. Detection wavelength: 220 nm. Collection: Based on the chromatogram, the fraction corresponding to the main peak was collected, and its molecular weight was confirmed by mass spectrometry (MS) to be consistent with the theoretical molecular weight of Gly-His-TXA-Cu. The qualified fractions from each batch were then combined.

[0071] Freeze-drying: Pre-freezing: Place the combined purified solution from the reversed-phase high-performance liquid chromatography (RP-HPLC) at -40°C for 4 hours to ensure complete solidification. First drying (sublimation): Activate the vacuum system to achieve a chamber vacuum of 100 mTorr. Programmatically increase the plate temperature to -10°C (0.2°C per minute) and maintain this temperature for 24 hours until all ice crystals have sublimated. Second drying (desorption): After the first drying, further programmatically increase the plate temperature to 25°C (0.1°C per minute) and maintain this temperature for 10 hours to remove residual water bound to the product, yielding a fluffy, homogeneous Gly-His-TXA-Cu blue powder with less than 2% residual moisture.

[0072] The above-mentioned Gly-His-TXA-Cu blue powder was dissolved in propylene glycol to obtain a 1% Gly-His-TXA-Cu solution.

[0073] Example 2 This invention provides an anhydrous essence, comprising the following components by mass percentage: 75.5% propylene glycol, 5% ethoxydiethylene glycol, 5% squalane, 1% salicylic acid, 8% niacinamide, 5% Gly-His-TXA-Cu solution (3% by mass), and 0.5% dipotassium glycyrrhizate. The solvent for the Gly-His-TXA-Cu solution is propylene glycol.

[0074] The method for preparing the anhydrous essence in this embodiment is as follows: S1. Weigh each component according to the above-mentioned proportions of the anhydrous essence.

[0075] S2. Propylene glycol (a non-aqueous solvent), ethoxydiethylene glycol (a penetration enhancer), and squalane (a skin moisturizer) are stirred and mixed evenly at 400 rpm at room temperature to obtain phase A.

[0076] S3. Add the acidic substance salicylic acid, the active substance nicotinamide, the Gly-His-TXA-Cu solution, and the anti-inflammatory and soothing agent dipotassium glycyrrhizate to phase A. Stir at 400 rpm at 45°C until all components are completely dissolved, forming a clear and transparent homogeneous phase, thus obtaining the anhydrous essence.

[0077] The preparation method of Gly-His-TXA-Cu is as described in Example 1. After obtaining the blue powder of Gly-His-TXA-Cu, it is dissolved in propylene glycol to obtain a Gly-His-TXA-Cu solution with a mass fraction of 3%.

[0078] Example 3 This invention provides an anhydrous essence, comprising the following components by mass percentage: 70% propylene glycol, 15% ethoxydiethylene glycol, 10% squalane, 1.5% salicylic acid, 2% niacinamide, 1% (by mass) Gly-His-TXA-Cu solution, and 0.5% dipotassium glycyrrhizate. The solvent for the Gly-His-TXA-Cu solution is propylene glycol.

[0079] The method for preparing the anhydrous essence in this embodiment is as follows: S1. Weigh each component according to the above-mentioned proportions of the anhydrous essence.

[0080] S2. Propylene glycol (a non-aqueous solvent), ethoxydiethylene glycol (a penetration enhancer), and squalane (a skin moisturizer) are stirred and mixed evenly at 300 rpm at room temperature to obtain phase A.

[0081] S3. Add the acidic substance salicylic acid, the active substance nicotinamide, the Gly-His-TXA-Cu solution, and the anti-inflammatory and soothing agent dipotassium glycyrrhizate to phase A. Stir at 300 rpm at 45°C until all components are completely dissolved, forming a clear and transparent homogeneous phase, thus obtaining the anhydrous essence.

[0082] The preparation method of Gly-His-TXA-Cu is as described in Example 1. After obtaining the blue powder of Gly-His-TXA-Cu, it is dissolved in propylene glycol to obtain a Gly-His-TXA-Cu solution with a mass fraction of 3%.

[0083] Example 4 This invention provides an anhydrous essence, comprising the following components by mass percentage: 54.4% butylene glycol, 10% isosorbide dimethyl ether, 15% caprylic / capric triglyceride, 0.5% azelaic acid, 10% retinaldehyde, 10% Gly-His-TXA-Cu solution (0.5% by mass), and 0.1% 4-tert-butylcyclohexanol. The solvent for the Gly-His-TXA-Cu solution is butylene glycol.

[0084] The method for preparing the anhydrous essence in this embodiment is as follows: S1. Weigh each component according to the above-mentioned proportions of the anhydrous essence.

[0085] S2. Butylene glycol (a non-aqueous solvent), isosorbide dimethyl ether (a penetration enhancer), and caprylic / capric triglyceride (a skin emollient) are stirred and mixed evenly at 600 rpm at room temperature to obtain phase A.

[0086] S3. Add the acidic substance azelaic acid, the active substance retinaldehyde, Gly-His-TXA-Cu solution, and the anti-inflammatory and soothing agent 4-tert-butylcyclohexanol to phase A. Stir at 600 rpm at 40°C until all components are completely dissolved, forming a clear and transparent homogeneous phase, thus obtaining the anhydrous essence.

[0087] The preparation method of Gly-His-TXA-Cu is as described in Example 1. After obtaining the blue powder of Gly-His-TXA-Cu, it is dissolved in butanediol to obtain a Gly-His-TXA-Cu solution with a mass fraction of 0.5%.

[0088] Example 5 This invention provides an anhydrous essence, comprising the following components by mass percentage: 66.5% 1,3-propanediol, 20% pentanediol, 5% isononyl isononanoate, 2% mandelic acid, 5% psoralen, 0.5% Gly-His-TXA-Cu solution (5% by mass), and 1% bisabolol. The solvent for the Gly-His-TXA-Cu solution is 1,3-propanediol.

[0089] The method for preparing the anhydrous essence in this embodiment is as follows: S1. Weigh each component according to the above-mentioned proportions of the anhydrous essence.

[0090] S2. The non-aqueous solvent 1,3-propanediol, the penetration enhancer pentanediol, and the emollient isononyl isononanoate are stirred and mixed evenly at room temperature and a speed of 500 rpm to obtain phase A.

[0091] S3. Add the acidic substance mandelic acid, the active substance psoralen, the Gly-His-TXA-Cu solution, and the anti-inflammatory and soothing agent bisabolol to phase A. Stir at 500 rpm at 50°C until all components are completely dissolved, forming a clear and transparent homogeneous phase, thus obtaining the anhydrous essence.

[0092] The preparation method of Gly-His-TXA-Cu is as described in Example 1. After obtaining the blue powder of Gly-His-TXA-Cu, it is dissolved in 1,3-propanediol to obtain a Gly-His-TXA-Cu solution with a mass fraction of 5%.

[0093] Comparative Example 1 Compared with the anhydrous essence in Example 1, the only difference is that the 1% Gly-His-TXA-Cu solution is replaced by an equal amount of a mixed solution of copper peptide and tranexamic acid (solvent is propylene glycol), in which the mass fraction of copper peptide and tranexamic acid is 1%.

[0094] Comparative Example 2 Compared with the anhydrous essence in Example 1, the only difference is that salicylic acid is replaced by an equal amount of 1% Gly-His-TXA-Cu solution (solvent is propylene glycol).

[0095] Comparative Example 3 The only difference between this and the anhydrous essence in Example 1 is that the 1% Gly-His-TXA-Cu solution is replaced with an equal amount of salicylic acid.

[0096] Test case Subjects: Eighty healthy volunteers aged 18 to 40 years with moderate acne vulgaris on the face and significant post-inflammatory erythema (PIE) and hyperpigmentation (PIH) were recruited.

[0097] Experimental Methods: Subjects were randomly divided into 8 groups, each using the anhydrous essences described in Examples 1-5 and Comparative Examples 1-3, respectively. Subjects applied 1 mL of the designated product evenly to their faces after cleansing morning and evening. During the experiment, all subjects used a uniformly provided gentle facial cleanser.

[0098] The subjects' skin condition was assessed under standardized conditions of constant temperature and humidity before the start of the trial (D0) and at week 8 after the start of the trial (D56), and the changes in the subjects' skin condition after 8 weeks were calculated.

[0099] The inflammatory and non-inflammatory skin lesions of the subjects were counted in a blinded manner by dermatologists.

[0100] Assessing red acne marks (PIE): Measure the skin redness value of the target acne mark area using a chromameter. ).

[0101] Assessment of acne scars (PIH): Individual type angle of the target acne scar area is measured using a colorimeter. This value is positively correlated with skin brightness.

[0102] The results are shown in Table 1.

[0103] Table 1 From the above results, we can conclude that: In Examples 1-5, Examples 1-3 showed PIE (pigmentation index) in inflammatory lesions and post-inflammatory erythema. The best performance was seen in improving non-inflammatory lesions (blackheads) and inflammatory pigmentation (PIH - Individual Type Opacity). Examples 4-5 showed the best improvement in non-inflammatory lesions (blackheads) and inflammatory pigmentation (PIH - Individual Type Opacity). The improvement is more prominent because retinaldehyde in Example 4 and psoralen in Example 5 have advantages in promoting keratin metabolism and whitening. The overall effect is slightly different from that of Examples 1-3, but equally excellent.

[0104] Comparative Example 1: After replacing the 1% Gly-His-TXA-Cu solution in Example 1 with an equal amount of a mixed solution of copper peptide and tranexamic acid, the reduction rates of both inflammatory and non-inflammatory skin lesions decreased, and the skin redness value of the acne scar area decreased. The changes in α and β angle (ITA°) were both reduced, indicating that the Gly-His-TXA-Cu solution of the present invention has a better effect on improving facial acne, post-inflammatory erythema (PIE) and post-inflammatory hyperpigmentation (PIH) compared with the physical mixture solution of copper peptide and tranexamic acid, proving the superiority of the Gly-His-TXA-Cu complex.

[0105] In Comparative Example 2, after replacing an equal amount of salicylic acid in Example 1 with a 1% (w / w) Gly-His-TXA-Cu solution, the reduction rates of both inflammatory and non-inflammatory skin lesions were significantly reduced, and the skin redness value of the acne scar area ( ) changes decreased, individual type angle value ( The change was significantly reduced; in Comparative Example 3, after replacing the 1% Gly-His-TXA-Cu solution in Example 1 with an equal amount of salicylic acid, the reduction rates of both inflammatory and non-inflammatory skin lesions decreased, and the skin redness value of the acne scar area ( ) changes, individual type angle values ​​( The significant reduction in changes demonstrates the synergistic effect of the Gly-His-TXA-Cu solution and acidic substances in this invention. The acidic substances open the hair follicle channels, allowing Gly-His-TXA-Cu to reach the inflammatory sites of acne more effectively, further reducing inflammatory and non-inflammatory skin lesions in the subjects, and effectively improving post-inflammatory erythema and post-inflammatory hyperpigmentation.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An anhydrous essence, characterized in that, The anhydrous essence comprises, by weight percentage, the following components: 50%–80% non-aqueous solvent, 1%–20% penetration enhancer, 5%–15% emollient, 0.5%–2% acidic substances, 2%–10% active ingredients, 0.5%–10% Gly-His-TXA-Cu solution, and 0.1%–1.0% anti-inflammatory and soothing agent; The structural formula of the Gly-His-TXA-Cu is shown in formula (I): Formula (I).

2. The anhydrous essence according to claim 1, characterized in that, The anhydrous essence comprises the following components by weight percentage: 60%~80% non-aqueous solvent, 5%~15% penetration enhancer, 5%~10% emollient, 1%~2% acidic substances, 2%~8% active substances, 1%~5% Gly-His-TXA-Cu solution, and 0.1%~1.0% anti-inflammatory soothing agent.

3. The anhydrous essence according to claim 1 or 2, characterized in that, The solvent for the Gly-His-TXA-Cu solution is the non-aqueous solvent; and / or, The mass concentration of Gly-His-TXA-Cu in the Gly-His-TXA-Cu solution is 0.5% to 5%.

4. The anhydrous essence according to claim 1 or 2, characterized in that, The acidic substance is at least one of salicylic acid, azelaic acid, or mandelic acid.

5. The anhydrous essence according to claim 1 or 2, characterized in that, The penetration enhancer is at least one of ethoxydiethylene glycol, isosorbide dimethyl ether, or pentylene glycol.

6. The anhydrous essence according to claim 1 or 2, characterized in that, The emollient is at least one of squalane, caprylic / capric triglyceride, or isononyl isononanoate; and / or The non-aqueous solvent is at least one of propylene glycol, butanediol, or 1,3-propanediol; and / or, The active substance is at least one of nicotinamide, retinaldehyde, or bakuchiol; and / or, The anti-inflammatory and soothing agent is at least one of dipotassium glycyrrhizate, bisabolol, or 4-tert-butylcyclohexanol.

7. A method for preparing an anhydrous essence, characterized in that, The preparation of the anhydrous essence according to any one of claims 1 to 6 comprises the following steps: Weigh each component according to the component ratio of the anhydrous essence according to any one of claims 1 to 6; A phase A is obtained by mixing a non-aqueous solvent, a penetration enhancer, and a skin emollient. The acidic substance, active substance, Gly-His-TXA-Cu solution, and anti-inflammatory soothing agent are added to phase A and stirred to dissolve, thus obtaining the anhydrous essence.

8. The method for preparing anhydrous essence according to claim 7, characterized in that, The mixing speed for non-aqueous solvents, penetration enhancers, and emollients is 300 rpm to 600 rpm.

9. The method for preparing anhydrous essence according to claim 7 or 8, characterized in that, The acidic substances, active substances, Gly-His-TXA-Cu solution, and anti-inflammatory soothing agents are added to phase A and stirred to dissolve. The temperature is 40℃~50℃ and the stirring speed is 300rpm~600rpm.

10. The use of the anhydrous essence according to any one of claims 1 to 6 in improving acne-induced post-inflammatory hyperpigmentation.

Images