A hydroxytyrosol supramolecular complex, and a preparation method and application thereof

Abstract

The application provides a hydroxytyrosol supramolecular complex and a preparation method and application thereof, and belongs to the fields of cosmetics and biological medicine technology.The supramolecular complex contains the following chemical components in mass fraction: 0.01-10% of hydroxytyrosol, 2-45% of an amino acid compound, 2-20% of alpha hydroxy acid, 2-30% of urea and 15-92% of water.The supramolecular complex can promote the transdermal delivery of hydroxytyrosol and keep the biological activity of hydroxytyrosol, and meanwhile, the supramolecular complex has high biocompatibility and can be applied to product development in the fields of antibacterial, regeneration and anti-aging.

Description

Technical Field

[0001] This invention belongs to the fields of cosmetics and biomedicine, and specifically relates to a hydroxytyrosol supramolecular complex, its preparation method, and its application. Background Technology

[0002] Hydroxytyrosol (HT), also known as oleanolic acid, has the chemical name 3,4-dihydroxyphenylethanol and the molecular formula C8H. 10 O3, with a relative molecular weight of 154.16, is one of the main antioxidant components in olive oil and possesses good pharmacological activity. Hydroxytyrosol, with high water solubility and moderate lipid solubility, is a natural small-molecule phenolic compound mainly found in olive fruits and leaves. Considered one of the strongest antioxidants, it also possesses various biological activities such as antibacterial, anti-inflammatory, lipid-regulating, anti-atherosclerotic, anti-pathogenic microorganism, bone protection, and osteoporosis prevention, making it a natural product with significant application and development value. Currently, hydroxytyrosol is widely used in pharmaceuticals, food, daily chemicals, and animal feed. However, hydroxytyrosol is unstable to light and heat, easily oxidizing and discoloring during use, gradually losing its activity. Furthermore, its direct transdermal absorption efficiency is low, limiting its application in cosmetics, food, and pharmaceuticals. Currently, co-crystallization technology, inclusion technology, or the addition of antioxidants are mainly used to improve the stability of hydroxytyrosol. These methods can improve the stability and melting point of hydroxytyrosol to a certain extent, expanding its application scenarios.

[0003] Regarding cocrystallization technology, Chinese patent application CN202110244109, "Hydroxytyrosol-Betaine Cocrystallization, Preparation Method and Composition Thereof," discloses a method for preparing hydroxytyrosol-betaine cocrystallization and its application. The hydroxytyrosol-betaine cocrystallization of this invention has a high melting point, is non-hygroscopic, and has good stability, greatly improving the ease of use and chemical stability of hydroxytyrosol. Chinese patent application CN202511072347, "A Hydroxytyrosol-Ergothione Cocrystallization, Its Preparation Method and Application Thereof," discloses a hydroxytyrosol-ergothione cocrystallization, its preparation method and application. This hydroxytyrosol-ergothione cocrystallization improves the stability of hydroxytyrosol and enhances the stability, antioxidant properties, and anti-aging effects of both hydroxytyrosol and ergothione. While hydroxytyrosol cocrystallization technology can improve the melting point and stability of hydroxytyrosol to a certain extent, the preparation process involves the use of organic solvents, making the process relatively cumbersome.

[0004] Regarding inclusion technology, Chinese patent application No. 201910315938.5, "A Hydroxy Ester Alcohol Inclusion Complex and Its Preparation Method and Uses," utilizes hydroxypropyl-β-cyclodextrin inclusion technology, further adding protective agents (vitamin C, vitamin E, glutathione, acetylcysteine, etc.) to improve the stability of hydroxy ester alcohols to light and high temperatures, expanding their application range. The resulting hydroxy ester alcohol inclusion complex can be used in the preparation of food, health food, functional food, and cosmetics. Regarding hydroxytyrosol inclusion technology, currently, cyclodextrin or its derivatives are mainly used for inclusion of hydroxytyrosol. The main problem is low inclusion efficiency; simple inclusion has a weak effect on improving the long-term stability of hydroxytyrosol in water. Even when the inclusion technology is combined with antioxidants (such as vitamin C), it still undergoes severe discoloration after long-term storage in aqueous solution at high temperatures.

[0005] Regarding the technology of stabilizing hydroxytyrosol with antioxidants, Chinese patent application No. 20241032477.X, "A Composition, Application and Preparation Method for Preventing Oxidative Discoloration of Hydroxytyrosol," discloses a hydroxytyrosol-reducing agent composition, its preparation method, and its application. Reducing agents such as sodium metabisulfite, L-cysteine, and vitamin C can improve the stability of hydroxytyrosol. However, the combined use of sodium metabisulfite and vitamin C with hydroxytyrosol still cannot improve the long-term stability of hydroxytyrosol in aqueous solution. Furthermore, when L-cysteine ​​is used in combination with hydroxytyrosol, long-term storage presents the problem of oxidation and precipitation, limiting its application. Summary of the Invention

[0006] To address the issues of low transdermal absorption efficiency and low stability of hydroxytyrosol, this invention provides a hydroxytyrosol supramolecular complex. This supramolecular complex can promote the transdermal delivery of hydroxytyrosol while maintaining its bioactivity, and exhibits high biocompatibility. It can be applied to product development in the fields of antibacterial, regenerative, and anti-aging.

[0007] The present invention also provides a method for preparing and applying a hydroxytyrosol supramolecular complex.

[0008] This invention is achieved through the following technical solution:

[0009] This invention provides a hydroxytyrosol supramolecular complex, which, by mass fraction, comprises the following chemical components:

[0010] Hydroxytyrosol: 0.01–10%, amino acid compounds: 2–45%, α-hydroxy acids: 2–20%, urea: 2–30%, water: 15–92%.

[0011] Furthermore, the amino acid compounds include at least one of glycine, arginine, serine, threonine, glutamine, valine, acetylglutamine, cysteine, acetylcysteine, carboxymethylcysteine, betaine, L-carnitine, and ergothioneine;

[0012] The α-hydroxy acid includes at least one of lactic acid, citric acid, malic acid, succinic acid, ketoglutaric acid, tartaric acid, and mandelic acid.

[0013] Based on the same inventive concept, this invention provides a method for preparing a hydroxytyrosol supramolecular complex, the method comprising:

[0014] Under an inert atmosphere, α-hydroxy acids are heated and melted, then amino acid compounds and urea are added, and after homogenization, a reaction mixture is obtained.

[0015] The temperature of the reaction mixture was adjusted to 20–90°C, water and hydroxytyrosol were added, and the mixture was homogenized to obtain a mixed solution.

[0016] Add a pH adjuster to adjust the pH of the mixed solution to 4-8 to obtain a hydroxytyrosol supramolecular complex;

[0017] The mass fractions of each chemical component in the hydroxytyrosol supramolecular complex are as follows:

[0018] Hydroxytyrosol: 0.01–10%, amino acid compounds: 2–45%, α-hydroxy acids: 2–20%, urea: 2–30%, water: 15–92%.

[0019] Furthermore, the amino acid compounds include at least one of glycine, arginine, serine, threonine, glutamine, valine, acetylglutamine, cysteine, acetylcysteine, carboxymethylcysteine, betaine (trimethylglycine), L-carnitine, and ergothioneine;

[0020] The α-hydroxy acid includes at least one of lactic acid, citric acid, malic acid, succinic acid, ketoglutaric acid, tartaric acid, and mandelic acid.

[0021] Furthermore, the process of heating and melting the α-hydroxy acid under an inert atmosphere, followed by the addition of an amino acid compound and urea, and then homogenizing the mixture to obtain a reaction mixture specifically includes:

[0022] Under nitrogen protection, α-hydroxy acids were heated and melted, and then amino acid compounds and urea were slowly added. After homogenization for 0.5 to 4 hours, a reaction mixture was obtained.

[0023] The process of adjusting the temperature of the reaction mixture to 20–90°C, adding water and hydroxytyrosol, and homogenizing to obtain a mixed solution specifically includes:

[0024] Adjust the temperature of the reaction mixture to 20–90°C, add water and hydroxytyrosol, and homogenize for 0.5–12 h to obtain a mixed solution.

[0025] Furthermore, the pH adjuster includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, disodium citrate, trisodium citrate, disodium hydrogen phosphate, triethanolamine, ammonia, citric acid, acetic acid, and hydrochloric acid.

[0026] Based on the same inventive concept, this invention provides the application of a hydroxytyrosol supramolecular complex in the preparation of any one of antibacterial reagents, anti-inflammatory reagents, hair regeneration reagents, anti-aging reagents, and antioxidant reagents.

[0027] Based on the same inventive concept, this invention provides the application of a hydroxytyrosol supramolecular complex in the preparation of hair regeneration drugs or skin care products.

[0028] Based on the same inventive concept, the present invention also provides a hair regeneration drug or skin care product, wherein the hair regeneration drug or skin care product contains the above-mentioned hydroxytyrosol supramolecular complex.

[0029] Furthermore, the dosage form of the hair regeneration drug or the skin care product includes any one of the following: spray, paste, cream, patch, soluble microneedle, gel, and ointment.

[0030] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0031] 1. This invention discloses a hydroxytyrosol supramolecular complex comprising an amino acid compound, an α-hydroxy acid, urea, water, and hydroxytyrosol. The amino acid compound acts as a hydrogen bond acceptor with extremely high affinity and extremely low sensitization rate. The α-hydroxy acid acts as a hydrogen bond donor, exhibiting softening and removal of excessive keratinization in the skin layer, and also promoting collagen synthesis and enhancing skin moisturizing ability. The supramolecular system composed of the amino acid compound and the α-hydroxy acid can instantaneously disrupt the highly ordered lipid layer structure of the stratum corneum, thereby enhancing the penetration of hydroxytyrosol. In addition, the extensive hydrogen bond network inside the supramolecular structure can act as a barrier to oxygen diffusion, effectively protecting hydroxytyrosol from oxidation, thereby promoting transdermal delivery of hydroxytyrosol and maintaining its biological activity, while exhibiting high biocompatibility. Urea can effectively reduce the viscosity of the system and enhance fluidity by weakening intermolecular attraction, which can further promote drug penetration.

[0032] 2. This invention discloses a hydroxytyrosol supramolecular complex, which effectively improves the long-term stability of hydroxytyrosol in aqueous solution through supramolecular interaction. Even under high temperature conditions for up to 3 months, the hydroxytyrosol aqueous solution at a concentration of ≥1% can still maintain color stability for a long time. On the other hand, the preparation of this supramolecular complex does not involve complex reaction steps, and the supramolecular system used is green, safe, and economical. It can effectively reduce skin irritation and has good skin affinity and penetration-enhancing effects. This supramolecular complex can effectively improve the antibacterial activity, antioxidant capacity, and hair regeneration effect of hydroxytyrosol, and can be applied to product development in the fields of antibacterial, regeneration, and anti-aging. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 Color changes and stability of hydroxytyrosol supramolecular complexes from different embodiments after being placed at room temperature (20°C) and high temperature (45°C) for 60 days.

[0035] Figure 2 The color changes and stability of hydroxytyrosol supramolecular complexes with different ratios were analyzed after being placed at room temperature (20℃) and high temperature (45℃) for 3 days.

[0036] Figure 3 The color changes and stability of hydroxytyrosol supramolecular complexes with different ratios were analyzed after being placed at room temperature (20℃) and high temperature (45℃) for 15 days.

[0037] Figure 4Design and characterization of supramolecular complexes: (A) Preparation process of BUL; (B) Photographs of different combinations of betaine, urea and lactic acid; (C) Absorbance changes of HT@H2O (Comparative Example 1) and HT@BUL (Example 11) after six days of outdoor exposure; (D) Shear stress-shear rate relationship of BU, BL and BUL; (E) Shear viscosity-shear rate relationship of BU, BL and BUL; (F) Proton nuclear magnetic resonance (1H NMR) spectrum (400 MHz, DMSO-d6); (G) Fourier transform infrared (FTIR) spectrum of BUL and its components; (H) Differential scanning calorimetry (DSC) thermogram of BUL and its components; (I) Differential thermogravimetric (DTG) curves of BUL and its components; (J) Thermogravimetric analysis (TGA) of BUL and its components; (K) X-ray diffraction (XRD) pattern of BUL and its components.

[0038] Figure 5 Antioxidant and skin penetration analysis of Example 11 (HT@BUL) and Comparative Example 1 (HT@H2O): (B) ABTS and (A) DPPH free radical scavenging activities of Example 11 and Comparative Example 1 (n=3); (C) Schematic diagram of contact angles of HT@H2O and HT@BUL coated on the oil surface; (D) Statistical graph of contact angles (n=3); (E) Longitudinal cross-sectional fluorescence images of RhB@H2O and RhB@BUL coated on the skin; (F) Statistical graph of RhB fluorescence penetration depth in the skin (n=10 observation fields).

[0039] Figure 6 For Example 10 (HT@BUL-L) and Example 11 (HT@BUL-H) in vivo hair regeneration experiments: (A) Schematic diagram of the AGA model constructed by daily treatment with testosterone for 16 days, HT, minoxidil, HT@BUL-L, HT@BUL-H, and BUL were administered daily for up to 16 days; (B) Photographs of mouse back hair from day 1 to day 16; (C) Scanning electron micrographs of regenerated hair; (D) Statistical results of hair regeneration area in mice on day 16 (n=5 mice); (E) Statistical results of the growth rate of melanin coverage area in mouse skin from day 1 to day 16; (F) Diameter of regenerated hair (n = 20 hairs).

[0040] Figure 7Results of hair growth induction in Examples 10 and 11: (A) HE staining images of treated mouse skin on day 16; (B) Statistical results of the number of hair follicles in treated mouse skin (n = 5 mice); (C) Masson staining images of treated mouse skin on day 16; (D) Statistical results of the thickness of treated mouse skin (n = 5 mice).

[0041] Figure 8 To illustrate the effects of Examples 10 and 11 on reducing oxidative stress and promoting HFSC proliferation: (A) Representative images of SOX9 in different skin tissues on day 16 post-hair removal (red for SOX9, blue for DAPI); (B) Representative images of EDU and SOX9 in different skin tissues on day 16 post-hair removal (red for SOX9, green for EDU; blue for DAPI); (C) Representative images of 8-ohdg in different skin tissues on day 16 post-hair removal (red for 8-ohdg; blue for DAPI); (D) SOX9 positive cell count (n = 5 mice); (E) EDU and SOX9 positive cell count (n = 5 mice); (F) Quantitative analysis of 8-ohdg intensity (n = 5 mice).

[0042] Figure 9 The results of the biocompatibility test of HT@BUL are as follows: (AF) the relative survival rate of cells after co-incubation with HDPC for 24 hours and 48 hours with different concentrations of HT, BUL, and HT@BUL (n = 3); (G) images of HDPC treated with HT, BUL, and HT@BUL for 24 hours and 48 hours, respectively, with double staining of calcein-AM (live cells, green) / PI (dead cells, red) in the cytoplasm.

[0043] Figure 10The effects of HT@BUL on the proliferation, antioxidant and anti-aging effects of human dermal papillary cells (HDPC) were investigated. The results included: (A) Ki67 images of cells treated with HT, BUL, and HT@BUL for 24 hours (green represents Ki67, blue represents DAPI); (B) Statistical analysis of the percentage of Ki67-positive cells in HDPC cells treated with H2O2 after co-incubation with HT, BUL, and HT@BUL for 24 hours (n=3); (C) Quantitative analysis of the fluorescence intensity of DHE in cells measured by a microplate reader (n=5); (D) DHE images of cells; (E) β-Gal images of cells; enzyme marker measurement of β-Gal images; and (F) Statistical analysis of the percentage of β-Gal cells (n=3). Detailed Implementation

[0044] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0045] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0046] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0047] The overall concept of this invention is as follows:

[0048] Hydroxytyrosol is a natural polyphenol compound with potent antioxidant activity, proven to protect skin cells from oxidative stress-induced inflammation. However, its clinical application is limited by its high hydrophilicity, which hinders its ability to penetrate the skin's lipid barrier, leading to poor skin absorption. Furthermore, hydroxytyrosol is easily oxidized, further impacting its efficacy.

[0049] To address the issues of low transdermal absorption efficiency and low stability of hydroxytyrosol, this invention proposes a hydroxytyrosol supramolecular complex and its preparation method. The complex comprises an amino acid compound, an α-hydroxy acid, urea, water, and hydroxytyrosol. This supramolecular complex promotes transdermal delivery of hydroxytyrosol while maintaining its biological activity and exhibits high biocompatibility.

[0050] Supramolecular systems, composed of hydrogen bond donor and acceptor pairs, offer several advantages in their preparation, including ease of preparation, biodegradability, low toxicity, and high thermal stability. The applicant discovered that supramolecular systems can transiently disrupt the highly ordered lipid layer structure of the stratum corneum, thereby enhancing transdermal drug penetration. Furthermore, the extensive hydrogen bond network within the supramolecular system acts as a barrier to oxygen diffusion, effectively protecting the encapsulated drug from oxidation. Therefore, as an "active stabilizer" against easily oxidized agents, supramolecular systems provide a feasible solution for improving the local delivery and stability of antioxidants such as hydroxytyrosol.

[0051] The applicant discovered that among various supramolecular systems, natural supramolecular systems (amino acids, α-hydroxy acids) possess excellent biocompatibility and transdermal absorption-enhancing effects. Amino acids, as important hydrogen bond acceptors, are the basic units constituting skin proteins and natural moisturizing factors, exhibiting extremely high affinity and very low sensitization rates. On the other hand, α-hydroxy acids, as important hydrogen bond donors, soften and remove excessive keratinization in the skin layer, promote collagen synthesis, and enhance skin moisturizing ability. The applicant's experiments showed that urea can effectively reduce system viscosity and enhance fluidity by weakening intermolecular attraction. Previous research also found that urea, as an important dissociative agent, can effectively promote the retention and penetration of drugs such as peptides in the mucosal layer. Based on these complementary properties, this invention proposes a supramolecular system composed of amino acid compounds, urea, α-hydroxy acids, water, and hydroxytyrosol. This formulation combines a dense hydrogen bond network, mild acidity, and low viscosity, providing ideal conditions for efficient and biocompatible transdermal delivery while improving the stability of hydroxytyrosol aqueous solutions.

[0052] Specifically, this invention proposes a hydroxytyrosol supramolecular complex, which, by mass fraction, comprises the following chemical components:

[0053] Hydroxytyrosol: 0.01–10%, amino acid compounds: 2–45%, α-hydroxy acids: 2–20%, urea: 2–30%, water: 15–92%.

[0054] In this invention, the mass fraction of hydroxytyrosol is 0.01-10%. A suitable concentration range can better exert its antioxidant activity, while excessively high concentrations of hydroxytyrosol cannot guarantee the safety of its biological applications.

[0055] The mass fraction of amino acid compounds is 2-45%. As hydrogen bond acceptors (HBAs), excessive content of these compounds will lead to an increase in "free" HBAs in the system, while the total number of hydrogen bonds will decrease. This can easily cause problems such as crystallization or phase separation upon cooling, a sudden increase in viscosity, and a decrease in conductivity, which is not conducive to transdermal absorption.

[0056] The mass fraction of α-hydroxy acid is 2-20%. As a hydrogen bond donor (HBD) in the system, if the content is too low, hydroxytyrosol will be difficult to dissolve and the hydrogen bond structure formed in the system will be unstable, resulting in poor thermal stability.

[0057] Urea has a mass fraction of 2-30%. As a hydrogen bond donor in the system, it can form certain hydrogen bonds with amino acid compounds. At the same time, it can effectively reduce the viscosity of the system and enhance its fluidity. However, as a liquid release agent, if the content is too high, it will increase the irritation of the system, and if it is too low, it will lead to excessively high viscosity of the system.

[0058] This invention also proposes a method for preparing a hydroxytyrosol supramolecular complex, the method comprising:

[0059] S1. Under an inert atmosphere, α-hydroxy acid is heated and melted, then amino acid compounds and urea are added, and after homogenization, a reaction mixture is obtained;

[0060] S2. Adjust the temperature of the reaction mixture to 20-90°C, add water and hydroxytyrosol, and homogenize to obtain a mixed solution;

[0061] S3. Add a pH adjuster to adjust the pH of the mixed solution to 4-8 to obtain a hydroxytyrosol supramolecular complex;

[0062] The mass fractions of each chemical component in the hydroxytyrosol supramolecular complex are as follows:

[0063] Hydroxytyrosol: 0.01–10%, amino acid compounds: 2–45%, α-hydroxy acids: 2–20%, urea: 2–30%, water: 15–92%.

[0064] In this invention, the advantage of first preparing a reaction mixture by mixing α-hydroxy acid, amino acid compounds and urea before adding water is that it can prevent water molecules from prematurely associating with small molecules to form hydrogen bonds.

[0065] Adjust the temperature of the reaction mixture to 20–90°C to dissolve the solid components and make it easier for the components to associate and form hydrogen bonds.

[0066] In this invention, adjusting the pH of the mixed solution to 4-8 has the advantage of preventing hydroxytyrosol from being easily oxidized at high pH.

[0067] The following will provide a detailed description of a hydroxytyrosol supramolecular complex, its preparation method, and its application, in conjunction with embodiments and experimental data.

[0068] I. Example

[0069] The chemical composition and weight parts of the hydroxytyrosol supramolecular complexes of Examples 1-11 are shown in Table 1 below, and the detailed preparation methods are as follows:

[0070] For the hydroxytyrosol supramolecular complex of Example 1, based on the preparation of 100 parts by weight of the supramolecular complex, under nitrogen protection, 6 parts by weight of lactic acid (Shanghai Maclean Biochemical Technology Co., Ltd.) with a melting point of approximately 54°C were prepared and heated to 60°C to prepare a solution. 4 parts by weight of glycine (Shanghai Maclean Biochemical Technology Co., Ltd.) and 10 parts by weight of urea (Shanghai Maclean Biochemical Technology Co., Ltd.) were added to the above lactic acid solution, and the temperature was maintained while stirring at 100 rpm for 2 hours to ensure complete mixing and reaction. The temperature of the mixture was adjusted to 70°C, and 1 part by weight of hydroxytyrosol (Shaanxi Fuheng Biotechnology Co., Ltd.) and 76.6 parts by weight of pure water were slowly added, along with 2.4 parts by weight of pure NaOH powder. The pH of the solution was adjusted to 5.8 until the total mass of the supramolecular complex was 100 parts by weight. The final product was a hydroxytyrosol supramolecular complex containing 4 parts by weight of glycine, 6 parts by weight of lactic acid, 10 parts by weight of urea, 1 part by weight of hydroxytyrosol, 76.6 parts by weight of pure water, with the remainder being NaOH.

[0071] The hydroxytyrosol supramolecular complexes of Examples 2-3 were based on a total of 100 parts by weight of supramolecular complexes. The 1 part by weight of hydroxytyrosol in Example 1 was replaced with 2 parts by weight and 5 parts by weight, respectively, while the other preparation processes remained unchanged. In this case, Example 2 contained 4 parts by weight of glycine, 6 parts by weight of lactic acid, 10 parts by weight of urea, 75.3 parts by weight of purified water, and 2 parts by weight of hydroxytyrosol; Example 3 contained 4 parts by weight of glycine, 6 parts by weight of lactic acid, 10 parts by weight of urea, 72.1 parts by weight of purified water, and 5 parts by weight of hydroxytyrosol.

[0072] The supramolecular complexes in Examples 4-7 were prepared by replacing 4 parts by weight of glycine (Shanghai Maclean Biotechnology Co., Ltd.), valine (Shanghai Maclean Biotechnology Co., Ltd.), arginine (Shanghai Maclean Biotechnology Co., Ltd.), and serine (Shanghai Maclean Biotechnology Co., Ltd.) with 4 parts by weight of glycine (Shanghai Maclean Biotechnology Co., Ltd.), valine (Shanghai Maclean Biotechnology Co., Ltd.), and serine (Shanghai Maclean Biotechnology Co., Ltd.) in Example 1, while simultaneously changing the content of purified water and NaOH. Specific component and content details are shown in Table 1.

[0073] For the hydroxytyrosol supramolecular complexes of Examples 8 and 9, based on 100 parts by weight of the supramolecular complex, 6 parts by weight of lactic acid in Example 1 were replaced with 6 parts by weight of malic acid (melting point 130℃, Shanghai Maclean Biochemical Technology Co., Ltd.) and 6 parts by weight of anhydrous citric acid (melting point 159℃, Shanghai Maclean Biochemical Technology Co., Ltd.), while simultaneously changing the content of purified water and NaOH. Specific component and content details are shown in Table 1.

[0074] For the hydroxytyrosol supramolecular complex of Example 10, based on 100 parts by weight of the supramolecular complex, 16 parts by weight of lactic acid with a melting point of approximately 54°C were prepared under nitrogen protection and heated to 75°C to prepare a solution. 42 parts by weight of betaine (Shanghai Maclean Biochemical Technology Co., Ltd.) and 25 parts by weight of urea were added to the above lactic acid solution. The temperature was then maintained and the mixture was stirred at 150 rpm for 2 hours to allow complete reaction. At this temperature, 0.5 parts by weight of hydroxytyrosol and 16.5 parts by weight of pure water were added until the total mass of the supramolecular complex was 100 parts by weight. The mixture was then homogenized at 150 rpm for 2 hours to obtain a supramolecular complex loaded with a low concentration of hydroxytyrosol, named HT@BUL-L.

[0075] It is worth noting that, in order to prepare a supramolecular complex of betaine / urea / lactic acid / pure water without hydroxytyrosol, named BUL, it is of great significance to study its physicochemical properties separately. The preparation process of BUL is as follows: Based on 100 parts by weight of the BUL complex, 16 parts by weight of lactic acid with a melting point of approximately 54°C were prepared under nitrogen protection and heated to 75°C to prepare a solution. 42 parts by weight of betaine and 25 parts by weight of urea were added to the above lactic acid solution, and homogenized at 150 rpm for 2 h. Subsequently, 17 parts by weight of pure water were added, and homogenized at 150 rpm for 2 h to allow complete reaction and prepare BUL.

[0076] For the hydroxytyrosol supramolecular complex of Example 11, based on 100 parts by weight of the supramolecular complex, 0.5 parts by weight of hydroxytyrosol in Example 10 were replaced with 1.0 parts by weight, while the other preparation conditions remained unchanged, to obtain the sample of Example 11, which was named HT@BUL (in...). Figure 6-8 In order to distinguish it from HT@BUL-L, it is named HT@BUL-H.

[0077] Table 1. Composition, weight parts, and pH parameters of the hydroxytyrosol supramolecular complexes in Examples 1-11.

[0078]

[0079] II. Comparative Example

[0080] The chemical composition and weight parts of the hydroxyl tyrosine complexes of Comparative Examples 1-11 are shown in Table 2 below. The detailed preparation process is as follows:

[0081] Comparative Example 1: Hydroxytyrosol / pure water complex group (HT@H2O). Using 100 parts by weight of the hydroxytyrosol complex as a baseline, under nitrogen protection and at room temperature, 1 part by weight of hydroxytyrosol was added to 97.9 parts by weight of pure water, and 1.1 parts by weight of NaOH was added to adjust the pH to 5.7. Then, the temperature was maintained and the mixture was stirred at 2000 rpm for 2 h to completely dissolve it, thus preparing a weakly acidic aqueous solution of hydroxytyrosol.

[0082] Comparative Example 2: Hydroxytyrosol / urea / pure water complex group. Using 100 parts by weight of the hydroxytyrosol complex as a baseline, 25 parts by weight of urea were added to a homogenizer containing a certain amount of pure water at 75°C under nitrogen protection. The mixture was stirred at 2000 rpm for 2 hours while maintaining the temperature until completely dissolved. After cooling to room temperature, 1 part by weight of hydroxytyrosol was added, followed by titration with pure water to a total of 100 parts by weight.

[0083] Comparative Example 3: Hydroxytyrosol / Lactic Acid / Pure Water Complex. Using 100 parts by weight of the hydroxytyrosol complex as a baseline, 16 parts by weight of lactic acid were added to an appropriate amount of pure water at 75°C under nitrogen protection. The temperature was maintained, and the mixture was stirred at 100 rpm for 2 hours until completely dissolved. After cooling to room temperature, 1 part by weight of hydroxytyrosol was added, and the mixture was stirred for 1 hour. Subsequently, a pH adjuster was added to bring the pH of the complex to a slightly acidic level, and finally, pure water was added and titrated to 100 parts by weight.

[0084] Comparative Example 4: Hydroxytyrosol / betaine / pure water complex. Using 100 parts by weight of the hydroxytyrosol complex as a baseline, 42 parts by weight of betaine were added to an appropriate amount of pure water at 75°C under nitrogen protection. The temperature was maintained, and the mixture was stirred at 150 rpm for 2 hours until completely dissolved. While maintaining this temperature, 1 part by weight of hydroxytyrosol was added, and the mixture was stirred for 1 hour. Subsequently, a pH adjuster was added to bring the pH of the complex to a slightly acidic level, and finally, pure water was added and titrated to 100 parts by weight.

[0085] Comparative Example 5: Hydroxytyrosol / Lactic Acid / Urea / Pure Water Complex. Using 100 parts by weight of the hydroxytyrosol complex as a baseline, 16 parts by weight of lactic acid were heated to melt at 75°C under nitrogen protection. 25 parts by weight of urea were added to the solution, and the mixture was stirred at 200 rpm for 2 hours to ensure complete reaction. Subsequently, 1 part by weight of hydroxytyrosol and pure water were added at the same temperature, and the mixture was homogenized at 200 rpm for 1 hour. Finally, a pH adjuster was added to bring the pH of the complex to a slightly acidic level.

[0086] Comparative Example 6: Hydroxytyrosol / Lactic Acid / Betaine / Pure Water Complex. Using 100 parts by weight of the hydroxytyrosol complex as a baseline, 16 parts by weight of lactic acid were heated to melt at 75°C under nitrogen protection. 42 parts by weight of betaine were added to the solution, and the mixture was stirred at 200 rpm for 2 h. Subsequently, while maintaining the temperature, 1 part by weight of hydroxytyrosol and an appropriate amount of pure water were added, and the mixture was stirred at 200 rpm for 2 h to allow for complete reaction. Finally, a pH adjuster was added to bring the pH of the complex to a slightly acidic level.

[0087] To prepare a betaine / lactic acid / pure water complex without hydroxytyrosol, named BL, its physicochemical properties were investigated separately to provide insights into the mechanism of supramolecular complexes. The preparation process of BL is as follows: Based on 100 parts by weight of the BL complex, under nitrogen protection, 16 parts by weight of lactic acid were heated to 75°C and reacted completely for 2 h. Subsequently, 42 parts by weight of betaine and 42 parts by weight of pure water were added, and the temperature was maintained while stirring at 200 rpm for 2 h to ensure complete reaction and obtain BL.

[0088] Comparative Example 7: Hydroxytyrosol / betaine / urea / pure water complex group. Based on 100 parts by weight of the hydroxytyrosol complex. Under nitrogen protection, at 75°C, 42 parts by weight of betaine and 25 parts by weight of urea were added to an appropriate amount of pure water. The temperature was maintained and the mixture was stirred at 200 rpm for 2 hours until completely dissolved. After cooling to room temperature, 1 part by weight of hydroxytyrosol was added, and the mixture was stirred at 200 rpm for 1 hour.

[0089] Similarly, to prepare a betaine / urea / pure water complex without hydroxytyrosol, named BU, its physicochemical properties were studied separately to provide insights into the mechanism of supramolecular complexes. The preparation process of BU is as follows: Based on 100 parts by weight of the BU complex, under nitrogen protection, 42 parts by weight of betaine and 25 parts by weight of urea were added to 33 parts by weight of pure water. The mixed solution was heated to 75°C, maintained at the temperature, and stirred at 200 rpm for 2 h to ensure complete reaction and obtain BU.

[0090] Comparative Example 8: Hydroxytyrosol / proline / pure water complex group. Using 100 parts by weight of the hydroxytyrosol complex as a baseline, 4 parts by weight of proline were added to an appropriate amount of pure water at 75°C under nitrogen protection. The temperature was maintained, and the mixture was stirred at 200 rpm for 2 hours until completely dissolved. After cooling to room temperature, 1 part by weight of hydroxytyrosol was added, and the mixture was stirred for 1 hour. Subsequently, a pH adjuster was added to bring the pH of the complex to a slightly acidic level.

[0091] Comparative Example 9: Hydroxytyrosol / glycine / pure water complex group. Using 100 parts by weight of the hydroxytyrosol complex as a baseline, 4 parts by weight of glycine were added to an appropriate amount of pure water at 75°C under nitrogen protection. The temperature was maintained, and the mixture was stirred at 200 rpm for 2 hours until completely dissolved. After cooling to room temperature, 1 part by weight of hydroxytyrosol was added, and the mixture was stirred at 200 rpm for 1 hour. Finally, pure water was added and titrated to a final volume of 100 parts by weight.

[0092] Comparative Example 10: Hydroxytyrosol / glycine / urea / pure water complex group. Using 100 parts by weight of the hydroxytyrosol complex as a baseline, 4 parts by weight of glycine and 10 parts by weight of urea were added to an appropriate amount of pure water at 75°C under nitrogen protection. The temperature was maintained, and the mixture was stirred at 200 rpm for 2 hours until completely dissolved. After cooling to room temperature, 1 part by weight of hydroxytyrosol was added, and the mixture was stirred at 200 rpm for 1 hour. Finally, pure water was added and titrated to 100 parts by weight.

[0093] Comparative Example 11: Hydroxytyrosol / glycine / lactic acid / pure water complex group. Based on 100 parts by weight of the hydroxytyrosol complex. At 75°C under nitrogen protection, 6 parts by weight of lactic acid were added to a reaction flask, followed by 4 parts by weight of glycine and pure water. The mixture was stirred at 200 rpm for 2 hours until completely homogeneous. After cooling to room temperature, 1 part by weight of hydroxytyrosol was added, and the mixture was stirred at 200 rpm for 1 hour. A pH adjuster was then added to bring the solution to a slightly acidic pH.

[0094] Table 2. Components, weight parts, and pH parameters of the hydroxytyrosol complexes in Comparative Examples 1-11.

[0095]

[0096] III. Effect Verification

[0097] Characterization of hydroxytyrosol supramolecular weights: Difference scanning calorimetry (DSC 214, NETZSCH, Germany) was performed at 10 °C / min. -1 The thermal behavior of BUL was analyzed by measuring the heating rate. A TG209F1 Libra thermogravimetric analyzer (NETZSCH, Germany) was used in a nitrogen atmosphere at a heating rate of 10 °C·min. -1 Thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DTG) were performed at the specified rates. A Spectrum Two spectrometer (PerkinElmer, USA) was used at 4000–400 cm⁻¹. -1 Within 4 cm -1Fourier transform infrared (FTIR) spectra were acquired at a high spectral resolution. Proton nuclear magnetic resonance (¹H NMR) spectra were recorded using an Avance III spectrometer (Bruker, Germany) with deuterated DMSO (DMSO-d6) as the solvent and δ = 2.5 ppm as the reference. X-ray diffraction (XRD) patterns were obtained at an angular frequency of 5 rad·s. -1 Data were acquired in oscillation amplitude scan mode (strain 0.1–100%). A rheometer (MCR302, Anton Paar, UK) was used in the range of 0.1–100 s. -1 Viscosity was determined within the shear rate range. Before measurement, the sample was equilibrated at 25°C for 5 minutes using a 20 mm parallel plate clamp with a 1 mm gap. The pH value was measured using a FE20 Plus pH meter (Mettler Toledo, USA) after calibration with two-point standard buffer. DPPH free radical scavenging experiment: The DPPH free radical scavenging ability was evaluated using a commercial kit (Yuanye, R27137) and according to the manufacturer's instructions. The simplified steps are as follows: nitrogen free radicals, sample solution, DPPH reagent, and anhydrous ethanol were added sequentially to the sample tube and control tube. After incubation at room temperature in the dark for 30 minutes, the absorbance value was measured at 517 nm and recorded as A0 (blank), A1 (sample), and A2 (sample control), respectively. ABTS free radical scavenging experiment: The ABTS free radical scavenging activity was determined using a total antioxidant capacity assay kit (T-AOC, S0119, Beyotime) and according to the manufacturer's instructions. An equal volume of ABTS reagent was mixed with an oxidant solution and incubated in the dark for 16 hours to prepare ABTS. + The solution was then diluted 30-fold with ethanol to achieve an absorbance of 0.70 ± 0.05 at 734 nm. For detection, 7 µL of sample or pure water (blank) was diluted with 280 µL of ABTS. + The solutions were mixed and reacted at room temperature for 6 minutes. The absorbance at 734 nm was then measured.

[0098] Statistical analysis: All experiments were performed in at least three replicates per group, and in vitro experiments were independently repeated at least three times. Data are expressed as mean ± standard error. One-way ANOVA was used for comparisons among multiple groups, and Student's t-test was used for comparisons between two groups. GraphPad Prism 8 software was used for statistical analysis, and the significance threshold was set at P < 0.05.

[0099] Figure 1-3 The results show that Examples 1-11 can effectively improve the color stability of HT in aqueous solution, and can prevent HT from being further oxidized and discolored in aqueous solution for a long time in both room temperature and high temperature environments.

[0100] The composite diagram of BUL and the resulting macroscopic photograph are as follows: Figure 4 A and Figure 4 As shown in Figure B, the absorbance measurements at 425 nm of HT@BUL (Example 11) and HT@H2O (Comparative Example 1) after being placed at room temperature for different times further verify that HT@BUL can significantly improve the stability of HT. Figure 4 C). Hydrogen nuclear magnetic resonance (1H NMR) spectrum ( Figure 4 D) Confirmed the existence of hydrogen bonding between B, Urea, and Lactic Acid. Using the methylene proton (B, –CH2–) data in betaine as a reference, the chemical shift of the hydrogen atom shifted from 3.54 (B) to 3.63 (B′), and the peak areas at A, C, and D decreased to A′, C′, and D′, respectively. The interaction between betaine, urea, and lactic acid was further investigated using Fourier transform infrared spectroscopy (FT-IR). Figure 4 E). The results showed that the characteristic absorption peaks of betaine, urea, and lactic acid were retained in BUL, reflecting information about the functional groups involved in the interaction. The absorption peak of the carbonyl group (C=O) in betaine is located at 1687 cm⁻¹. -1 The C=O stretching vibration peak in lactic acid is located at 1723 cm⁻¹. -1 The N–H stretching vibration peaks in urea are located at 3342 cm⁻¹. -1 and 3433 cm -1 A depth of 3348 cm can be observed in BUL. -1 and 1610 cm -1 The strong absorption bands at these locations correspond to the stretching vibrations of C=O and N–H, respectively. FTIR analysis revealed shifts in the C=O and N–H stretching vibrations, which can be attributed to electron density rearrangement caused by hydrogen bonding interactions between the C=O groups in betaine / lactic acid and the N–H groups in urea. Structural characterization of the synthesized BUL confirmed the formation of a broad hydrogen bond network among the betaine, urea, and lactic acid components.

[0101] The rheological properties of BUL have been optimized for local drug delivery; compared with binary supramolecular systems, the introduction of urea significantly reduces the viscosity of the system. Figure 4 F, Figure 4 G). DSC analysis showed thermal transitions at 91.6℃, 125.3℃, and 185.1℃, with a decomposition initiation temperature of 125℃ and a maximum decomposition rate corresponding to 200℃. Figure 4 HJ), confirming its thermal stability suitable for skin formulations. The XRD pattern shows broadened, diffuse peaks, consistent with the characteristics of the amorphous-nanocrystalline composite structure. Figure 4 These properties—including a broad hydrogen bond network, good thermal stability, amorphous structure, and low viscosity—combined indicate that BUL is suitable for transdermal drug delivery applications.

[0102] Both DPPH and ABTS experiments confirmed that BUL can effectively maintain the antioxidant activity of HT. Figure 5 A, Figure 5 B).

[0103] Skin permeability assay: The skin permeability efficiency of BUL was assessed using an inverted fluorescence microscope. Rhodamine B fluorescently labeled formulations were prepared using H₂O or BUL as solvents, applied to freshly avulsed mouse skin, and left for 2 hours. After rinsing with physiological saline, the samples were embedded in OCT embedding medium, sectioned to a thickness of 10 µm, and images were acquired using a DMi8 fluorescence microscope (Leica, Germany).

[0104] HT@BUL exhibits stronger lipophilicity, as evidenced by its reduced contact angle on the coated surface. Figure 5 C, Figure 5 D), indicating enhanced skin permeability. Rhodamine B permeation experiments show that when BUL is used as a carrier, its penetration depth in the skin is greater ( Figure 5 E, Figure 5 (F), demonstrating that BUL, as a transdermal drug delivery carrier, has good compatibility and effectiveness.

[0105] In vivo validation: After a one-week acclimatization period, seven-week-old healthy male mice were anesthetized, and a 2 cm × 2 cm area on their backs was treated with an electric shaver, followed by application of depilatory cream for 3 minutes. Mice were randomly assigned to groups. During the 16-day experimental period, except for the control group, all other groups received daily topical application of 200 µL of testosterone solution (0.5%, v / v) to induce androgenetic alopecia (AGA). Fifty minutes after application, the treatment groups received 200 µL of the corresponding formulation (H2O, HT solution, HT@BUL, or BUL). Histological and immunofluorescence analysis: On day 16, mice were sacrificed, and full-thickness skin samples were collected from the backs and fixed in 4% paraformaldehyde (PFA) for 24 hours. The fixed tissues were processed, embedded in paraffin, and sectioned to a thickness of 3–5 µm. For histological analysis, the sections were dewaxed, hydrated, and stained with hematoxylin and eosin (H&E). For immunofluorescence staining, dewaxed sections were incubated overnight at 4°C with primary antibodies (anti-Sox9 antibody, Huaan Biotechnology; anti-8-OHdG antibody, Bio-Sens) followed by incubation at room temperature with the corresponding fluorescent secondary antibody (Jackson). Cell nuclei were counterstained with DAPI (Beyotime). Cell proliferation was detected by the EdU incorporation assay using the Click-it EdU Alexa Fluor 488 imaging kit (Thermo Fisher Scientific, C10337). Images of all stained sections were acquired using a laser confocal microscope (FV3000RS, Olympus, Japan) and a fluorescence microscope (DMi8, Leica, Germany). Positive staining areas for each marker were quantitatively analyzed using ImageJ software.

[0106] To evaluate the in vivo efficacy of HT@BUL, we used a low concentration of HT (5 mg / mL). -1 ) and high concentrations of HT (10 mg·mL) -1 The BUL supramolecular complexes, namely HT@BUL-L (Example 10) and HT@BUL-H (Example 11), were used to treat the AGA model. Figure 6 A). On day 16, the Example 10 group showed the most significant hair growth effect, while the Example 11 group showed the same effect as HT (10 mg / mL). -1 The aqueous solution group was comparable, indicating the existence of a dose-dependent optimal effect range. Figure 6 B). However, BUL alone had minimal effect. Quantitative melanin analysis and scanning electron microscopy confirmed that the mice in Example 10 group had thicker and more structurally intact hair. Figure 6 (CF). The results in summary indicate that the 10th group of examples optimally promoted hair growth in terms of both hair coverage and diameter.

[0107] H&E and Masson staining of skin tissue treated on day 16 showed that the number of hair follicles increased in group 10, and the proportion of hair follicles located in the deep dermis was higher. Figure 7 AB). Skin thickness changes with the hair follicle cycle—thickening during the anagen phase and thinning during the telogen phase. Results showed that Group 10 (promoted anagen phase induction) contrasted with the AGA group and the pure water treatment group, which exhibited predominantly telogen follicles. Figure 7 CD). Histological analysis showed that the supramolecular complex carrying hydroxytyrosol could promote hair follicles to enter the growth phase, thereby achieving better hair regeneration results.

[0108] Immunofluorescence staining using the hair follicle stem cell (HFSC) marker SOX9 revealed a significant increase in the number of SOX9-positive cells in the Example 10 group. Figure 8 A, D). We used EdU (5-ethynyl-2'-deoxyuridine) staining to label newly synthesized strands during DNA replication. EdU incorporation experiments confirmed that the proliferation activity of HFSCs in Example 10 group was enhanced ( Figure 8 B, E). To evaluate the in vivo antioxidant effect of the Example 10 group, we measured the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage. The results showed that the fluorescence intensity of 8-OHdG in the Example 10 treatment group was significantly reduced (B, E). Figure 8 (C, F). These results indicate that the 10 group effectively alleviated oxidative DNA damage while creating a microenvironment conducive to the growth of hair follicle stem cells.

[0109] Cell compatibility assay: Immortalized human dermal papillary cells (HDPCs) were cultured at 1.6 × 10⁶ cells per well. 4HDPCs were seeded at a density of 1,000 cells / well in 96-well plates and treated with different concentrations of HT, BUL, or HT@BUL for 24 hours. Cell viability was assessed using the CCK-8 assay according to the manufacturer's instructions. Reactive oxygen species (ROS) levels were detected using the oxidation-sensitive fluorescent probe dihydroethidium (DHE, APExBIO). HDPCs were pretreated with HT, BUL, or HT@BUL for 24 hours and then exposed to H2O2 for 2 hours to induce oxidative stress. They were then incubated with 15 µM DHE in the dark for 30 minutes, washed with PBS, and fluorescence intensity was detected using a Varioskan LUX microplate reader (Thermo Scientific, USA) at 506 / 618 nm (excitation / emission wavelengths). The experiment was repeated three times. Live / dead cell staining: HDPCs were seeded at 1.6 × 10⁶ cells / well. 4 Cells were seeded in 96-well plates and cultured for 24 hours, followed by treatment with HT, BUL, or HT@BUL for 24 or 48 hours, respectively. Cells were stained using a live-dead cell double staining kit (ApexBIO) at 37°C in the dark for 25 minutes, and images were acquired using a DMi8 fluorescence microscope (Leica, Germany). Ki67 immunostaining of HDPCs: After 24 hours of drug pretreatment, HDPCs cultured in 96-well plates were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. After a second PBS wash, the cells were blocked with antibody dilution buffer for 30 minutes, and then incubated overnight at 4°C with rabbit Ki67 primary antibody (catalog number #9129, Cell Signaling Technology). After washing, the cells were incubated with fluorescent secondary antibody for 45 minutes, and the nuclei were counterstained with DAPI. Observation and recording were performed using a fluorescence microscope (DMi8, Leica, Germany). Senescence-related β-galactosidase staining: Cell senescence was detected using an SA-β-gal staining kit (Beyotime). HDPCs were fixed in 4% paraformaldehyde for 15 minutes, and then reacted with a solution containing 0.05 mg / mL. -1 X-gal staining buffer was incubated overnight at 37°C.

[0110] In the mammalian life cycle, hair follicles undergo a cyclical process of anagen (growth), catagen (regression), and telogen (resting) phases, driven by the periodic activation of hair follicle stem cells (HFSCs). Dermal papillary cells (DPCs) are one of the key cell types regulating HFSC behavior. DPCs guide HFSCs into different stages of the hair follicle cycle by secreting various cytokines and help maintain a stable number of hair follicles in the microenvironment. Therefore, the physiological state of DPCs is closely related to the overall health of the hair follicle.

[0111] Biocompatibility is a fundamental prerequisite for the application of all therapeutic agents or biomaterials in biological systems. HDPCs treated with HT, BUL, or HT@BUL for 24-48 hours maintained high cell viability as confirmed by CCK-8 assay and live / dead cell staining. Figure 9 (AG), indicating that the formulation has excellent biocompatibility.

[0112] This invention further verified the proliferative effects of HT, BUL, and HT@BUL on HDPCs. Ki67 staining showed that HT and HT@BUL enhanced HDPC proliferation, while BUL alone showed no activity. Figure 10 A, Figure 10 B). To establish an oxidative stress model, we induced oxidative damage in HDPCs using H2O2 treatment. First, HDPCs were treated with different concentrations of H2O2 for 2 hours, and ROS levels were detected by DHE staining and quantified using a microplate reader. Subsequently, to investigate the in vitro antioxidant effect of HT, HDPCs were pretreated with HT, BUL, and HT@BUL for 24 hours, respectively, and then oxidative stress was induced with 440 μM H2O2. ROS levels were measured under the same conditions to evaluate the protective effects of each agent. The results showed that in cells pretreated with HT, BUL, or HT@BUL for 24 hours, HT alleviated oxidative stress, with HT@BUL showing a more significant effect. Figure 10 C, Figure 10 D). β-galactosidase staining further showed that HT@BUL could significantly reduce the level of cellular senescence ( Figure 10 E, Figure 10 These results collectively demonstrate that HT@BUL promotes HDPC proliferation while mitigating oxidative stress and cellular senescence, thereby creating a favorable microenvironment for hair regeneration.

[0113] Based on existing evidence, this invention not only confirms that HT can serve as an effective agent for alleviating oxidative stress and promoting AGA hair regeneration, but also develops a supramolecular, multifunctional, and tunable transdermal drug delivery platform. By combining the antioxidant advantages of HT with the enhanced permeability and stability of the amino acid / organic acid / urea / pure water supramolecular formulation, the hydroxytyrosol supramolecular formulation exhibits significant therapeutic potential, demonstrating superior efficacy compared to conventional therapies in preclinical models. Further optimization of hydration levels and active compound concentrations allows for precise control of efficacy, safety, and applicability. These findings establish an ideal foundation for the development of next-generation topical therapeutic agents using the hydroxytyrosol / amino acid / organic acid / urea / pure water supramolecular formulation, with potential applications in dermatology, pharmaceuticals, cosmetics, and biocatalysis.

[0114] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0115] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0116] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A hydroxytyrosol supramolecular complex, characterized in that, The supramolecular complex comprises the following chemical components by mass fraction: Hydroxytyrosol: 0.01–10%, amino acid compounds: 2–45%, α-hydroxy acids: 2–20%, urea: 2–30%, water: 15–92%; The amino acid compounds include any one of glycine, arginine, serine, threonine, valine, and betaine; The α-hydroxy acid includes any one of lactic acid, citric acid, and malic acid.

2. The method for preparing a hydroxytyrosol supramolecular complex as described in claim 1, characterized in that, The preparation method includes: Under an inert atmosphere, α-hydroxy acids are heated and melted, then amino acid compounds and urea are added, and after homogenization, a reaction mixture is obtained. The temperature of the reaction mixture was adjusted to 20–90°C, water and hydroxytyrosol were added, and the mixture was homogenized to obtain a mixed solution. A pH adjuster was added to adjust the pH of the mixed solution to 4-8, yielding a hydroxytyrosol supramolecular complex. The mass fractions of each chemical component in the hydroxytyrosol supramolecular complex are as follows: Hydroxytyrosol: 0.01–10%, amino acid compounds: 2–45%, α-hydroxy acids: 2–20%, urea: 2–30%, water: 15–92%; The amino acid compounds include any one of glycine, arginine, serine, threonine, valine, and betaine; The α-hydroxy acid includes any one of lactic acid, citric acid, and malic acid.

3. The method for preparing a hydroxytyrosol supramolecular complex according to claim 2, characterized in that, The process involves heating and melting an α-hydroxy acid under an inert atmosphere, then adding an amino acid compound and urea, followed by homogenization to obtain a reaction mixture, specifically comprising: Under nitrogen protection, α-hydroxy acids were heated and melted, and then amino acid compounds and urea were slowly added. After homogenization for 0.5 to 4 hours, a reaction mixture was obtained. The process of adjusting the temperature of the reaction mixture to 20–90°C, adding water and hydroxytyrosol, and homogenizing to obtain a mixed solution specifically includes: Adjust the temperature of the reaction mixture to 20–90°C, add water and hydroxytyrosol, and homogenize for 0.5–12 h to obtain a mixed solution.

4. The method for preparing a hydroxytyrosol supramolecular complex according to claim 2, characterized in that, The pH adjuster includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, disodium citrate, trisodium citrate, disodium hydrogen phosphate, triethanolamine, and ammonia.

5. The use of the hydroxytyrosol supramolecular complex as described in claim 1 in the preparation of any one of antibacterial reagents, anti-inflammatory reagents, hair regeneration reagents, anti-aging reagents, and antioxidant reagents.

6. The use of the hydroxytyrosol supramolecular complex as described in claim 1 in the preparation of hair regeneration drugs or skin care products.

7. A hair regeneration drug or skin care product, characterized in that, The hair regeneration drug or the skin care product contains a hydroxytyrosol supramolecular complex as described in claim 1.

8. A hair regeneration drug or skin care product according to claim 7, characterized in that, The dosage form of the hair regeneration drug or the skin care product includes any one of the following: spray, paste, cream, patch, soluble microneedle, gel, and ointment.

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